Role of LrpC from Bacillus subtilis in DNA transactions during DNA repair and recombination

Abstract

Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein, which binds both single-stranded (ss) and double-stranded (ds) DNA and facilitates the formation of higher order protein-DNA complexes in vitro. LrpC binds at different sites within the same DNA molecule promoting intramolecular ligation. When bound to separate molecules, it promotes intermolecular ligation, and joint molecule formation between a circular ssDNA and a homologous ssDNA-tailed linear dsDNA. LrpC binding showed a higher affinity for 4-way (Holliday) junctions in their open conformation, when compared with curved dsDNA. Consistent with these biochemical activities, an lrpC null mutant strain rendered cells sensitive to DNA damaging agents such as methyl methanesulfonate and 4-nitroquinoline-1-oxide, and showed a segregation defect. These findings collectively suggest that LrpC may be involved in DNA transactions during DNA repair and recombination.

Figure 1

Effect of Mg²⁺ in the binding of LrpC to DNA. The binding of increasing concentrations of LrpC to 10 nM pGEM-3Zf(+) circular ssDNA (A), pGEM-3Zf(+) EcoRI-linearized dsDNA (B) and pGEM-3Zf(+) supercoiled dsDNA (C) was assayed in the presence of 5 mM EDTA or 5 mM MgC1₂ by agarose gel electrophoresis followed by ethidium bromide staining. LrpC concentrations are 50, 100, 150, 200 and 400 nM in (A); 100, 200, 400, 500, 600 and 700 nM in (B); 200, 400, 500, 600, 700 and 800 nM in (C). In the last lane of all the parts, the maximal LrpC concentration was incubated with DNA followed by addition of 1% SDS.

Figure 2

LrpC promotes looped and transcomplexes on dsDNA. EcoRI-linearized pUC18 dsDNA [1.8 nM (A) or 0.3 nM (B)] was incubated with increasing concentrations of LrpC in the presence of 3 U of T4 DNA ligase for 3 h a room temperature. The deproteinized DNA was analyzed by agarose gel electrophoresis followed by ethidium bromide staining. LrpC concentrations are 20–80 nM in (A) (lanes 3–5 and 7–9); 80 and 160 nM in (B) (lanes 2 and 3) and 10–160 nM (lanes 5–9). Positions of Form II, Form III and multimers are indicated. In (A), the controls are shown (Form I and II in lane 1, and molecular marker in lane 10).

Figure 3

LrpC promotes ssDNA-bridging. pGEM-3Zf(+) and introduce a space between pGEM and ssDNA (5 nM) was incubated with increasing concentrations of LrpC (12.5–500 nM) in the presence of 5 mM EDTA or 5 mM MgC1₂ for 10 min on ice, and then centrifuged at 12 000 g for 20 min. After addition of 0.5% SDS, the supernatant (upper panel) and the pellet (lower panel) were analyzed in agarose gel electrophoresis followed by ethidium bromide staining.

Figure 4

DNA annealing by LrpC. (A) linearized pGEM-3Zf(+) ssDNA (5 nM) and circular pGEM-3Zf(−) ssDNA (5 nM) were incubated with increasing concentrations of LrpC (22–180 nM) in the presence of buffer B containing 0.1 mM MgCl₂ for 15 min at 30°C. The deproteinized DNA was analyzed by agarose gel electrophoresis followed ethidium bromide staining. (B) HincII-linearized pGEM-3Zf(+) was treated, when indicated, with 7 nM of the SPP1-encoded 5′–3′ exonuclease G34.1P for 45 s to generate 3′-tailed dsDNA. Then the enzyme was inactivated, and the DNA was incubated with circular pGEM-3Zf(+) ssDNA (5 nM) and increasing concentrations of LrpC (lane 5, 1.5 µM; lanes 6–11, 45–1.5 µM) for 15 min at 30°C in buffer C containing 2.5 mM MgCl₂. As controls, the SPP1-encoded G35P ATP-independent recombinase, that anneals only 3′-tailed DNA (700 nM, lanes 3 and 4) and the B.subtilis RecA protein, that performs strand exchange on blunt-ended DNA (2 µM, lane 13, and 4 µM lane 14) were used. The deproteinized DNA was analyzed by agarose gel electrophoresis followed ethidium bromide staining. Running positions of the joint molecules (jm), Form I, II and III DNA, and ssDNA (circular and lineal) are indicated. Plus and minus denote the presence and absence of the indicated protein.

Figure 5

DNA-binding specificity of LrpC. (A) Holliday-junction DNA (0.5 nM) was incubated with increasing concentrations of LrpC (6–50 nM) protein in presence of 5 mM of MgCl₂ or 5 mM EDTA. Protein–DNA complexes were visualized by 6% native PAGE followed autoradiography. (B) The LrpC concentration to reach half-saturation (K_app) with the different DNA substrates used at 5 mM MgCl₂ or 5 mM EDTA is indicated. The values are the average of at least three independent experiments.

Figure 6

LrpC is involved in DNA repair. Survival of B.subtilis strains following exposure to 10 mM MMS (A) and to 100 µM 4NQO (B). The survival of wild-type YB886 (closed triangles), recQ (crosses), addA5addB72 (open triangles), ΔlrpC (closed circles), hbs4755 (open diamonds), recF15 (open squares), ΔrecU (closed squares), ΔrecU ΔlrpC (open circles) and ΔrecA (closed diamond) is shown.

Figure 7

Nucleoid morphologies of ΔlrpC cells. Exponentially growing cells were fixed, stained with DAPI and analyzed by fluorescence microscopy to visualize the nucleoids. The white arrows point to misplaced and linked nucleoids, anucleate cells, and cells with a guillotine phenotype. In the lower panel, wild-type cells (wt) are shown as a control.