Hin-mediated DNA knotting and recombining promote replicon dysfunction and mutation

Abstract

Background: The genetic code imposes a dilemma for cells. The DNA must be long enough to encode for the complexity of an organism, yet thin and flexible enough to fit within the cell. The combination of these properties greatly favors DNA collisions, which can knot and drive recombination of the DNA. Despite the well-accepted propensity of cellular DNA to collide and react with itself, it has not been established what the physiological consequences are.

Results: Here we analyze the effects of recombined and knotted plasmids in E. coli using the Hin site-specific recombination system. We show that Hin-mediated DNA knotting and recombination (i) promote replicon loss by blocking DNA replication; (ii) block gene transcription; and (iii) cause genetic rearrangements at a rate three to four orders of magnitude higher than the rate for an unknotted, unrecombined plasmid.

Conclusion: These results show that DNA reactivity leading to recombined and knotted DNA is potentially toxic and may help drive genetic evolution.

Figure 1

Physiological effects of Hin-mediated recombination/knotting. (A) Assay for the effect of knotting on the function of a gene. The ovals represent E. coli cells. The Hin expression vector, pHIN, and plasmid substrates pBR, pREC and pKNOT containing the bla gene (encoding β-lactamase) are depicted. Wild-type recombination sites are depicted as black arrows. The mutant hix site is shown as a grey arrow. (B) Effect of DNA knotting on ampicillin sensitivity of E. coli strain W3110 containing pHIN and either pBR, pREC or pKNOT. Single colonies were streaked from left to right across LB-agar that contained an ampicillin gradient and constant IPTG (1 mM) and spectinomycin (50 μg/ml) for Hin overexpression and maintenance. The experiment was repeated five times in either strain C600 or W3110, and was carried out either from high to low or from low to high ampicillin concentration with identical results. (C) Ampicillin sensitivity (MIC₅₀) was quantified using the plate dilution method.

Figure 2

Hin-mediated effect on β-lactamase protein levels. Cultures of C600 (left) and parCts (right) were grown in rich (LB) or minimal (M9) medium. Results here are from a typical experiment performed at 42°C. Immunoblotting was performed on total cellular lysates. Blots were probed with anti-AcrA, anti-β-lactamase, anti-ParC or anti-ParE antibodies. Shown below the blot from cells grown in LB is the mean of four independent experiments (except for parCts pREC, which was performed three times) and standard deviations. The values below the M9 experiment show the quantification of that blot, but the M9 experiment was repeated with the same results.

Figure 3

Hin-mediated effect on plasmid replication. (A) Plasmid DNA was isolated from strains C600 or parCts containing the plasmids indicated. The DNA was linearized with HindIII, which cuts all the plasmids once, including pHIN. Shown is a representative ethidium bromide-stained agarose gel from an experiment performed at 42°C. Given below the gel image are the mean plasmid level values of three independent experiments with standard deviations. (B) Plasmid DNA was isolated 50 min. after IPTG induction of Hin, nicked, displayed by high-resolution gel electrophoresis and visualized by Southern blotting. Shown is an autoradiogram. All lanes contain plasmid DNA from the pHIN-harboring parCts cells with pBR, pREC or pKNOT. The positions of nicked dimer (), linear dimer () catenanes (e.g., ) are indicated.

Figure 4

Potential models for the Hin-mediated effect. Plasmid pKNOT is recombined by Hin to knot the DNA (a single line represents the central axis of the double helix). In the roadblock model, the knot (or possibly Hin bound to or cleaving DNA) is impassable and stalls polymerase. Alternatively, in the breakage model, knots may break DNA as a result of forces on the plasmid.

Figure 5

Hin-mediated mutagenesis. (A) Ampicillin resistant colonies growing in the zone of clearance around a filter containing 4 mg ampicillin. (B) Quantitation of ampicillin resistance of individual colonies. (C) Ethidium bromide-stained gel of plasmid DNA isolated from mutant colonies growing within the zone of clearance and separated by agarose gel electrophoresis. Lane a is a supercoiled molecular weight standard. Lanes b, c and d contain plasmid DNA from the parental strains harboring pHIN and either pBR, pREC or pKNOT, respectively. Lanes e-j contain plasmid DNA isolated from mutant pKNOT colonies. (D) Total cell lysates of mutants grown in 1 mM IPTG were separated by SDS-PAGE and submitted to immunoblotting. Immunoblots were probed with anti-AcrA antibodies (for a loading control) or anti-β-lactamase antibodies. Shown below the blot are signal intensities in arbitrary units. AcrA and anti-β-lactamase levels for C600 strains containing pHIN and either pBR, pREC or pKNOT are shown for comparison.