Non-intertwined strands of plasmid DNA contradict the Watson and Crick model of DNA structure

According to Watson and Crick (W/C) model of DNA structure, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although W/C model is accepted widely, some researchers have raised questions against it and proposed alternative structures for DNA. In the present study, we examined W/C model using plasmid DNA. It was hypothesized that two strands of plasmid will remain intertwined (and not separate from each other) under denaturing conditions if it follows W/C model. To test this, plasmid DNA was denatured using sodium hydroxide (NaOH) and analyzed by agarose gel electrophoresis. It was observed that addition of 0.5 N NaOH to pUC19 or pBR322 plasmids resulted in a new form of DNA having higher electrophoretic mobility in agarose gel. Higher electrophoretic mobility DNA (HmDNA) in NaOH-denatured pUC19 was digestible with S1 nuclease, but not with HindIII and ‘exonuclease I + alkaline phosphatase’. These results demonstrated that HmDNA is single-stranded circular DNA, formed due to separation of two strands of NaOH-denatured plasmid. Single-stranded and circular nature of HmDNA was corroborated by its comparable electrophoretic mobility with purified top and bottom strands of plasmid DNA. Next, we examined whether HmDNA can re-anneal into the native plasmid. Interestingly, when subjected to renaturing conditions, HmDNA from NaOH-denatured pUC19 re-annealed to form native pUC19 plasmid, which was digestible with HindIII and induced ampicillin resistance in Escherichia coli . These findings demonstrated the reversible separation of two strands of plasmid DNA and contradicted the W/C model of DNA structure.