Kinetic and Structural Analysis of Ribonucleotide Extension by DNA Polymerase η

DNA polymerases catalyze DNA synthesis with high fidelity, which is essential for all life. Extensive kinetic and structural efforts have been executed in exploring mechanisms of DNA polymerases, surrounding their kinetic pathway, catalytic mechanisms, and factors that dictate polymerase fidelity. Recent time-resolved crystallography studies on DNA polymerase η (Pol η) and β have revealed essential transient events during DNA synthesis reaction, such as mechanisms of primer deprotonation, separated roles of the three metal ions, and conformational changes that disfavor incorporation of the incorrect substrate. DNA-embedded ribonucleotides (rN) are the most common lesion on DNA and a major threat to genome integrity. While kinetics of rN incorporation has been explored and structural studies have revealed that DNA polymerases have a steric gate that destabilizes rNTP binding, mechanism of extension upon rN addition remains poorly characterized. Using steady-state kinetics, static and time-resolved X-ray crystallography with Pol η as a model system, we showed that the extra hydroxyl group on the primer terminus does not reduce the catalytic efficiency of Pol η. However, rN ended primers alter the dynamics of the polymerase active site as well as the catalysis and fidelity of DNA synthesis. During rN extension, Pol η fidelity drops significantly across different sequence context. Systematic structural studies suggest that the rN at the primer end improved primer alignment and reduced barriers in C2’-endo to C3’-endo sugar conformation change. Our work provides important insights for rN extension and implicates a possible mechanism for rN removal.