RuvAB is essential for replication forks reversal in certain replication mutants

Abstract

Inactivated replication forks may be reversed by the annealing of leading- and lagging-strand ends, resulting in the formation of a Holliday junction (HJ) adjacent to a DNA double-strand end. In Escherichia coli mutants deficient for double-strand end processing, resolution of the HJ by RuvABC leads to fork breakage, a reaction that we can directly quantify. Here we used the HJ-specific resolvase RusA to test a putative role of the RuvAB helicase in replication fork reversal (RFR). We show that the RuvAB complex is required for the formation of a RusA substrate in the polymerase III mutants dnaEts and holD, affected for the Pol III catalytic subunit and clamp loader, and in the helicase mutant rep. This finding reveals that the recombination enzyme RuvAB targets forks in vivo and we propose that it directly converts forks into HJs. In contrast, RFR occurs in the absence of RuvAB in the dnaNts mutant, affected for the processivity clamp of Pol III, and in the priA mutant, defective for replication restart. This suggests alternative pathways of RFR.

Figure 1

The RFR model (adapted from Seigneur et al, 1998). In the first step (A), the replication fork is arrested by inactivation of dnaE or dnaN protein, causing fork reversal. The reversed fork forms a four-arm structure (HJ, two alternative representations of this structure are shown, open X and parallel stacked X). RecBC is essential for the resetting of a fork, either by RecA-dependent homologous recombination (B, C) or by DNA degradation (B–D). In the absence of RecBCD (E), resolution of the HJ causes chromosome linearization. We use here the RusA resolvase to test a putative role or RuvAB in reversing forks. Continuous lines: parental chromosome. Dashed lines: newly synthesized strands. Circle: RuvAB. Incised circle: RecBCD.

Figure 2

RusA cleaves forks in the absence of RuvABC in a dnaNts mutant, but not in a dnaEts mutant. The histograms indicate the percentage of linear DNA in cultures propagated for 3 h at restrictive temperature (42°C) or semipermissive temperature (37°C). Bars indicate standard deviations. Strain genotypes are indicated below the blocks (full genotypes are described in Supplementary Table S1). (A) RusA cleaves blocked forks in the dnaNts recBC ruvABC mutant. Hatched blocks 37°C, white blocks 42°C: Ruv⁺, JJC1221 (dnaNts recBC); ruv⁻, JJC1323 (dnaNts recBC ruvABC); ruv⁻ rus-1, JJC2648 (dnaNts recBC ruvABC rus-1). (B) RusA does not cleave blocked forks in the dnaEts recBC ruvABC mutant. White blocks, dnaEts strains, 42°C: Ruv⁺, JJC1983 (dnaEts recBCts); ruv⁻, JJC1541 (dnaEts recBCts ruvABC); ruv⁻ rus-1, JJC2624/JJC2671 (dnaEts recBCts ruvABC rus-1). Gray block, DnaE⁺ recBCts ruvABC rus-1 mutant at 42°C (JJC2712/JJC2641).

Figure 3

RusA does not cleave forks in dnaEts recBCts ruvABC rus-1 mutants regardless of the presence or absence of RecA or RecF. The histograms indicate the percentage of linear DNA in cultures propagated for 3 h at restrictive temperature (42°C). Bars indicate standard deviations. Strain genotypes are indicated below the blocks (full genotypes are described in Supplementary Table S1). (A) recA mutants. White blocks, dnaEts: Ruv⁺, JJC1394 (dnaEts recBC recA); ruv⁻, JJC1396 (dnaEts recBC recA ruvABC); ruv⁻ rus-1, JJC2663 (dnaEts recBC recA ruvABC rus-1). Gray blocks, DnaE⁺:Ruv⁺, JJC1105 (recBC recA); ruv⁻, JJC1152 (recBC recA ruvABC); ruv⁻ rus-1, JJC1282 (recBC recA ruvABC rus-1). (B) recF(O) mutants. White blocks, dnaEts: Ruv⁺, JJC1476 (dnaEts recBCts recF); ruv⁻, JJC2217 (dnaEts recBCts recF ruvABC); ruv⁻ rus-1, JJC2625 (dnaEts recBCts recF ruvABC rus-1). Gray block, DnaE⁺ recBCts ruvABC rus-1 recO (JJC2685).

Figure 4

RFR is not required for growth of the dnaEts mutant at semipermissive temperature. Cells propagated at 30°C for 2 h were shifted to 37°C and appropriate dilutions were plated at the indicated times; plates were counted after 48-h incubation at 30°C. (A) Inactivation of recA allows growth of the dnaEts ruvABC mutant at 37°C: JJC1954, dnaEts (circles); JJC2024, dnaEts recA (crosses); JJC2745, dnaEts recA ruvABC (stars); JJC2654, dnaEts ruvABC (triangles), JJC2650 dnaEts recB (squares). (B) Inactivation of recF allows growth of the dnaEts ruvABC mutant: JJC2750, dnaEts recF (circles); JJC2758, dnaEts recF ruvABC (triangles). Inactivation of ruvABC improves growth of the dnaEts recF recB mutant: JJC2864, dnaEts recF ruvABC recB (crosses); JJC2807, dnaEts recF recB (squares).

Figure 5

RusA cleaves forks in holD recBCts ruvABC rus-1 mutants and rep recBCts ruvABC rus-1, but not in priA recB ruvABC rus-1 cells. The histograms indicate the percentage of linear DNA in cultures propagated for 3 h at 42°C. Bars indicate standard deviations. Strain genotypes are indicated below the blocks (full genotypes are described in Supplementary Table S1). White blocks Ruv⁺, JJC2716 cured of pAM-holD⁺ (holD recBCts), JJC1401 cured of pAM-priA⁺ (priA recB), JJC505 and JJC790 (rep recBCts). Gray blocks, ruvABC rus-1, JJC2689 cured of pAM-holD⁺ (holD recBCts ruvABC rus-1), JJC2667 cured of pAM-priA⁺ (priA recB ruvABC rus-1) and JJC2730 cured of pAM-rep⁺ (rep recBCts ruvABC rus-1).

Figure 6

Model of action of RuvAB at blocked forks. In the first step, a RuvA tetramer binds to the fork and drives the assembly of a RuvB hexamer on the template strands. The translocase action of this RuvB hexamer pulls the leading and lagging strands into the RuvA complex (direction of migration of DNA is indicated by arrows) and results in the formation of a HJ. This HJ is bound by a second RuvB hexamer forming a bona fide branch migration complex (direction of translocation of DNA is indicated by arrows; it is unclear at present whether the active form of the branch migration complex in vivo carries one or, as drawn here, two tetramers of RuvA). The HJ is then resolved by RuvC resulting in a cleaved replication fork. For simpliclty, RuvABC and DNA are shown alone, but the complexity of the replication fork factory suggests that additional proteins could play a role in this process.