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

Abbreviations

DNA, Deoxyribonucleic acid

NaOH, Sodium hydroxide

HCl, Hydrochloric acid

HmDNA, Higher electrophoretic mobility DNA.

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 side-by-side positioning of two strands in a DNA molecule, has generated significant interest in the scientific community².

In the present study, 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 was found to be single-stranded and circular, suggesting the separation of two strands of plasmid DNA. Under suitable conditions, higher electrophoretic mobility DNA (HmDNA) re-annealed to form native pUC19 plasmid. These results demonstrated the reversible separation of plasmid DNA strands 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 alkali⁵. In the present study, approximately 5 µg of plasmid DNA was denatured using NaOH solution of increasing concentration. It was observed that addition of 0.5 N NaOH to pUC19 resulted in a new form of DNA having higher electrophoretic mobility in agarose gel (Figure 1a). Similar results were obtained with pBR322 added with 0.5 N NaOH (Figure 1b). In keeping with these findings, formation of higher electrophoretic mobility DNA (HmDNA) in NaOH-denatured pBR322 has also been reported previously⁶.

Figure 1. Formation of higher electrophoretic mobility DNA (HmDNA) in NaOH-denatured plasmid and its characterization using DNA modifying enzymes.

Approximately 5 µl plasmid DNA was added with an equal volume of NaOH solution of indicated concentration and after ~5 min, run on 1% agarose gel. A distinct band of HmDNA (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 0.5 N HCl followed by incubation with different enzymes for 30 min. Native pUC19, but not HmDNA was digested by HindIII, which acts on double-stranded DNA (c). S1 nuclease, which recognizes single-stranded DNA, degraded HmDNA but not native pUC19 plasmid (d). Exonuclease I (Exo I) and alkaline phosphatase (AP), which would eliminate single-stranded linear DNA, digested neither of native pUC19 or HmDNA (e). Electrophoretic mobility of top and bottom strands of pCMV6-AN-HA was compared with that of its HmDNA. Both HmDNA and purified strands of pCMV6-AN-HA exhibited comparable migration in agarose gel (f). Representative data of 2–3 independent experiments are shown.

HmDNA in NaOH-denatured pUC19 was characterized using DNA modifying enzymes. HindIII, which acts on double-stranded DNA, digested pUC19 plasmid but not HmDNA (Figure 1c). S1 nuclease, which digests single-stranded DNA, degraded HmDNA but not pUC19 plasmid (Figure 1d). Exonuclease I and alkaline phosphatase, which would digest single-stranded linear DNA, degraded neither of pUC19 or HmDNA (Figure 1e) but a mix of primers (experimental control). Digested primers did not form product in polymerase chain reaction (Supplementary Figure 1). To rule out the possibility that HmDNA might be denatured but unevenly intertwined plasmid DNA, its electrophoretic mobility was compared with those of individual strands of plasmid. Interestingly, top and bottom strands of pMCV6-AN-HA and its HmDNA exhibited comparable electrophoretic mobilities in agarose gel (Figure 1f). These results showed that HmDNA is single-stranded and circular DNA formed with the separation of two strands in NaOH-denatured plasmid.

Next, we examined whether HmDNA from NaOH-denatured pUC19 can reanneal to form the native plasmid. Interestingly, it was observed that neutralization of NaOH in denatured plasmid with 0.25 N HCl resulted in the appearance of pUC19 plasmid (Figure 2a). Similar results were obtained with HmDNA extracted from agarose gel. Gel-extracted HmDNA re-annealed to form pUC19, which exhibited electrophoretic mobility comparable with that of the native plasmid (Figure 2b). Further, we observed that similar to the native plasmid, pUC19 formed by gel-extracted HmDNA was degraded by HindIII but not by S1 nuclease (Figure 2c).

Figure 2. Formation of native plasmid by higher electrophoretic mobility DNA (HmDNA) and its characterization.

pUC19 plasmid was denatured using 0.5 NaOH as described above. After ~5 min, NaOH in denatured pUC19 was neutralized using hydrochloric acid (HCl) solution of indicated concentration and plasmid DNA was run on agarose gel. Native pUC19 DNA reappeared in denatured plasmid DNA solution added with 0.25 N HCl (a). HmDNA was extracted from agarose gel and approximately 1–3 µg of it was rerun on 1% agarose gel. Gel-extracted HmDNA re-annealed to form pUC19, with its electrophoretic mobility comparable with that of the native plasmid (b). Gel-extracted HmDNA from NaOH-denatured pUC19 was incubated with HindIII and S1 nuclease. pUC19 plasmid formed by HmDNA was degraded by HindIII but not by S1 nuclease (c). E. coli strain DH5-α was transformed with gel-extracted pUC19 HmDNA by heat-shock method. HmDNA-transformed bacteria formed colonies on ampicillin-X-Gal-nutrient agar plates (d). No colonies were formed by non-transformed bacteria (e). Representative data of 2–3 independent experiments are shown.

Functionality of pUC19 formed by gel-extracted HmDNA was demonstrated by its ability to induce bacterial transformation. HmDNA-transformed E. coli acquired ampicillin resistance and formed colonies on ampicillin-nutrient agar plates (Figure 2d). No colonies were formed by non-transformed bacteria (Figure 2e). These results showed that pUC19 plasmid formed by re-annealing of HmDNA is structurally and functionally similar to native plasmid DNA.

Concludingly, reversible separation of two strands of plasmid DNA into single-stranded circular 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