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

Abbreviations

DNA, Deoxyribonucleic acid

NaOH, Sodium hydroxide

HCl, Hydrochloric acid

hmP19, higher electrophoretic mobility band of pUC19 plasmid.

Introduction

DNA is the genetic material of all organisms, with the exception of some viruses. The currently accepted model of DNA structure was proposed by James Watson and Francis Crick in 1953¹. According to this model, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although the Watson and Crick (W/C) model is accepted widely, some researchers have raised questions against it and proposed alternative models for DNA structure^2,3. Among these, Rodley’s model which envisaged that two strands of a DNA molecule are held side-by-side has generated significant interest and curiosity in the scientific community².

In the present study, the W/C model of DNA structure was examined with the help of plasmid DNA. It was hypothesized that two strands of a plasmid will remain intertwined and not separate into single-stranded circular DNA molecules under denaturing conditions, if it follows the W/C model. To test this, pUC19 and pBR322 plasmids were denatured using sodium hydroxide (NaOH) and analyzed by gel electrophoresis. Interestingly, addition of NaOH to pUC19 and pBR322 plasmids resulted in new form of DNA showing higher electrophoretic mobility in agarose gel. DNA corresponding to higher electrophoretic mobility band of pUC19 (hmP19) was found to be single-stranded and circular, suggesting the separation of two strands of plasmid DNA. Under suitable conditions, hmP19 DNA re-annealed to form native pUC19 plasmid. These results showed that two strands of a DNA molecule are not intertwined with each other and contradicted the W/C model of DNA structure.

Methods

Results and discussion

Two strands of a DNA molecule are held together by non-covalent interactions, which can be disrupted by increasing the pH of DNA solution⁵. In the present study, approximately 5 µg of plasmid DNA was denatured by adding NaOH solution of increasing concentration. It was observed that addition of 0.5 N NaOH to pUC19 resulted in a new form of DNA showing higher electrophoretic mobility in agarose gel (Figure 1a, Underlying data⁶). Similar results were obtained with pBR322 plasmid added with 0.5 N NaOH (Figure 1b, Underlying data⁷). Formation of higher electrophoretic mobility DNA in pBR322 plasmid added with NaOH has also been reported previously⁸.

Figure 1. Double-stranded plasmid is denatured by NaOH into single-stranded circular DNA.

Approximately 5 µg plasmid DNA was added with an equal volume (5 µl) of NaOH solution of indicated concentration (normality, N) and run on 1% agarose gel. A distinct band of higher electrophoretic mobility DNA (marked by arrows) was observed in pUC19 (a) and pBR322 (b) plasmids added with 0.5 N NaOH. For enzymatic digestion, NaOH in denatured plasmid was neutralized using 0.5M HCl, followed by incubation with different enzymes. Native pUC19, but not hmP19 DNA was digested by Hind III, which acts on double-stranded DNA (c). S1 nuclease, which recognizes single-stranded DNA, degraded hmP19 DNA but not native pUC19 plasmid (d). Exonuclease I (Exo I) and alkaline phosphatase (AP), which eliminate single-stranded linear DNA, digested neither of native pUC19 or hmP19 DNA (e). Representative data of at least three independent experiments are shown.

DNA corresponding to higher electrophoretic mobility band of pUC19 (hmP19) was characterized using DNA modifying enzymes in next experiments. Incubation with Hind III, which acts on double-stranded DNA, digested pUC19 plasmid but not hmP19 DNA (Figure 1c, Underlying data⁹). S1 nuclease, which digests single-stranded DNA, degraded hmP19 DNA but not pUC19 plasmid (Figure 1d, Underlying data¹⁰). Exonuclease I and alkaline phosphatase, which would digest single-stranded linear DNA, degraded neither of pUC19 or hmP19 DNA (Figure 1e, Underlying data¹¹). These results showed that hmP19 DNA is single-stranded and circular. Exonuclease I- and alkaline phosphatase-digested primers (experimental control) did not form product in PCR reaction (Supplementary Figure 1, Extended data¹²).

We asked whether hmP19 DNA was generated by separation of two strands of pUC19 or breakage of one strand followed by release of another one. Formation of a single band of higher electrophoretic mobility by denatured pUC19 (instead of two, which would have been the case when one strand was linearized and another was circular) suggested that hmP19 DNA was generated by separation of two strands of the plasmid (Figure 1a, b). To confirm this notion, hmP19 DNA was subjected to renaturing conditions. If hmP19 DNA were formed due to separation of two strands of pUC19, it would reanneal to form the native plasmid under suitable conditions. Interestingly, neutralization of NaOH with 0.25 M HCl resulted in the appearance of pUC19 plasmid in denatured DNA solution (Figure 2a, Underlying data¹³). To further confirm this, hmP19 DNA was extracted from gel and re-examined by gel electrophoresis. Both, gel-extracted hmP19 DNA and native plasmid DNA exhibited similar band patterns in the agarose gel (Figure 2b). These results showed that single-stranded circular hmP19 DNA was formed due to reversible separation of two strands of pUC19 plasmid DNA.

Figure 2. Single-stranded circular hmP19 DNA annealed to form double-stranded pUC19 plasmid.

pH of denatured pUC19 plasmid DNA was normalized using HCl solution of indicated concentration. Agarose gel electrophoresis showed the formation of native pUC19 plasmid DNA in denatured plasmid DNA solution added with 0.25 M HCl (a). hmP19 DNA was extracted from agarose gel and approximately 3 µg of it was rerun on 1% agarose gel. Band pattern of pUC19 plasmid formed by extracted hmP19 DNA was same as that of native pUC19 plasmid (b). Gel-extracted hmP19 DNA was incubated with Hind III and run on 1% agarose gel. Hind III digested the pUC19 plasmid formed by gel-extracted hmP19 DNA (c). E. coli strain DH5-alpha was transformed with gel-extracted hmP19 DNA by heat-shock method. hmP19 DNA-transformed (d), but not non-transformed (e) bacteria formed colonies on ampicillin-X-Gal-nutrient agar plates. Representative data of two-three independent experiments are shown.

Next, we characterized pUC19 plasmid formed by reannealing of gel-extracted hmP19 DNA. Interestingly, similar to the native plasmid, pUC19 plasmid formed by gel-extracted hmP19 DNA was also degraded by Hind III (Figure 2c). Functionality of pUC19 formed by gel-extracted hmP19 DNA was demonstrated by its ability to transform E. coli. hmP19 DNA-transformed bacteria acquired ampicillin resistance and formed colonies on ampicillin-nutrient agar plates supplemented with X-Gal (Figure 2d, Underlying data¹⁴). No colonies were formed by non-transformed bacteria (Figure 2e). These results showed that pUC19 plasmid formed by re-annealing of hmP19 DNA was structurally and functionally similar to native plasmid DNA.

Concludingly, reversible separation of two strands of plasmid DNA into single-stranded circular DNA molecules shows that DNA strands are not intertwined with each other. These findings contradict the W/C model of DNA structure and provide evidence for the side-by-side structure of DNA.

Data availability