In-silico studies on hydration in EcoRI-cognate DNA complex

In-silico studies on hydration in EcoRI-cognate DNA complex | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In-silico studies on hydration in EcoRI-cognate DNA complex Sasthi Charan Mandal, Jaydeb Chakrabarti This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2691161/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Restriction endonucleases (REs) cleave DNA at specific site in presence of Mg 2+ ion. Experiments further emphasize the role of hydration in metal ion specificity and sequence specificity of DNA cleavage. However, the relation between hydration and specificity has not been understood till date. This leads us to study via all-atom molecular dynamics (MD) simulations how the hydration around the scissile phosphate group changes in presence of Mg 2+ and Ca 2+ and depend on the DNA sequence. We observe the least number of hydrogen bonds around the scissile phosphate group in presence of Mg 2+ ion. We further find that the hydrogen bonds decrease at the scissile phosphate on mutating one base pair in the cleavage region of the DNA in Mg 2+ loaded EcoRI-DNA complex which makes the scissile phosphate group more accessible for the non-hydrogen bonded water molecules. We also perform steered MD simulations and observe that the rate of decrease of hydrogen bonds is slower in the mutated complex than the unmutated complex. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction REs are found in a wide range of bacteria, archaea, and viruses of certain unicellular algae. They protect the host cell from the incoming foreign DNA by cleaving it into fragments. REs are also used in gene cloning and protein expression. The DNA cleavage by REs is extraordinarily specific to DNA sequence, the type of metal ion cofactor and their positions in the RE. The microscopic mechanism of the cleavage and the specificities are largely not well-understood. Several experimental and theoretical studies suggest that Mg 2+ plays pivotal roles: It might aid in the binding and positioning of DNA, as well as partially neutralizing the extra negative charge in the transition state and protonate the leaving group after cleavage by providing a water molecule in its coordination sphere [ 1 ]. Our recent molecular dynamics (MD) simulation studies show that replacement of Mg 2+ ions by Ca 2+ ions in RE-DNA complex makes the cleavage region unstable and disordered due to enhanced fluctuations of the catalytically active residues and the cleavage base pairs of the DNA [ 2 – 6 ]. The other divalent metal ions such as Mn 2+ and Co 2+ also make the cleavage probability significantly lower [ 6 , 7 ]. The cleavage activity of RE involves specific contacts between DNA bases and amino acids of the enzyme. A change of a single base in the recognition sequence of REs lower the cleavage probability by more than a million-fold [ 8 ]. It is reported that under certain buffer conditions, such as changes in pH, very high enzyme to substrate ratio, presence of divalent metal cations and so on, REs lose specificity for their recognition sequence [ 9 – 11 ]. In such cases REs cleave at noncognate sequences which differ by a single base pair from the cognate sequence. The role of water molecules in DNA cleavage by RE has been emphasized in several experiments [ 12 , 13 ]. The cleavage of DNA by REs is shown to involve activation of a water molecule, followed by nucleophilic attack at the phosphodiester bond in presence of Mg 2+ . In particular, experimental data exist on how hydration changes during cleavage and recognition activities of EcoRI, a type II RE. EcoRI is a dimer, having two identical chains A and B where each chain consists of 261 residues. The recognition sequence of EcoRI is GAATTC, and the cleavage occurs between G and A on both strands in presence of Mg 2+ ions [ 12 ]. The residues ASP75 and GLU95 in EcoRI hold the metal ion [ 1 , 6 ]. Changes in hydration during binding of DNA to EcoRI show sensitivity to the recognition and cleavage of DNA [ 13 ]. It has been shown that the pro-Rp phosphoryl oxygen of the phosphate group on the 3' side of the scissile bond activates the attacking water molecule [ 1 ]. Experiments further suggest that EcoRI binding to the cognate sequence (GAATTC) releases 70 fewer water molecules than binding to a noncognate sequence (TAATTC) [ 13 ]. Moreover, the cleavage reaction is accompanied by the binding of 30 water molecules in cognate sequence [ 13 ]. Molecular dynamics simulations propose that one of the crystal water coordinated with Mg 2+ attacks the scissile phosphate, making Mg 2+ to behave as a Lewis acid. It is reported that one of the water molecule in the DNA recognition sequence, involved in hydrogen bond between ARG 200 and ARG 203 is released out of the EcoRI-DNA interface due to increase in osmotic pressure. This may be responsible for loss of specificity at cognate sequence, leading to cleave at the other sequence differ by a single base pair from the cognate sequence. However, the role of water molecules in the metal ion and sequence specificities is far from understood. The experimental observations call for detailed understanding of hydration patterns in EcoRI-DNA complexes. We perform classical all-atom Molecular Dynamics simulations on EcoRI-DNA complexes in various conditions. We focus in particular on how the distribution of water molecules and their hydrogen bonding pattern depends on the type of metal ion and the DNA sequence. We observe that the number of hydrogen bonded water molecules is the least around the scissile phosphate in the sequence of the Mg 2+ -EcoRI-DNA complex, and increases upon substitution of Mg 2+ by Ca 2+ . We mutate one of the cleavage base pair (AT) by GC in Mg 2+ -EcoRI-DNA complex and observe that the fraction of hydrogen bonded water molecules around the scissile phosphate group increases in the mutated complex compared to that in Mg 2+ -EcoRI-DNA. We also extend our calculations to steered molecular dynamics (SMD) simulations and stopped SMD simulations to investigate the dynamic evolution of hydration in DNA cleavage by EcoRI. We observe a lower rate of decrease of the fraction of hydrogen bonds in the mutated complex compared to that of Mg 2+ -EcoRI-DNA, indicating lower reaction kinetics in the mutated complex. Materials And Methods System preparations : The initial structure of free DNA that contains EcoRI recognition sequence (GAATTC) is modelled by using the NUCGEN software [ 14 ]. The DNA bases sequence is 5’-dCGC GAATTC GCG-3’, with the recognition sequence shown in bold, in 5’-3’ direction and the complementary sequence in 3’-5’ direction. The initial structure of EcoRI is obtained from protein data bank (PDB) (PDB ID: 1ERI). The crystal structure of EcoRI bound with DNA (EcoRI-DNA) is also taken from PDB with PDB ID 1ERI. Figure 1 (a) shows the numbering of phosphate, sugar and bases of DNA. The recognition sequence is shown by the box in dotted line. There are two active sites in EcoRI-DNA complex, one containing G 4 :C 21 and A 5 :T 20 base pairs, and ASP75 and GLU95 residues in chain A, and another one containing T 8 :A 17 and C 9 :G 16 base pairs, and ASP75 and GLU95 residues in chain B. The arrows in the figure shows the cleavage points. The complexes of metal ions, such as Mg 2+ and Ca 2+ loaded EcoRI-DNA are not available. To model these complexes, we follow the similar protocol as explained in our earlier publication [ 15 ]. We model Mg 2+ -EcoRI-DNA complex by inserting one Mg 2+ ion in the vicinity of each active site of EcoRI-DNA. Similarly, we model Ca 2+ -EcoRI-DNA complex by placing each Ca 2+ ion in the vicinity of each active site of EcoRI-DNA. As the two active sites are symmetrical in all the complexes, the calculations are averaged over two strands. Molecular dynamics simulations : The force-field of Amber99ff14SB [ 16 ] is used for the protein and Amberff99bsc0 [ 17 ] for the DNA. We use Ambertools 15 package [ 18 ] for the preparations of the systems. First, the systems are solvated in a cubic water box containing TIP3P water molecules in such a way that the solute is surrounded by at least 15 Å thick layers of water molecules with the periodic boundary conditions (PBC) are applied in all directions. Subsequently, the systems are neutralized with required number of Na + and Cl − ions. We use both steepest-descent and conjugate-gradient algorithms for the energy minimizations of the systems. The cut off of 10 Å is used for both Lennard-Jones and short-ranged electrostatic interactions. For long-ranged electrostatic interactions, we employ the particle mesh Ewald (PME) summation technique with 1 Å grid spacing and 10 − 6 convergence criteria. Starting with the energy minimized structure, we carry out MD simulations using NAMD [ 19 ] at 310 K and 1 atmospheric pressure in an isothermal–isobaric (NPT) ensemble. We use Langevin thermostat to control the temperature, while the pressure is maintained by using Nosé-Hoover Langevin piston. For all calculations, we use a simulation time step of 1 fs. 1 µs simulation is carried out for all the systems. The data are analysed from the equilibrated trajectory of 500–1000 ns for all cases. Steered molecular dynamics (SMD) simulations : We carry out SMD [ 20 ] simulations by applying a force to one end of the DNA along the DNA helix, which is aligned along z axis. The α carbons of EcoRI residues are restrained by a harmonic potential of force constant, \({k}_{res}\) =10 kcal mol −1 nm −2 , while the protein can move freely along x and y directions. The SMD force that varies with time, is calculated by the following formula, \(F\left(t\right)={k}_{SMD}[{z}_{SMD}\left(t\right)-{z}_{COM}\left(t\right)]\) , where \({z}_{COM}\left(t\right)\) is the z coordinate of the center of mass (COM) of DNA, \({z}_{SMD}\left(t\right)={z}_{COM}\left(0\right)+{v}_{SMD}t\) . \({v}_{SMD}\) = 0.5 nm ns −1 is the SMD velocity, and \({k}_{SMD}\) = 50,000 kcal mol −1 nm −2 is the force constant. In addition to the SMD simulations, we carry out a series of stopped SMD simulations. A stopped SMD simulation is the continuation of a constant-velocity SMD with the velocity set to zero. A stopped SMD simulation is described by the reference SMD simulation time \(t\) where the complex of EcoRI-DNA is subjected to a high strain rate. We carry out both SMD and stopped SMD simulations on EcoRI-DNA, Mg 2+ -EcoRI-DNA and Ca 2+ -EcoRI-DNA complexes. Analysis : Calculation of hydrogen bonds : For calculations of hydrogen bonds (HBs), we consider the following protocol: (i) the distance between the donor O atom and acceptor O atom is less or equal to 0.34 nm and (ii) O-H (donor) – O (acceptor) angle is greater or equal to 150 0 . Conformational Thermodynamics The detailed calculations of the DNA step parameters and the calculations of the changes in conformational thermodynamics is discussed in the Methods section in our previous publication [ 15 ]. Displacements of nucleotides : The displacement of nucleotide from the interaction regime of EcoRI is characterized as follows: first, for each nucleotide, we calculate the distance between each atom of a particular nucleotide and each atom of EcoRI residues. If the distance is less than 0.45 nm, we make a list of the atom pairs ( \(ij\) ), where atom \(i\) belongs to EcoRI, and atom \(j\) belongs to DNA. We compute the average distance of the atom pairs for each nucleotide in a single snapshot, denoted as \({d}_{ij}\) . Finally, we calculate the average distance ( \(\frac{1}{n}{)\) of the atom pairs of each nucleotide over the last 10 ns of the equilibration. \(n\) is the number of pairs. we also calculate \(\frac{1}{n}{d}_{ij}\) from the SMD trajectory for each snapshot. Finally, the displacement of each nucleotide is computed as R \(=\frac{1}{n}\sum _{ij}({d}_{ij}-)\) . We consider a nucleotide being ruptured if R \(>\) 0.25 nm during the SMD simulation. We follow the reference [ 21 ] for calculating the displacements of nucleotides. Results And Discussion We study the following systems using all-atom molecular dynamics simulations: (1) EcoRI and DNA containing cognate sequence (EcoRI-DNA), (2) EcoRI-DNA in presence of Mg 2+ ions (Mg 2+ -EcoRI-DNA), (3) EcoRI-DNA in presence of Ca 2+ ions (Ca 2+ -EcoRI-DNA) (4) EcoRI with mutated DNA where A 5 :T 20 base pair in the DNA is mutated by G 5 :C 20 , in presence of Mg 2+ ions (Mg 2+ -EcoRI-mtDNA) and (5) Free DNA containing the cognate sequence. The co-ordinations of Mg 2+ in Mg 2+ -EcoRI-DNA complex, Ca 2+ in Ca 2+ -EcoRI-DNA and Mg 2+ in Mg 2+ -EcoRI-mtDNA obtained from MD trajectories are shown in Figs. 1 (b)-(d) respectively. As the co-ordinations of the divalent metal ions in the two active sites in each complex are identical, we only show those of the divalent metal ion in the first active site. If the average distance between any heavy atom of DNA or water or EcoRI and metal ion is less or equal to 3.0 Å, coordination is considered. OD1 of ASP75, OE1 of GLU95 and OP1 of P 5 participate in coordination with Mg 2+ . Similarly, OD1 and OD2 of ASP75, OE1 and OE2 of GLU95 and OP1 of P 5 participate in coordination with Ca 2+ . The co-ordinations of Mg 2+ in Mg 2+ -EcoRI-mtDNA are same as those of Mg 2+ in Mg 2+ -EcoRI-DNA. The rest of the co-ordinations of Mg 2+ and Ca 2+ are satisfied by the O atoms of water molecules. Equilibrium hydration pattern We calculate the water density around a given chemical moiety. First, we consider the metal ions. Figure 2 (a) shows the density of water, \({{\rho }}^{\text{M}\text{g}}\) (r) and \({{\rho }}^{\text{C}\text{a}}\) (r) around Mg 2+ in Mg 2+ -EcoRI-DNA complex and Ca 2+ in Ca 2+ -EcoRI-DNA complex respectively, where r is distance between the metal ion and water oxygen. For all cases, the distributions show two well defined peaks. The first peak in \({{\rho }}^{\text{M}\text{g}}\) (r) appears at r = 0.22 nm, while the second peak is at r = 0.29 nm. The density is observed to be maximum of 3.1 gm/cc at 0.22 nm. As the radius of Ca 2+ is greater than that of Mg 2+ , the peaks in \({{\rho }}^{\text{C}\text{a}}\) (r) is further away from Ca 2+ than those in \({{\rho }}^{\text{M}\text{g}}\) (r). The first peak arises at 0.25 nm, while the second peak at 0.32 nm. Here, the maximum density is 1.98 gm/cc which occurs at 0.25 nm. Thus, Mg 2+ is better hydrated than Ca 2+ due to smaller size and consequently, larger charge density. Next, we consider the hydration of the phosphate groups of the DNA in various cases. We calculate the density of water molecules, \({{\rho }}_{{\alpha }}^{0}\left(r\right)\) , \({{\rho }}_{{\alpha }}^{\text{c}}\left(r\right)\) , \({{\rho }}_{{\alpha }}^{\text{M}\text{g}}\left(r\right)\) , \({{\rho }}_{{\alpha }}^{\text{C}\text{a}}\left(r\right)\) and \({{\rho }}_{{\alpha }}^{Mut}\left(r\right)\) around phosphate group ( \(\alpha\) ) in free DNA, EcoRI-DNA, Mg 2+ -EcoRI-DNA, Ca 2+ -EcoRI-DNA and Mg 2+ -EcoRI-mtDNA complexes respectively where \(r\) is the distance between phosphorus atom and the oxygen of water. We compare the cases for α = P5 in the cleavage region and α = P7 in the non-cleavage region in Figs. 2 (b) and (c) respectively. \({{\rho }}_{\text{P}5}^{0}\left(r\right)\) in the free DNA shows a peak at 0.4 nm with maximum density 0.82 gm/cc, while the other peak at 0.6 nm has similar maximum density. \({{\rho }}_{\text{P}5}^{\text{c}}\left(r\right),\) in EcoRI-DNA complex without any metal ion shows a well-defined maximum at 0.43 nm, while it shows another broad peak around 0.48 nm. Similarly, \({{\rho }}_{\text{P}5}^{\text{M}\text{g}}\left(r\right)\) shows two well defined maxima. The position of the first maximum in \({{\rho }}_{\text{P}5}^{\text{M}\text{g}}\left(r\right)\) is smaller than that in \({{\rho }}_{\text{P}5}^{0}\left(r\right)\) . On the other hand, the first peak in \({{\rho }}_{\text{P}5}^{\text{C}\text{a}}\left(r\right)\) is comparable to that in \({{\rho }}_{\text{P}5}^{0}\left(r\right)\) . This indicates that P 5 is more hydrated in Mg 2+ -EcoRI-DNA complex than both EcoRI-DNA and Ca 2+ -EcoRI-DNA due to larger hydration around Mg 2+ ion. On the other hand, the density of water around P7 show two different peaks at 0.3 nm and 0.4 nm for all the cases. Thus, the hydration level of the phosphate groups at the cleavage site is sensitive to the type of metal ions but not so at the non-cleavage site. We calculate the fraction of water molecules in the first coordination shell, defined by the first peak in the density profile, having hydrogen bonds with O atoms of the phosphate groups in the recognition region. We denote the fraction by \({f}_{{\alpha }}^{0}\) , \({f}_{{\alpha }}^{\text{c}}\) , \({f}_{{\alpha }}^{\text{M}\text{g}}\) and \({f}_{{\alpha }}^{\text{C}\text{a}}\) in free DNA, EcoRI-DNA, Mg 2+ -EcoRI-DNA and Ca 2+ -EcoRI-DNA respectively. As the two strands are symmetric, the results shown in Fig. 2 (d) in the recognition region are averaged over both strands. For free DNA, the values of \({f}_{{\alpha }}^{0}\) for all the phosphate groups in recognition region are similar. However, for the complexes, the results show sequence specificity. In EcoRI-DNA complex, \({f}_{\text{P}5}^{\text{c}}\) shows a minimum value. The values of \({f}_{\alpha }^{\text{c}}\) for the other phosphate groups are similar to those in free DNA. In Mg 2+ -EcoRI-DNA complex, the minimum of \({f}_{\text{P}5}^{\text{M}\text{g}}\) is even deeper and a maximum at P 7 is observed unlike in the EcoRI-DNA complex. In Ca 2+ -EcoRI-DNA complex, \({f}_{\text{P}5}^{\text{C}\text{a}}\) has a minimum comparable to that in EcoRI-DNA complex. The maximum of \({f}_{\text{P}7}^{\text{C}\text{a}}\) is similar to that of \({f}_{\text{P}7}^{\text{M}\text{g}}\) . Thus, the data suggest that the hydrogen bond formation is specific to the kind of the metal ion. The number of hydrogen bonded water molecules at the cleavage site is the least in case of Mg 2+ -EcoRI-DNA complex. We compare the fluctuations of base pair step parameters in EcoRI-DNA, Mg 2+ -EcoRI-DNA and Ca 2+ -EcoRI-DNA complexes with those in free DNA. A given Watson-Crick paired bases is taken to define a base pair. The base pair step parameters define the relative displacement (shift, slide and rise) and orientations (tilt, roll and twist) of two successive base pairs.[ 22 ] For a given base pair step (i, i + 1), the distribution is denoted by \({\text{H}}_{\text{i},\text{i}+1}^{\text{f}}\) (b) in the free state and \({\text{H}}_{\text{i},\text{i}+1}^{\text{c}}\) (b) in the complex for a given base pair step parameter b where i and i + 1 are two successive bases in a given strand, omitting the WC paired bases. The distributions in free DNA, Mg 2+ -EcoRI-DNA and Ca 2+ -EcoRI-DNA complexes are denoted as \({\text{H}}_{\text{i},\text{i}+1}^{0}\) (b), \({\text{H}}_{\text{i},\text{i}+1}^{\text{M}\text{g}}\) (b) and \({\text{H}}_{\text{i},\text{i}+1}^{\text{C}\text{a}}\) (b) respectively. The base pair parameters are taken to be conformational variables for the DNA in our previous analysis [ 2 ]. Here, we illustrate the data for base pair step, (A 5 :T 20 , A 6 :T 19 ) in the cleavage region. We show the histograms in Supplementary Information (SI), Figs. S1(a)-(f). τ for all cases are broad single peaked (see SI, Fig. S1 (a)). However, the peak heights of \({\text{H}}_{\text{5,6}}^{0}\) (τ) and \({\text{H}}_{5,6}^{\text{C}\text{a}}\) (τ) are smaller than that of \({\text{H}}_{5,6}^{\text{M}\text{g}}\) (τ). The distributions of ρ (see SI, Fig. S1 (b)) and ω (see SI, Fig. S1 (c)) are all single peaked in all the cases. However, the peak height is smaller and broader in \({\text{H}}_{5,6}^{0}\) (ω) compared to the other complexes. Although, Dx shows single peaked distributions for all cases, the peak height for \({\text{H}}_{5,6}^{\text{M}\text{g}}\) (Dx) is larger than that of other complexes (see SI, Fig. S1 (d)). Similarly, the distributions of Dy (see SI, Fig. S1 (e)) and Dz (see SI, Fig. S1 (f)) are single peaked in all complexes. However, the peak height of the distributions of Dy is highest for \({\text{H}}_{5,6}^{\text{M}\text{g}}\) (Dy). Thus, we observe that the distributions of rotational parameters of the base pair steps are broader in all cases than those of translational parameters. We quantify the fluctuations in terms of the changes in conformational free energy and entropy of DNA in complexes with respect to the free DNA.[ 23 ] A negative change in the free energy denotes stabilization and that in entropy denotes enhanced order in the complexed DNA with respect to the reference free DNA. For a given step parameter, the changes in free energy and entropy of ( \(\text{i}, \text{i}+1\) ) step in the Mg 2+ -EcoRI-DNA complex with respect to the free DNA are denoted as \({{\Delta }\text{G}}_{\text{i},\text{i}+1}^{\text{M}\text{g}}\) (b) and \({\text{T}{\Delta }\text{S}}_{\text{i},\text{i}+1}^{\text{M}\text{g}}\) (b) respectively. Similarly, the analogous quantities for Ca 2+ -EcoRI-DNA complex with respect to the free DNA are denoted by \({{\Delta }\text{G}}_{\text{i},\text{i}+1}^{\text{C}\text{a}}\) (b) and \({\text{T}{\Delta }\text{S}}_{\text{i},\text{i}+1}^{\text{C}\text{a}}\) (b). The total free energy ( \({{\Delta }\text{G}}_{\text{i},\text{i}+1}^{\text{M}\text{g}}\) and \({{\Delta }\text{G}}_{\text{i},\text{i}+1}^{\text{C}\text{a}}\) ) and entropy changes ( \({\text{T}{\Delta }\text{S}}_{\text{i},\text{i}+1}^{\text{M}\text{g}}\) and \({\text{T}{\Delta }\text{S}}_{\text{i},\text{i}+1}^{\text{C}\text{a}}\) ) of a base pair step parameters of DNA in the complexes with respect to those in free DNA, adding the individual contributions, are shown in Figs. 2 (e) and (f) respectively. The base pair steps are highly stabilized in Mg 2+ -EcoRI-DNA. Figure 2 (e) shows that the base pair step \({{\Delta }\text{G}}_{\text{4,5}}^{\text{M}\text{g}}\) in cleavage region of Mg 2+ -EcoRI-DNA shows maximum conformational stabilization. Figure 2 (f) shows that \({\text{T}{\Delta }\text{S}}_{\text{4,5}}^{\text{M}\text{g}}\) has maximum order compared to the other base pair steps and that at \({\text{T}{\Delta }\text{S}}_{\text{6,7}}^{\text{M}\text{g}}\) pair has a minimum. In Ca 2+ -EcoRI-DNA complex (Fig. 2 (e)), the stabilization in \({{\Delta }\text{G}}_{\text{4,5}}^{\text{C}\text{a}}\) is maximum but it is lower compared to that in Mg 2+ -EcoRI-DNA. Here, \({{\Delta }\text{G}}_{\text{6,7}}^{\text{C}\text{a}}\) shows destabilization. The base pair steps in Ca 2+ -EcoRI-DNA are all ordered with a similar value of entropy change − 3.0 kJ/mol, except \({\text{T}{\Delta }\text{S}}_{\text{6,7}}^{\text{C}\text{a}}\) (Fig. 2 (f)). Next, we consider a mutated DNA sequence, denoted by Mg 2+ -EcoRI-mtDNA where we mutate the cleavage base pair A-T in the first active site by G-C in Mg 2+ -EcoRI-DNA complex. First, we show the density profile of water molecules, \({{\rho }}_{\text{P}5}^{\text{M}\text{u}\text{t}}\) and \({{\rho }}_{\text{P}7}^{\text{M}\text{u}\text{t}}\) in the first strand of the mutated complex in Figs. 2 (b) and (c) respectively. We observe that the density of water around P5 and P7 in the mutated complex are similar to those in Mg 2+ -EcoRI-DNA. The fraction of hydrogen bonds, \({\text{f}}_{{\alpha }}^{\text{M}\text{u}\text{t}}\) for different \({\alpha }\) of Mg 2+ -EcoRI-mtDNA is shown in Fig. 2 (d). It is observed that \({\text{f}}_{{\alpha }}^{\text{M}\text{u}\text{t}}\) has a minimum at P5. However, \({\text{f}}_{\text{P}5}^{\text{M}\text{u}\text{t}}\) > \({\text{f}}_{\text{P}5}^{\text{M}\text{g}}\) . Thus, the hydrogen bonding network is sequence specific and sensitive to the mutation of the DNA base. Time dependent hydration pattern We investigate the changes in hydration pattern, while the DNA gets unbound from the binding pocket of EcoRI. We show the forces \({\text{F}}^{0}\) , \({\text{F}}^{\text{M}\text{g}}\) and \({\text{F}}^{\text{C}\text{a}}\) experienced by the systems due to unbinding of DNA from EcoRI for EcoRI-DNA, Mg 2+ -EcoRI-DNA and Ca 2+ -EcoRI-DNA complexes respectively in Fig. 3 . For all the cases, the forces increase gradually until they reach a maximum, and after that they gradually decrease. The force is maximum when all the nucleotides of the DNA come out from the interaction regime of protein. This is in agreement to an earlier report [ 21 ]. The maximum of the force is larger in Mg 2+ -EcoRI-DNA than all other cases. This indicates that the interactions between EcoRI and DNA is strongest in Mg 2+ -EcoRI-DNA. We also follow the unbinding of the nucleotides. As time progresses, the Nβ nucleotide of the DNA fragment unbinds. The distances of the centre of mass of a nucleotide which contains a phosphate group, a sugar group and one of the four bases, with respect to the centre of mass of the binding residues of EcoRI are denoted by \({\text{R}}_{{\beta }}^{0}\) , \({\text{R}}_{{\beta }}^{\text{M}\text{g}}\) and \({\text{R}}_{{\beta }}^{\text{C}\text{a}}\) for EcoRI-DNA, Mg 2+ -EcoRI-DNA and Ca 2+ -EcoRI-DNA respectively. Here Nβ = N4-N9 for the first strand and Nβ = N16-N21 for the second strand. The data are shown in SI, Fig. S2. The jump in the displacement denote the unbinding of the corresponding nucleotide. Based on jumps in the distances with time, we show in Table 1 the times at which different nucleotides are liberated from the binding pocket. We first consider EcoRI-DNA, shown in SI, Figs. S2(a) and (b) for the first and second strands respectively. \(\text{N}20\) in the second strand breaks away first from the interaction regime of EcoRI around 1.35 ns. \(\text{N}19\) and \(\text{N}21\) break away at ⁓ 2.05 ns and 2.9 ns respectively. After that around 3.55 ns, N4 in the first strand leaves the binding site. In the range 4-5.5 ns, N5-N7 in the first strand and N18 in the second strand leave the binding site. All the nucleotides in the recognition region of first strand leave around 6.9 ns, while those in the second strand leave around 6.6 ns, yielding the total rapture time 6.9 ns. The sequence of nucleotide rupture is similar to the earlier study.[ 21 ] However, the rupture times are somewhat less in our system which may be due to differences in force fields in two cases. Next, we consider the Mg 2+ -EcoRI-DNA complex (SI, Figs. S2(c) and (d) for the first and second strands respectively). In EcoRI-DNA, the nucleotide starts breaking away at 1.35 ns, whereas in Mg 2+ -EcoRI-DNA complex, this time is 3 ns. Both N20 and N21 in the second strand are ruptured at 3 ns. N4 and N19 break away at 3.4 ns and 3.9 ns respectively. N18 breaks away at 4.95 ns. N4-N6 rupture gradually in a gap of ⁓ 1 ns, while both N7 and N8 break away approximately at the same time (⁓ 5.8 ns). N16 and N17 in the second strand leave the binding site around ⁓ 6.5 ns, while the remaining nucleotide N9 in the first strand breaks away around ⁓ 8 ns. Thus, the rupture time in Mg 2+ -EcoRI-DNA complex is ~ 8 ns. It indicates that the interactions between EcoRI and DNA is stronger in Mg 2+ -EcoRI-DNA than in EcoRI-DNA. The sequence of events in Ca 2+ -EcoRI-DNA complex is different. The data for the first and second strands are shown in SI, Figs. S2 (e) and (f) respectively. In this complex, N4, N20 and N21 leave the binding site approximately at the same time ⁓ 3.7 ns. N19 breaks away at ⁓ 4.05 ns. After that all four nucleotides N5, N6, N7 and N9 in the first strand break away at approximately same time (⁓ 5.1 ns) (SI, Fig. S2(e)). N18 breaks away at ⁓ 5.6 ns, while N8 breaks away around ⁓ 6 ns. Here, N9 is ruptured before N8 (SI, Fig. S2(e)). N16 comes out from the binding pocket around ⁓ 6 ns. N17 breaks away last at ⁓ 6.55 ns, while in Mg 2+ -EcoRI-DNA the last nucleotide is N9 that breaks away at ⁓ 8 ns, indicating Mg 2+ -EcoRI-DNA complex is more stable than Ca 2+ -EcoRI-DNA. Now we consider the mutated complex (SI, Figs. S2 (g) and (h) for the first and second strands respectively). N21 is ruptured first at around ⁓3.2 ns, while N4 at 3.6 ns in the first strand. In the range 4–6 ns, all the nucleotides except nucleotide 9 in the first strand, and N19 and N20 in the second strand break away. The nucleotides N9, N16-N18 all break away at approximately same time ⁓6.35 ns. The rapture time in Mg 2+ -EcoRI-mtDNA is ⁓6.35 ns. This indicates higher stability of the Mg 2+ -EcoRI-DNA complex compared to the Mg 2+ -EcoRI-mtDNA complex. We show histogram, H(t) of the breaking times, t of the nucleotides in different cases in Fig. 3 (b). The histogram shows three different maxima at 3.5 ns, 5 ns and 6 ns. Hence, we carry out stopped SMD simulations by considering snapshots at these times from the SMD trajectory for all the complexes. In addition to these times, we perform stopped SMD simulations of the snapshots at 4 ns corresponds to the first minimum of the histogram and at 6.5 ns where most of the nucleotides come out from the binding pocket of EcoRI. We calculate the fraction of hydrogen bonds, \({\text{f}}_{\alpha }^{\text{c}}\) , \({\text{f}}_{{\alpha }}^{\text{M}\text{g}}\) , \({\text{f}}_{{\alpha }}^{\text{C}\text{a}}\) and \({\text{f}}_{{\alpha }}^{\text{M}\text{u}\text{t}}\) at these times, shown in Fig. 4 . Let us consider the scissile phosphate P5 in Fig. 4 (a). \({\text{f}}_{\text{P}5}^{\text{c}}\) does not show significant changes with time. The values of \({\text{f}}_{\text{P}5}^{\text{C}\text{a}}\) at different times are similar to those of \({\text{f}}_{\text{P}5}^{\text{c}}\) . However, \({\text{f}}_{\text{P}5}^{\text{M}\text{g}}\) shows much lower values at lower times compared to those of \({\text{f}}_{\text{P}5}^{\text{c}}\) and approaches \({\text{f}}_{\text{P}5}^{\text{c}}\) at larger times. Similar time dependent growth is observed in \({\text{f}}_{\text{P}5}^{\text{M}\text{u}\text{t}}.\) In both cases the time dependences are linear as shown by the fitted curves with slopes 0.14 and 0.07 for Mg 2+ -EcoRI-DNA and Mg 2+ -EcoRI-mtDNA cases respectively. Thus, the growth for the mutated complex is not as rapid as that in the case of the wild type complex. On the other hand, \({\text{f}}_{\text{P}7}^{\text{c}}\) , \({\text{f}}_{\text{P}7}^{\text{M}\text{g}}\) , \({\text{f}}_{\text{P}7}^{\text{C}\text{a}}\) and \({\text{f}}_{\text{P}7}^{\text{M}\text{u}\text{t}}\) do not show significant changes with time as shown in Fig. 4 (b). It may be noted that the phosphodiester bond cleavage cannot be observed from classical MD simulations. However, our studies based on hydration pattern from classical MD trajectories give significant insight to the role of the metal ion and DNA sequence. The smaller metal ion like Mg 2+ is solvated more efficiently than larger ion like Ca 2+ . This is favourable from the stand point of activation of water molecules for cleavage [ 6 , 24 , 25 ] The overall fluctuations in the microscopic conformational variables in the cleavage region is less in Mg 2+ -EcoRI-DNA compared to the Ca 2+ -EcoRI-DNA. This scenario is quite analogous to that of EcoRV. The enhanced stability and order are pre-requisite for positioning the metal ion in the vicinity of the scissile phosphate and is more efficiently done in Mg 2+ -EcoRI-DNA than in Ca 2+ -EcoRI-DNA. It may be noted that the maximum stabilization and ordering in (4,5) step of Mg 2+ -EcoRI-DNA complex has the presence of lowest number of hydrogen bonded water molecules. On the other hand, the most destabilized and disordered (6,7) step has the maximum number of hydrogen bonded water molecules. Less abundance of H-bonds indicates that the scissile phosphate group in this complex is more accessible to have the nucleophilic attack by a non-hydrogen bonded water molecule. A single base pair mutation in Mg 2+ -EcoRI-DNA significantly reduces the fraction of hydrogen bonds around the scissile phosphate group. Time dependent changes in hydrogen bond fraction during the unbinding gives idea about the kinetics of the cleavage process. The increase in the fraction \({\text{f}}_{\text{P}5}^{\text{M}\text{u}\text{t}}\) and \({\text{f}}_{\text{P}5}^{\text{M}\text{g}}\) while the unbinding may also be taken to indicate decreases in the fraction as the DNA enters into the binding pocket. Thus, the slower rate of increase in mutated complex than that of Mg 2+ -EcoRI-DNA suggests a lower cleavage reaction in the mutated complex. Conclusion To summarize, we investigate hydration in EcoRI-cognate DNA complex. The data suggest that the water density around scissile phosphate group is higher in Mg 2+ -EcoRI-DNA complex. However, the hydrogen bond network around this phosphate group is less strong in this complex compared to those in other complexes, formed by replacing the ion or mutating the sequence. Time dependent changes in hydration indicates that the kinetics becomes slower after mutation of a single base pair in the cognate DNA sequence of Mg 2+ -EcoRI-DNA complex. Thus, our studies may be helpful for microscopic understanding of the cleavage process. The hydration pattern can be probed by spectroscopic measurements which may open up further understanding on specificity of DNA cleavage by RE. Declarations Acknowledgement SCM thanks to Council of Scientific & Industrial Research (CSIR) for providing the fellowship. SCM and JC thank the Thematic Unit of Excellence (TUE) and the Technical Research Centre (TRC) at S. N. Bose National Centre for Basic Sciences for computational facilities. Data availability Data are freely available upon request of the corresponding author. Author contributions SCM performed the calculations and the analyses, and wrote the paper. JC planned and supervised the project, and corrected the manuscript. Ethics Declarations Conflict of interest The authors declare no competing interests. References A. Jeltsch, J. Alves, G. Maass, A. Pingoud, FEBS letters , 1992 , 304 , 4–8. S. C. Mandal, L. Maganti, M. Mondal, J. Chakrabarti, Biopolymers , 2020 , 111 , e23396. W. A. Loenen, D. T. Dryden, E. A. Raleigh, G. G. Wilson, N. E. Murray, Nucleic acids research , 2013 , 42 , 3–19. A. Belkebir, H. Azeddoug, Microbiological research , 2013 , 168 , 99–105. I. B. Vipond, G. S. Baldwin, S. E. Halford, Biochemistry , 1995 , 34 , 697–704. A. Pingoud, A. Jeltsch, European Journal of Biochemistry , 1997 , 246 , 1–22. A. Pingoud, G. G. Wilson, W. Wende, Nucleic acids research , 2014 , 42 , 7489–7527. D. R. Lesser, M. R. Kurpiewski, L. Jen-Jacobson, Science , 1990 , 250 , 776–786. M. Hsu, P. Berg, Biochemistry , 1978 , 17 , 131–138. R. C. Gardner, A. J. Howarth, J. Messing, R. J. Shepherd, DNA , 1982 , 1 , 109–115. J. M. ROSENBERG, P. GREENE, Dna , 1982 , 1 , 117–124. A. Jeltsch, J. Alves, H. Wolfes, G. Maass, A. Pingoud, Proceedings of the National Academy of Sciences , 1993 , 90 , 8499–8503. C. R. Robinson, S. G. Sligar, Proceedings of the National Academy of Sciences , 1998 , 95 , 2186–2191. M. Bansal, D. Bhattacharyya, B. Ravi, Bioinformatics , 1995 , 11 , 281–287. S. C. Mandal, L. Maganti, M. Mondal, J. Chakrabarti, Biopolymers , e23396. J. A. Maier, C. Martinez, K. Kasavajhala, L. Wickstrom, K. E. Hauser, C. Simmerling, Journal of chemical theory and computation , 2015 , 11 , 3696–3713. A. Pérez, I. Marchán, D. Svozil, J. Sponer, T. E. Cheatham III, C. A. Laughton, M. Orozco, Biophysical journal , 2007 , 92 , 3817–3829. D. A. Case, J. T. Berryman, R. M. Betz, D. S. Cerutti, T. E. Cheatham III, T. A. Darden, R. E. Duke, T. J. Giese, H. Gohlke, A. W. Goetz, There is no corresponding record for this reference.[Google Scholar] , 2015 . J. C. Phillips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C. Chipot, R. D. Skeel, L. Kale, K. Schulten, Journal of computational chemistry , 2005 , 26 , 1781–1802. B. Isralewitz, S. Izrailev, K. Schulten, Biophysical journal , 1997 , 73 , 2972–2979. B. Dorvel, G. Sigalov, Q. Zhao, J. Comer, V. Dimitrov, U. Mirsaidov, A. Aksimentiev, G. Timp, Nucleic acids research , 2009 , 37 , 4170–4179. P. S. Ho, M. Carter, DNA structure: alphabet soup for the cellular soul, DNA Replication-Current Advances . IntechOpen, , 2011. A. Das, J. Chakrabarti, M. Ghosh, Biophysical journal , 2013 , 104 , 1274–1284. J. Heitman, BioEssays , 1992 , 14 , 445–454. A. Pingoud, M. Fuxreiter, V. Pingoud, W. Wende, Cellular and molecular life sciences , 2005 , 62 , 685. table Table 1. Time scales of breaking up the nucleotides. Nucleotides in recognition region 1 st strand (Time of break up) of DNA in 2 nd strand (Time of break up) of DNA in EcoRI-DNA (ns) Mg 2+ -EcoRI-DNA (ns) Ca 2+ -EcoRI-DNA (ns) Mg 2+ -EcoRI-mtDNA (ns) Nucleotides in recognition region EcoRI-DNA (ns) Mg2+-EcoRI-DNA (ns) Ca 2+ -EcoRI-DNA (ns) Mg 2+ -EcoRI-mtDNA (ns) N4 3.55 3.4 3.65 3.6 N21 2.9 3 3.55 3.2 N5 4.55 4.35 5.0 4.55 N20 1.35 3 3.85 5.1 N6 5.25 5.2 5.05 4.5 N19 2.05 3.9 4.05 4.45 N7 5.2 5.75 5.2 5.25 N18 3.8 4.95 5.6 6.3 N8 6.25 5.85 5.9 5.3 N17 6.6 6.55 6.55 6.35 N9 6.9 7.2 5.1 6.35 N16 4.75 6.4 6.1 6.35 Additional Declarations No competing interests reported. Supplementary Files SI.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2691161","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":184802059,"identity":"14a5e5a5-6cff-4417-8a86-ca724ba74f6f","order_by":0,"name":"Sasthi Charan Mandal","email":"","orcid":"","institution":"S. N. Bose National Centre for Basic Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sasthi","middleName":"Charan","lastName":"Mandal","suffix":""},{"id":184802060,"identity":"f77a348d-a7ec-47aa-abbf-c0dc462ac585","order_by":1,"name":"Jaydeb Chakrabarti","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACxgYGBgnGBpsEuAgbkVrSSNACAkAthxMIKUIA5vYzhjc+7jifx89/+ABzRUUdA590AwGH9eQYW848c7tYckZaAuOZM4cZ2GQOENDSkGMmzdt2O3HDDR4Dxsa2AwxsEgQcydj/xkz6b9u5xA3nz39gbPxXR4SWGUBbGNsOJG44kMPA2NjATIyWZ8WWvWeSQX4xONhw7DAPQS2G/ckbb/zcYQcKsYcPG2rq5ORnENLSwGEA5xwAYh786oFAnoH9AUFFo2AUjIJRMMIBAOBrROR4PF/7AAAAAElFTkSuQmCC","orcid":"","institution":"S. N. Bose National Centre for Basic Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jaydeb","middleName":"","lastName":"Chakrabarti","suffix":""}],"badges":[],"createdAt":"2023-03-14 10:14:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2691161/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2691161/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34653493,"identity":"a32c40ab-b577-4d68-a6f3-9b77c71232eb","added_by":"auto","created_at":"2023-03-22 14:37:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1091230,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Labelling of DNA functional groups. The recognition region is shown in dotted box. The arrows indicate where the cleavage occurs. Co-ordinations of (b) Mg\u003csup\u003e2+\u003c/sup\u003e in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA, (c) Ca\u003csup\u003e2+\u003c/sup\u003e in Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA and (d) Mg\u003csup\u003e2+\u003c/sup\u003e in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2691161/v1/6b900c9f07bf1d458cbaacfa.png"},{"id":34654874,"identity":"4f672341-c2af-4799-9370-bf7f5acebc06","added_by":"auto","created_at":"2023-03-22 14:45:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":782793,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2691161/v1/ab9b09931d454b2c24362cb7.jpg"},{"id":34653494,"identity":"e05df13b-48d0-4b4c-98fa-02a5d20fe5f3","added_by":"auto","created_at":"2023-03-22 14:37:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":216293,"visible":true,"origin":"","legend":"\u003cp\u003e(a) SMD force profiles for EcoRI-DNA (black), Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA (red) and Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA (blue) with SMD time. (b) Histogram, H(t) for different breaking times (t) of the nucleotides.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2691161/v1/9ba1e0858bec6fa871d0d93d.jpg"},{"id":34653496,"identity":"2283e65a-9875-4bb5-aa3f-45e4192659ae","added_by":"auto","created_at":"2023-03-22 14:37:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":284831,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2691161/v1/6b7c9b7dd1c81462aa9ad486.jpg"},{"id":34808789,"identity":"76bf1072-2f56-40ac-93c0-07ee9288d5a4","added_by":"auto","created_at":"2023-03-25 07:29:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1132197,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2691161/v1/42d5a9df-7ebc-46a8-a2b3-bfcca17f72c4.pdf"},{"id":34653497,"identity":"4b986929-40e4-421d-8d0a-cddd63b69337","added_by":"auto","created_at":"2023-03-22 14:37:12","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5295224,"visible":true,"origin":"","legend":"","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-2691161/v1/0ef26eb1b918a6c58513d6f2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"In-silico studies on hydration in EcoRI-cognate DNA complex","fulltext":[{"header":"Introduction","content":"\u003cp\u003eREs are found in a wide range of bacteria, archaea, and viruses of certain unicellular algae. They protect the host cell from the incoming foreign DNA by cleaving it into fragments. REs are also used in gene cloning and protein expression. The DNA cleavage by REs is extraordinarily specific to DNA sequence, the type of metal ion cofactor and their positions in the RE. The microscopic mechanism of the cleavage and the specificities are largely not well-understood.\u003c/p\u003e\n\u003cp\u003eSeveral experimental and theoretical studies suggest that Mg\u003csup\u003e2+\u003c/sup\u003e plays pivotal roles: It might aid in the binding and positioning of DNA, as well as partially neutralizing the extra negative charge in the transition state and protonate the leaving group after cleavage by providing a water molecule in its coordination sphere [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. Our recent molecular dynamics (MD) simulation studies show that replacement of Mg\u003csup\u003e2+\u003c/sup\u003e ions by Ca\u003csup\u003e2+\u003c/sup\u003e ions in RE-DNA complex makes the cleavage region unstable and disordered due to enhanced fluctuations of the catalytically active residues and the cleavage base pairs of the DNA [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]. The other divalent metal ions such as Mn\u003csup\u003e2+\u003c/sup\u003e and Co\u003csup\u003e2+\u003c/sup\u003e also make the cleavage probability significantly lower [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. The cleavage activity of RE involves specific contacts between DNA bases and amino acids of the enzyme. A change of a single base in the recognition sequence of REs lower the cleavage probability by more than a million-fold [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. It is reported that under certain buffer conditions, such as changes in pH, very high enzyme to substrate ratio, presence of divalent metal cations and so on, REs lose specificity for their recognition sequence [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. In such cases REs cleave at noncognate sequences which differ by a single base pair from the cognate sequence.\u003c/p\u003e\n\u003cp\u003eThe role of water molecules in DNA cleavage by RE has been emphasized in several experiments [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. The cleavage of DNA by REs is shown to involve activation of a water molecule, followed by nucleophilic attack at the phosphodiester bond in presence of Mg\u003csup\u003e2+\u003c/sup\u003e. In particular, experimental data exist on how hydration changes during cleavage and recognition activities of EcoRI, a type II RE. EcoRI is a dimer, having two identical chains A and B where each chain consists of 261 residues. The recognition sequence of EcoRI is GAATTC, and the cleavage occurs between G and A on both strands in presence of Mg\u003csup\u003e2+\u003c/sup\u003e ions [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. The residues ASP75 and GLU95 in EcoRI hold the metal ion [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]. Changes in hydration during binding of DNA to EcoRI show sensitivity to the recognition and cleavage of DNA [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. It has been shown that the pro-Rp phosphoryl oxygen of the phosphate group on the 3' side of the scissile bond activates the attacking water molecule [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. Experiments further suggest that EcoRI binding to the cognate sequence (GAATTC) releases 70 fewer water molecules than binding to a noncognate sequence (TAATTC) [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, the cleavage reaction is accompanied by the binding of 30 water molecules in cognate sequence [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Molecular dynamics simulations propose that one of the crystal water coordinated with Mg\u003csup\u003e2+\u003c/sup\u003e attacks the scissile phosphate, making Mg\u003csup\u003e2+\u003c/sup\u003e to behave as a Lewis acid. It is reported that one of the water molecule in the DNA recognition sequence, involved in hydrogen bond between ARG 200 and ARG 203 is released out of the EcoRI-DNA interface due to increase in osmotic pressure. This may be responsible for loss of specificity at cognate sequence, leading to cleave at the other sequence differ by a single base pair from the cognate sequence. However, the role of water molecules in the metal ion and sequence specificities is far from understood.\u003c/p\u003e\n\u003cp\u003eThe experimental observations call for detailed understanding of hydration patterns in EcoRI-DNA complexes. We perform classical all-atom Molecular Dynamics simulations on EcoRI-DNA complexes in various conditions. We focus in particular on how the distribution of water molecules and their hydrogen bonding pattern depends on the type of metal ion and the DNA sequence. We observe that the number of hydrogen bonded water molecules is the least around the scissile phosphate in the sequence of the Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex, and increases upon substitution of Mg\u003csup\u003e2+\u003c/sup\u003e by Ca\u003csup\u003e2+\u003c/sup\u003e. We mutate one of the cleavage base pair (AT) by GC in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex and observe that the fraction of hydrogen bonded water molecules around the scissile phosphate group increases in the mutated complex compared to that in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA. We also extend our calculations to steered molecular dynamics (SMD) simulations and stopped SMD simulations to investigate the dynamic evolution of hydration in DNA cleavage by EcoRI. We observe a lower rate of decrease of the fraction of hydrogen bonds in the mutated complex compared to that of Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA, indicating lower reaction kinetics in the mutated complex.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cem\u003eSystem preparations\u003c/em\u003e:\u003c/p\u003e\n\u003cp\u003eThe initial structure of free DNA that contains EcoRI recognition sequence (GAATTC) is modelled by using the NUCGEN software [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. The DNA bases sequence is 5\u0026rsquo;-dCGC\u003cstrong\u003eGAATTC\u003c/strong\u003eGCG-3\u0026rsquo;, with the recognition sequence shown in bold, in 5\u0026rsquo;-3\u0026rsquo; direction and the complementary sequence in 3\u0026rsquo;-5\u0026rsquo; direction. The initial structure of EcoRI is obtained from protein data bank (PDB) (PDB ID: 1ERI). The crystal structure of EcoRI bound with DNA (EcoRI-DNA) is also taken from PDB with PDB ID 1ERI. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(a) shows the numbering of phosphate, sugar and bases of DNA. The recognition sequence is shown by the box in dotted line. There are two active sites in EcoRI-DNA complex, one containing G\u003csub\u003e4\u003c/sub\u003e:C\u003csub\u003e21\u003c/sub\u003e and A\u003csub\u003e5\u003c/sub\u003e:T\u003csub\u003e20\u003c/sub\u003e base pairs, and ASP75 and GLU95 residues in chain A, and another one containing T\u003csub\u003e8\u003c/sub\u003e:A\u003csub\u003e17\u003c/sub\u003e and C\u003csub\u003e9\u003c/sub\u003e:G\u003csub\u003e16\u003c/sub\u003e base pairs, and ASP75 and GLU95 residues in chain B. The arrows in the figure shows the cleavage points.\u003c/p\u003e\n\u003cp\u003eThe complexes of metal ions, such as Mg\u003csup\u003e2+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e loaded EcoRI-DNA are not available. To model these complexes, we follow the similar protocol as explained in our earlier publication [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. We model Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex by inserting one Mg\u003csup\u003e2+\u003c/sup\u003e ion in the vicinity of each active site of EcoRI-DNA. Similarly, we model Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex by placing each Ca\u003csup\u003e2+\u003c/sup\u003e ion in the vicinity of each active site of EcoRI-DNA. As the two active sites are symmetrical in all the complexes, the calculations are averaged over two strands.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMolecular dynamics simulations\u003c/em\u003e:\u003c/p\u003e\n\u003cp\u003eThe force-field of Amber99ff14SB [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e] is used for the protein and Amberff99bsc0 [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e] for the DNA. We use Ambertools 15 package [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e] for the preparations of the systems. First, the systems are solvated in a cubic water box containing TIP3P water molecules in such a way that the solute is surrounded by at least 15 \u0026Aring; thick layers of water molecules with the periodic boundary conditions (PBC) are applied in all directions. Subsequently, the systems are neutralized with required number of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions. We use both steepest-descent and conjugate-gradient algorithms for the energy minimizations of the systems. The cut off of 10 \u0026Aring; is used for both Lennard-Jones and short-ranged electrostatic interactions. For long-ranged electrostatic interactions, we employ the particle mesh Ewald (PME) summation technique with 1 \u0026Aring; grid spacing and 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e convergence criteria. Starting with the energy minimized structure, we carry out MD simulations using NAMD [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e] at 310 K and 1 atmospheric pressure in an isothermal\u0026ndash;isobaric (NPT) ensemble. We use Langevin thermostat to control the temperature, while the pressure is maintained by using Nos\u0026eacute;-Hoover Langevin piston. For all calculations, we use a simulation time step of 1 fs. 1 \u0026micro;s simulation is carried out for all the systems. The data are analysed from the equilibrated trajectory of 500\u0026ndash;1000 ns for all cases.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSteered molecular dynamics (SMD) simulations\u003c/em\u003e:\u003c/p\u003e\n\u003cp\u003eWe carry out SMD [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e] simulations by applying a force to one end of the DNA along the DNA helix, which is aligned along z axis. The \u0026alpha; carbons of EcoRI residues are restrained by a harmonic potential of force constant, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({k}_{res}\\)\u003c/span\u003e\u003c/span\u003e=10 kcal mol\u003csup\u003e\u0026minus;1\u003c/sup\u003e nm\u003csup\u003e\u0026minus;2\u003c/sup\u003e, while the protein can move freely along x and y directions. The SMD force that varies with time, is calculated by the following formula, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(F\\left(t\\right)={k}_{SMD}[{z}_{SMD}\\left(t\\right)-{z}_{COM}\\left(t\\right)]\\)\u003c/span\u003e\u003c/span\u003e, where \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({z}_{COM}\\left(t\\right)\\)\u003c/span\u003e\u003c/span\u003e is the z coordinate of the center of mass (COM) of DNA, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({z}_{SMD}\\left(t\\right)={z}_{COM}\\left(0\\right)+{v}_{SMD}t\\)\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({v}_{SMD}\\)\u003c/span\u003e\u003c/span\u003e = 0.5 nm ns\u003csup\u003e\u0026minus;1\u003c/sup\u003e is the SMD velocity, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({k}_{SMD}\\)\u003c/span\u003e\u003c/span\u003e= 50,000 kcal mol\u003csup\u003e\u0026minus;1\u003c/sup\u003e nm\u003csup\u003e\u0026minus;2\u003c/sup\u003e is the force constant. In addition to the SMD simulations, we carry out a series of stopped SMD simulations. A stopped SMD simulation is the continuation of a constant-velocity SMD with the velocity set to zero. A stopped SMD simulation is described by the reference SMD simulation time \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(t\\)\u003c/span\u003e\u003c/span\u003e where the complex of EcoRI-DNA is subjected to a high strain rate. We carry out both SMD and stopped SMD simulations on EcoRI-DNA, Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA and Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complexes.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnalysis\u003c/em\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCalculation of hydrogen bonds\u003c/em\u003e:\u003c/p\u003e\n\u003cp\u003eFor calculations of hydrogen bonds (HBs), we consider the following protocol: (i) the distance between the donor O atom and acceptor O atom is less or equal to 0.34 nm and (ii) O-H (donor) \u0026ndash; O (acceptor) angle is greater or equal to 150\u003csup\u003e0\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConformational Thermodynamics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe detailed calculations of the DNA step parameters and the calculations of the changes in conformational thermodynamics is discussed in the Methods section in our previous publication [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDisplacements of nucleotides\u003c/em\u003e:\u003c/p\u003e\n\u003cp\u003eThe displacement of nucleotide from the interaction regime of EcoRI is characterized as follows: first, for each nucleotide, we calculate the distance between each atom of a particular nucleotide and each atom of EcoRI residues. If the distance is less than 0.45 nm, we make a list of the atom pairs (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(ij\\)\u003c/span\u003e\u003c/span\u003e), where atom \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(i\\)\u003c/span\u003e\u003c/span\u003e belongs to EcoRI, and atom \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(j\\)\u003c/span\u003e\u003c/span\u003e belongs to DNA. We compute the average distance of the atom pairs for each nucleotide in a single snapshot, denoted as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({d}_{ij}\\)\u003c/span\u003e\u003c/span\u003e. Finally, we calculate the average distance (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{1}{n}{\u0026lt;d}_{ij}\u0026gt;)\\)\u003c/span\u003e\u003c/span\u003e of the atom pairs of each nucleotide over the last 10 ns of the equilibration. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(n\\)\u003c/span\u003e\u003c/span\u003e is the number of pairs. we also calculate \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{1}{n}{d}_{ij}\\)\u003c/span\u003e\u003c/span\u003e from the SMD trajectory for each snapshot. Finally, the displacement of each nucleotide is computed as R\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(=\\frac{1}{n}\\sum _{ij}({d}_{ij}-\u0026lt;{d}_{ij}\u0026gt;)\\)\u003c/span\u003e\u003c/span\u003e. We consider a nucleotide being ruptured if R\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\u0026gt;\\)\u003c/span\u003e\u003c/span\u003e 0.25 nm during the SMD simulation. We follow the reference [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e] for calculating the displacements of nucleotides.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003eWe study the following systems using all-atom molecular dynamics simulations: (1) EcoRI and DNA containing cognate sequence (EcoRI-DNA), (2) EcoRI-DNA in presence of Mg\u003csup\u003e2+\u003c/sup\u003e ions (Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA), (3) EcoRI-DNA in presence of Ca\u003csup\u003e2+\u003c/sup\u003e ions (Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA) (4) EcoRI with mutated DNA where A\u003csub\u003e5\u003c/sub\u003e:T\u003csub\u003e20\u003c/sub\u003e base pair in the DNA is mutated by G\u003csub\u003e5\u003c/sub\u003e:C\u003csub\u003e20\u003c/sub\u003e, in presence of Mg\u003csup\u003e2+\u003c/sup\u003e ions (Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA) and (5) Free DNA containing the cognate sequence.\u003c/p\u003e\n\u003cp\u003eThe co-ordinations of Mg\u003csup\u003e2+\u003c/sup\u003e in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex, Ca\u003csup\u003e2+\u003c/sup\u003e in Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA and Mg\u003csup\u003e2+\u003c/sup\u003e in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA obtained from MD trajectories are shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(b)-(d) respectively. As the co-ordinations of the divalent metal ions in the two active sites in each complex are identical, we only show those of the divalent metal ion in the first active site. If the average distance between any heavy atom of DNA or water or EcoRI and metal ion is less or equal to 3.0 \u0026Aring;, coordination is considered. OD1 of ASP75, OE1 of GLU95 and OP1 of P\u003csub\u003e5\u003c/sub\u003e participate in coordination with Mg\u003csup\u003e2+\u003c/sup\u003e. Similarly, OD1 and OD2 of ASP75, OE1 and OE2 of GLU95 and OP1 of P\u003csub\u003e5\u003c/sub\u003e participate in coordination with Ca\u003csup\u003e2+\u003c/sup\u003e. The co-ordinations of Mg\u003csup\u003e2+\u003c/sup\u003e in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA are same as those of Mg\u003csup\u003e2+\u003c/sup\u003e in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA. The rest of the co-ordinations of Mg\u003csup\u003e2+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e are satisfied by the O atoms of water molecules.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEquilibrium hydration pattern\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe calculate the water density around a given chemical moiety. First, we consider the metal ions. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(a) shows the density of water, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e(r) and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e(r) around Mg\u003csup\u003e2+\u003c/sup\u003e in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex and Ca\u003csup\u003e2+\u003c/sup\u003e in Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex respectively, where r is distance between the metal ion and water oxygen. For all cases, the distributions show two well defined peaks. The first peak in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e(r) appears at r\u0026thinsp;=\u0026thinsp;0.22 nm, while the second peak is at r\u0026thinsp;=\u0026thinsp;0.29 nm. The density is observed to be maximum of 3.1 gm/cc at 0.22 nm. As the radius of Ca\u003csup\u003e2+\u003c/sup\u003e is greater than that of Mg\u003csup\u003e2+\u003c/sup\u003e, the peaks in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e(r) is further away from Ca\u003csup\u003e2+\u003c/sup\u003e than those in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e(r). The first peak arises at 0.25 nm, while the second peak at 0.32 nm. Here, the maximum density is 1.98 gm/cc which occurs at 0.25 nm. Thus, Mg\u003csup\u003e2+\u003c/sup\u003e is better hydrated than Ca\u003csup\u003e2+\u003c/sup\u003e due to smaller size and consequently, larger charge density.\u003c/p\u003e\n\u003cp\u003eNext, we consider the hydration of the phosphate groups of the DNA in various cases. We calculate the density of water molecules, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{{\\alpha }}^{0}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{{\\alpha }}^{\\text{c}}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{{\\alpha }}^{\\text{M}\\text{g}}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{{\\alpha }}^{\\text{C}\\text{a}}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{{\\alpha }}^{Mut}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e around phosphate group (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\alpha\\)\u003c/span\u003e\u003c/span\u003e) in free DNA, EcoRI-DNA, Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA, Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA and Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA complexes respectively where \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(r\\)\u003c/span\u003e\u003c/span\u003e is the distance between phosphorus atom and the oxygen of water. We compare the cases for \u0026alpha;\u0026thinsp;=\u0026thinsp;P5 in the cleavage region and \u0026alpha;\u0026thinsp;=\u0026thinsp;P7 in the non-cleavage region in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(b) and (c) respectively. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{\\text{P}5}^{0}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e in the free DNA shows a peak at 0.4 nm with maximum density 0.82 gm/cc, while the other peak at 0.6 nm has similar maximum density. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{\\text{P}5}^{\\text{c}}\\left(r\\right),\\)\u003c/span\u003e\u003c/span\u003e in EcoRI-DNA complex without any metal ion shows a well-defined maximum at 0.43 nm, while it shows another broad peak around 0.48 nm. Similarly, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{\\text{P}5}^{\\text{M}\\text{g}}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e shows two well defined maxima. The position of the first maximum in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{\\text{P}5}^{\\text{M}\\text{g}}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e is smaller than that in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{\\text{P}5}^{0}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e. On the other hand, the first peak in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{\\text{P}5}^{\\text{C}\\text{a}}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e is comparable to that in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{\\text{P}5}^{0}\\left(r\\right)\\)\u003c/span\u003e\u003c/span\u003e. This indicates that P\u003csub\u003e5\u003c/sub\u003e is more hydrated in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex than both EcoRI-DNA and Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA due to larger hydration around Mg\u003csup\u003e2+\u003c/sup\u003e ion. On the other hand, the density of water around P7 show two different peaks at 0.3 nm and 0.4 nm for all the cases. Thus, the hydration level of the phosphate groups at the cleavage site is sensitive to the type of metal ions but not so at the non-cleavage site.\u003c/p\u003e\n\u003cp\u003eWe calculate the fraction of water molecules in the first coordination shell, defined by the first peak in the density profile, having hydrogen bonds with O atoms of the phosphate groups in the recognition region. We denote the fraction by \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{{\\alpha }}^{0}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{{\\alpha }}^{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{{\\alpha }}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{{\\alpha }}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e in free DNA, EcoRI-DNA, Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA and Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA respectively. As the two strands are symmetric, the results shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(d) in the recognition region are averaged over both strands. For free DNA, the values of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{{\\alpha }}^{0}\\)\u003c/span\u003e\u003c/span\u003e for all the phosphate groups in recognition region are similar. However, for the complexes, the results show sequence specificity. In EcoRI-DNA complex, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{\\text{P}5}^{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e shows a minimum value. The values of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{\\alpha }^{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e for the other phosphate groups are similar to those in free DNA. In Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex, the minimum of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{\\text{P}5}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e is even deeper and a maximum at P\u003csub\u003e7\u003c/sub\u003e is observed unlike in the EcoRI-DNA complex. In Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{\\text{P}5}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e has a minimum comparable to that in EcoRI-DNA complex. The maximum of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{\\text{P}7}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e is similar to that of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({f}_{\\text{P}7}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e. Thus, the data suggest that the hydrogen bond formation is specific to the kind of the metal ion. The number of hydrogen bonded water molecules at the cleavage site is the least in case of Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex.\u003c/p\u003e\n\u003cp\u003eWe compare the fluctuations of base pair step parameters in EcoRI-DNA, Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA and Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complexes with those in free DNA. A given Watson-Crick paired bases is taken to define a base pair. The base pair step parameters define the relative displacement (shift, slide and rise) and orientations (tilt, roll and twist) of two successive base pairs.[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e] For a given base pair step (i, i\u0026thinsp;+\u0026thinsp;1), the distribution is denoted by \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{\\text{i},\\text{i}+1}^{\\text{f}}\\)\u003c/span\u003e\u003c/span\u003e(b) in the free state and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{\\text{i},\\text{i}+1}^{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e(b) in the complex for a given base pair step parameter b where i and i\u0026thinsp;+\u0026thinsp;1 are two successive bases in a given strand, omitting the WC paired bases. The distributions in free DNA, Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA and Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complexes are denoted as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{\\text{i},\\text{i}+1}^{0}\\)\u003c/span\u003e\u003c/span\u003e(b), \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{\\text{i},\\text{i}+1}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e(b) and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{\\text{i},\\text{i}+1}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e(b) respectively. The base pair parameters are taken to be conformational variables for the DNA in our previous analysis [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eHere, we illustrate the data for base pair step, (A\u003csub\u003e5\u003c/sub\u003e:T\u003csub\u003e20\u003c/sub\u003e, A\u003csub\u003e6\u003c/sub\u003e:T\u003csub\u003e19\u003c/sub\u003e) in the cleavage region. We show the histograms in Supplementary Information (SI), Figs. S1(a)-(f). \u0026tau; for all cases are broad single peaked (see SI, Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e(a)). However, the peak heights of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{\\text{5,6}}^{0}\\)\u003c/span\u003e\u003c/span\u003e(\u0026tau;) and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{5,6}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e(\u0026tau;) are smaller than that of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{5,6}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e(\u0026tau;). The distributions of \u0026rho; (see SI, Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e(b)) and \u0026omega; (see SI, Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e(c)) are all single peaked in all the cases. However, the peak height is smaller and broader in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{5,6}^{0}\\)\u003c/span\u003e\u003c/span\u003e(\u0026omega;) compared to the other complexes. Although, Dx shows single peaked distributions for all cases, the peak height for \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{5,6}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e(Dx) is larger than that of other complexes (see SI, Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e(d)). Similarly, the distributions of Dy (see SI, Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e (e)) and Dz (see SI, Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e(f)) are single peaked in all complexes. However, the peak height of the distributions of Dy is highest for \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{H}}_{5,6}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e(Dy). Thus, we observe that the distributions of rotational parameters of the base pair steps are broader in all cases than those of translational parameters.\u003c/p\u003e\n\u003cp\u003eWe quantify the fluctuations in terms of the changes in conformational free energy and entropy of DNA in complexes with respect to the free DNA.[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e] A negative change in the free energy denotes stabilization and that in entropy denotes enhanced order in the complexed DNA with respect to the reference free DNA. For a given step parameter, the changes in free energy and entropy of (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{i}, \\text{i}+1\\)\u003c/span\u003e\u003c/span\u003e) step in the Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex with respect to the free DNA are denoted as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\Delta }\\text{G}}_{\\text{i},\\text{i}+1}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e(b) and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{T}{\\Delta }\\text{S}}_{\\text{i},\\text{i}+1}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e(b) respectively. Similarly, the analogous quantities for Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex with respect to the free DNA are denoted by \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\Delta }\\text{G}}_{\\text{i},\\text{i}+1}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e(b) and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{T}{\\Delta }\\text{S}}_{\\text{i},\\text{i}+1}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e(b). The total free energy (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\Delta }\\text{G}}_{\\text{i},\\text{i}+1}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\Delta }\\text{G}}_{\\text{i},\\text{i}+1}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e) and entropy changes (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{T}{\\Delta }\\text{S}}_{\\text{i},\\text{i}+1}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{T}{\\Delta }\\text{S}}_{\\text{i},\\text{i}+1}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e) of a base pair step parameters of DNA in the complexes with respect to those in free DNA, adding the individual contributions, are shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(e) and (f) respectively. The base pair steps are highly stabilized in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(e) shows that the base pair step \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\Delta }\\text{G}}_{\\text{4,5}}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e in cleavage region of Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA shows maximum conformational stabilization. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(f) shows that \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{T}{\\Delta }\\text{S}}_{\\text{4,5}}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e has maximum order compared to the other base pair steps and that at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{T}{\\Delta }\\text{S}}_{\\text{6,7}}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e pair has a minimum. In Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(e)), the stabilization in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\Delta }\\text{G}}_{\\text{4,5}}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e is maximum but it is lower compared to that in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA. Here, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\Delta }\\text{G}}_{\\text{6,7}}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e shows destabilization. The base pair steps in Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA are all ordered with a similar value of entropy change \u0026minus;\u0026thinsp;3.0 kJ/mol, except \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{T}{\\Delta }\\text{S}}_{\\text{6,7}}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(f)).\u003c/p\u003e\n\u003cp\u003eNext, we consider a mutated DNA sequence, denoted by Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA where we mutate the cleavage base pair A-T in the first active site by G-C in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex. First, we show the density profile of water molecules, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{\\text{P}5}^{\\text{M}\\text{u}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\rho }}_{\\text{P}7}^{\\text{M}\\text{u}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e in the first strand of the mutated complex in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(b) and (c) respectively. We observe that the density of water around P5 and P7 in the mutated complex are similar to those in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA. The fraction of hydrogen bonds, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{{\\alpha }}^{\\text{M}\\text{u}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e for different \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\alpha }\\)\u003c/span\u003e\u003c/span\u003e of Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(d). It is observed that \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{{\\alpha }}^{\\text{M}\\text{u}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e has a minimum at P5. However, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{M}\\text{u}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e \u0026gt;\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e. Thus, the hydrogen bonding network is sequence specific and sensitive to the mutation of the DNA base.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTime dependent hydration pattern\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe investigate the changes in hydration pattern, while the DNA gets unbound from the binding pocket of EcoRI. We show the forces \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{F}}^{0}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{F}}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{F}}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e experienced by the systems due to unbinding of DNA from EcoRI for EcoRI-DNA, Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA and Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complexes respectively in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. For all the cases, the forces increase gradually until they reach a maximum, and after that they gradually decrease. The force is maximum when all the nucleotides of the DNA come out from the interaction regime of protein. This is in agreement to an earlier report [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. The maximum of the force is larger in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA than all other cases. This indicates that the interactions between EcoRI and DNA is strongest in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA.\u003c/p\u003e\n\u003cp\u003eWe also follow the unbinding of the nucleotides. As time progresses, the N\u0026beta; nucleotide of the DNA fragment unbinds. The distances of the centre of mass of a nucleotide which contains a phosphate group, a sugar group and one of the four bases, with respect to the centre of mass of the binding residues of EcoRI are denoted by \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{R}}_{{\\beta }}^{0}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{R}}_{{\\beta }}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{R}}_{{\\beta }}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e for EcoRI-DNA, Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA and Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA respectively. Here N\u0026beta;\u0026thinsp;=\u0026thinsp;N4-N9 for the first strand and N\u0026beta;\u0026thinsp;=\u0026thinsp;N16-N21 for the second strand. The data are shown in SI, Fig. S2. The jump in the displacement denote the unbinding of the corresponding nucleotide.\u003c/p\u003e\n\u003cp\u003eBased on jumps in the distances with time, we show in Table\u0026nbsp;1 the times at which different nucleotides are liberated from the binding pocket. We first consider EcoRI-DNA, shown in SI, Figs. S2(a) and (b) for the first and second strands respectively. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{N}20\\)\u003c/span\u003e\u003c/span\u003e in the second strand breaks away first from the interaction regime of EcoRI around 1.35 ns. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{N}19\\)\u003c/span\u003e\u003c/span\u003eand \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{N}21\\)\u003c/span\u003e\u003c/span\u003e break away at ⁓ 2.05 ns and 2.9 ns respectively. After that around 3.55 ns, N4 in the first strand leaves the binding site. In the range 4-5.5 ns, N5-N7 in the first strand and N18 in the second strand leave the binding site. All the nucleotides in the recognition region of first strand leave around 6.9 ns, while those in the second strand leave around 6.6 ns, yielding the total rapture time 6.9 ns. The sequence of nucleotide rupture is similar to the earlier study.[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e] However, the rupture times are somewhat less in our system which may be due to differences in force fields in two cases.\u003c/p\u003e\n\u003cp\u003eNext, we consider the Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex (SI, Figs. S2(c) and (d) for the first and second strands respectively). In EcoRI-DNA, the nucleotide starts breaking away at 1.35 ns, whereas in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex, this time is 3 ns. Both N20 and N21 in the second strand are ruptured at 3 ns. N4 and N19 break away at 3.4 ns and 3.9 ns respectively. N18 breaks away at 4.95 ns. N4-N6 rupture gradually in a gap of ⁓ 1 ns, while both N7 and N8 break away approximately at the same time (⁓ 5.8 ns). N16 and N17 in the second strand leave the binding site around ⁓ 6.5 ns, while the remaining nucleotide N9 in the first strand breaks away around ⁓ 8 ns. Thus, the rupture time in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex is ~\u0026thinsp;8 ns. It indicates that the interactions between EcoRI and DNA is stronger in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA than in EcoRI-DNA.\u003c/p\u003e\n\u003cp\u003eThe sequence of events in Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex is different. The data for the first and second strands are shown in SI, Figs. S2 (e) and (f) respectively. In this complex, N4, N20 and N21 leave the binding site approximately at the same time ⁓ 3.7 ns. N19 breaks away at ⁓ 4.05 ns. After that all four nucleotides N5, N6, N7 and N9 in the first strand break away at approximately same time (⁓ 5.1 ns) (SI, Fig. S2(e)). N18 breaks away at ⁓ 5.6 ns, while N8 breaks away around ⁓ 6 ns. Here, N9 is ruptured before N8 (SI, Fig. S2(e)). N16 comes out from the binding pocket around ⁓ 6 ns. N17 breaks away last at ⁓ 6.55 ns, while in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA the last nucleotide is N9 that breaks away at ⁓ 8 ns, indicating Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex is more stable than Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA.\u003c/p\u003e\n\u003cp\u003eNow we consider the mutated complex (SI, Figs. S2 (g) and (h) for the first and second strands respectively). N21 is ruptured first at around ⁓3.2 ns, while N4 at 3.6 ns in the first strand. In the range 4\u0026ndash;6 ns, all the nucleotides except nucleotide 9 in the first strand, and N19 and N20 in the second strand break away. The nucleotides N9, N16-N18 all break away at approximately same time ⁓6.35 ns. The rapture time in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA is ⁓6.35 ns. This indicates higher stability of the Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex compared to the Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA complex.\u003c/p\u003e\n\u003cp\u003eWe show histogram, H(t) of the breaking times, t of the nucleotides in different cases in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(b). The histogram shows three different maxima at 3.5 ns, 5 ns and 6 ns. Hence, we carry out stopped SMD simulations by considering snapshots at these times from the SMD trajectory for all the complexes. In addition to these times, we perform stopped SMD simulations of the snapshots at 4 ns corresponds to the first minimum of the histogram and at 6.5 ns where most of the nucleotides come out from the binding pocket of EcoRI. We calculate the fraction of hydrogen bonds, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\alpha }^{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{{\\alpha }}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{{\\alpha }}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{{\\alpha }}^{\\text{M}\\text{u}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e at these times, shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. Let us consider the scissile phosphate P5 in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e(a). \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e does not show significant changes with time. The values of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e at different times are similar to those of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e. However, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e shows much lower values at lower times compared to those of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e and approaches \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e at larger times. Similar time dependent growth is observed in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{M}\\text{u}\\text{t}}.\\)\u003c/span\u003e\u003c/span\u003e In both cases the time dependences are linear as shown by the fitted curves with slopes 0.14 and 0.07 for Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA and Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA cases respectively. Thus, the growth for the mutated complex is not as rapid as that in the case of the wild type complex. On the other hand, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}7}^{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}7}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e,\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}7}^{\\text{C}\\text{a}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}7}^{\\text{M}\\text{u}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e do not show significant changes with time as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e(b).\u003c/p\u003e\n\u003cp\u003eIt may be noted that the phosphodiester bond cleavage cannot be observed from classical MD simulations. However, our studies based on hydration pattern from classical MD trajectories give significant insight to the role of the metal ion and DNA sequence. The smaller metal ion like Mg\u003csup\u003e2+\u003c/sup\u003e is solvated more efficiently than larger ion like Ca\u003csup\u003e2+\u003c/sup\u003e. This is favourable from the stand point of activation of water molecules for cleavage [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/p\u003e\n\u003cp\u003eThe overall fluctuations in the microscopic conformational variables in the cleavage region is less in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA compared to the Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA. This scenario is quite analogous to that of EcoRV. The enhanced stability and order are pre-requisite for positioning the metal ion in the vicinity of the scissile phosphate and is more efficiently done in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA than in Ca\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA. It may be noted that the maximum stabilization and ordering in (4,5) step of Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex has the presence of lowest number of hydrogen bonded water molecules. On the other hand, the most destabilized and disordered (6,7) step has the maximum number of hydrogen bonded water molecules. Less abundance of H-bonds indicates that the scissile phosphate group in this complex is more accessible to have the nucleophilic attack by a non-hydrogen bonded water molecule. A single base pair mutation in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA significantly reduces the fraction of hydrogen bonds around the scissile phosphate group.\u003c/p\u003e\n\u003cp\u003eTime dependent changes in hydrogen bond fraction during the unbinding gives idea about the kinetics of the cleavage process. The increase in the fraction \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{M}\\text{u}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{f}}_{\\text{P}5}^{\\text{M}\\text{g}}\\)\u003c/span\u003e\u003c/span\u003e while the unbinding may also be taken to indicate decreases in the fraction as the DNA enters into the binding pocket. Thus, the slower rate of increase in mutated complex than that of Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA suggests a lower cleavage reaction in the mutated complex.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo summarize, we investigate hydration in EcoRI-cognate DNA complex. The data suggest that the water density around scissile phosphate group is higher in Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex. However, the hydrogen bond network around this phosphate group is less strong in this complex compared to those in other complexes, formed by replacing the ion or mutating the sequence. Time dependent changes in hydration indicates that the kinetics becomes slower after mutation of a single base pair in the cognate DNA sequence of Mg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA complex. Thus, our studies may be helpful for microscopic understanding of the cleavage process. The hydration pattern can be probed by spectroscopic measurements which may open up further understanding on specificity of DNA cleavage by RE.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSCM thanks to Council of Scientific \u0026amp; Industrial Research (CSIR) for providing the fellowship. SCM and JC thank the Thematic Unit of Excellence (TUE) and the Technical Research Centre (TRC) at S. N. Bose National Centre for Basic Sciences for computational facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are freely available upon request of the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSCM performed the calculations and the analyses, and wrote the paper. JC planned and supervised the project, and corrected the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eA. Jeltsch, J. Alves, G. Maass, A. Pingoud, \u003cem\u003eFEBS letters\u003c/em\u003e, \u003cstrong\u003e1992\u003c/strong\u003e, \u003cem\u003e304\u003c/em\u003e, 4\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eS. C. Mandal, L. Maganti, M. Mondal, J. Chakrabarti, \u003cem\u003eBiopolymers\u003c/em\u003e, \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e111\u003c/em\u003e, e23396.\u003c/li\u003e\n\u003cli\u003eW. A. Loenen, D. T. Dryden, E. A. Raleigh, G. G. Wilson, N. E. 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Ghosh, \u003cem\u003eBiophysical journal\u003c/em\u003e, \u003cstrong\u003e2013\u003c/strong\u003e, \u003cem\u003e104\u003c/em\u003e, 1274\u0026ndash;1284.\u003c/li\u003e\n\u003cli\u003eJ. Heitman, \u003cem\u003eBioEssays\u003c/em\u003e, \u003cstrong\u003e1992\u003c/strong\u003e, \u003cem\u003e14\u003c/em\u003e, 445\u0026ndash;454.\u003c/li\u003e\n\u003cli\u003eA. Pingoud, M. Fuxreiter, V. Pingoud, W. Wende, \u003cem\u003eCellular and molecular life sciences\u003c/em\u003e, \u003cstrong\u003e2005\u003c/strong\u003e, \u003cem\u003e62\u003c/em\u003e, 685.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"table","content":"\u003cp\u003eTable 1. Time scales of breaking up the nucleotides.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" \u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"9.393063583815028%\"\u003e\n \u003cp\u003eNucleotides in recognition region\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"38.72832369942196%\"\u003e\n \u003cp\u003e1\u003csup\u003est\u003c/sup\u003e strand (Time of break up) of DNA in\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" width=\"51.87861271676301%\"\u003e\n \u003cp\u003e2\u003csup\u003end\u003c/sup\u003e strand (Time of break up) of DNA in\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.569377990430622%\"\u003e\n \u003cp\u003eEcoRI-DNA (ns)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.121212121212121%\"\u003e\n \u003cp\u003eMg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA (ns)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.047846889952153%\"\u003e\n \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA (ns)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.004784688995215%\"\u003e\n \u003cp\u003eMg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA (ns)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.556618819776714%\"\u003e\n \u003cp\u003eNucleotides in recognition region\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.090909090909092%\"\u003e\n \u003cp\u003eEcoRI-DNA (ns)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.526315789473685%\"\u003e\n \u003cp\u003eMg2+-EcoRI-DNA (ns)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.526315789473685%\"\u003e\n \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e-EcoRI-DNA (ns)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.556618819776714%\"\u003e\n \u003cp\u003eMg\u003csup\u003e2+\u003c/sup\u003e-EcoRI-mtDNA (ns)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.393063583815028%\"\u003e\n \u003cp\u003eN4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.670520231213873%\"\u003e\n \u003cp\u003e3.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.982658959537572%\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.104046242774567%\"\u003e\n \u003cp\u003e3.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.971098265895954%\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.283236994219653%\"\u003e\n \u003cp\u003eN21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.236994219653178%\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.53757225433526%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.53757225433526%\"\u003e\n \u003cp\u003e3.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.283236994219653%\"\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.393063583815028%\"\u003e\n \u003cp\u003eN5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.670520231213873%\"\u003e\n \u003cp\u003e4.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.982658959537572%\"\u003e\n \u003cp\u003e4.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.104046242774567%\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.971098265895954%\"\u003e\n \u003cp\u003e4.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.283236994219653%\"\u003e\n \u003cp\u003eN20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.236994219653178%\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.53757225433526%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.53757225433526%\"\u003e\n \u003cp\u003e3.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.283236994219653%\"\u003e\n \u003cp\u003e5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.393063583815028%\"\u003e\n \u003cp\u003eN6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.670520231213873%\"\u003e\n \u003cp\u003e5.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.982658959537572%\"\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.104046242774567%\"\u003e\n \u003cp\u003e5.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.971098265895954%\"\u003e\n 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valign=\"top\" width=\"9.393063583815028%\"\u003e\n \u003cp\u003eN9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.670520231213873%\"\u003e\n \u003cp\u003e6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.982658959537572%\"\u003e\n \u003cp\u003e7.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.104046242774567%\"\u003e\n \u003cp\u003e5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.971098265895954%\"\u003e\n \u003cp\u003e6.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.283236994219653%\"\u003e\n \u003cp\u003eN16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.236994219653178%\"\u003e\n \u003cp\u003e4.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.53757225433526%\"\u003e\n \u003cp\u003e6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.53757225433526%\"\u003e\n \u003cp\u003e6.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.283236994219653%\"\u003e\n \u003cp\u003e6.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2691161/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2691161/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRestriction endonucleases (REs) cleave DNA at specific site in presence of Mg\u003csup\u003e2+\u003c/sup\u003e ion. Experiments further emphasize the role of hydration in metal ion specificity and sequence specificity of DNA cleavage. However, the relation between hydration and specificity has not been understood till date. This leads us to study via all-atom molecular dynamics (MD) simulations how the hydration around the scissile phosphate group changes in presence of Mg\u003csup\u003e2+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e and depend on the DNA sequence. We observe the least number of hydrogen bonds around the scissile phosphate group in presence of Mg\u003csup\u003e2+\u003c/sup\u003e ion. We further find that the hydrogen bonds decrease at the scissile phosphate on mutating one base pair in the cleavage region of the DNA in Mg\u003csup\u003e2+\u003c/sup\u003e loaded EcoRI-DNA complex which makes the scissile phosphate group more accessible for the non-hydrogen bonded water molecules. We also perform steered MD simulations and observe that the rate of decrease of hydrogen bonds is slower in the mutated complex than the unmutated complex.\u003c/p\u003e","manuscriptTitle":"In-silico studies on hydration in EcoRI-cognate DNA complex","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-22 14:37:07","doi":"10.21203/rs.3.rs-2691161/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f991dec7-478b-4b65-9b99-f5c51baf579e","owner":[],"postedDate":"March 22nd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-03-25T07:29:10+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-22 14:37:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2691161","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2691161","identity":"rs-2691161","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}