RuvAB is essential for replication forks reversal in certain replication mutants
2006; Springer Nature; Volume: 25; Issue: 3 Linguagem: Inglês
10.1038/sj.emboj.7600941
ISSN1460-2075
AutoresZeynep Baharoglu, Mirjana Petranović, M.J. Flores, Bénédicte Michel,
Tópico(s)Chromosomal and Genetic Variations
ResumoArticle19 January 2006free access RuvAB is essential for replication forks reversal in certain replication mutants Zeynep Baharoglu Zeynep Baharoglu Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas Cedex, France Present address: Centre de génétique Moléculaire, CNRS Bâtiment 26, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France Search for more papers by this author Mirjana Petranovic Mirjana Petranovic Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas Cedex, FrancePresent address: Department of Molecular Genetics, Ruder Boskovic Institute, 10001 Zagreb, Croatia Search for more papers by this author Maria-Jose Flores Maria-Jose Flores Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas Cedex, FranceUnité d'Ecologie et de Physiologie du Système Digestif INRA 78352, Jouy-en-Josas Cedex, France Search for more papers by this author Bénédicte Michel Corresponding Author Bénédicte Michel Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas Cedex, France Present address: Centre de génétique Moléculaire, CNRS Bâtiment 26, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France Search for more papers by this author Zeynep Baharoglu Zeynep Baharoglu Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas Cedex, France Present address: Centre de génétique Moléculaire, CNRS Bâtiment 26, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France Search for more papers by this author Mirjana Petranovic Mirjana Petranovic Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas Cedex, FrancePresent address: Department of Molecular Genetics, Ruder Boskovic Institute, 10001 Zagreb, Croatia Search for more papers by this author Maria-Jose Flores Maria-Jose Flores Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas Cedex, FranceUnité d'Ecologie et de Physiologie du Système Digestif INRA 78352, Jouy-en-Josas Cedex, France Search for more papers by this author Bénédicte Michel Corresponding Author Bénédicte Michel Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas Cedex, France Present address: Centre de génétique Moléculaire, CNRS Bâtiment 26, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France Search for more papers by this author Author Information Zeynep Baharoglu1,2, Mirjana Petranovic1, Maria-Jose Flores1 and Bénédicte Michel 1,2 1Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas Cedex, France 2Present address: Centre de génétique Moléculaire, CNRS Bâtiment 26, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France *Corresponding author. Centre de génétique Moléculaire, CNRS Bâtiment 26, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. Tel.: +33 1 69 82 32 29; Fax: +33 1 69 82 31 40; E-mail: [email protected] The EMBO Journal (2006)25:596-604https://doi.org/10.1038/sj.emboj.7600941 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Replication fork progression may be impeded by the encounter of obstacles such as DNA-bound proteins, or by a transient deprivation of an essential replication component. The consequences of replication fork arrest has been the subject of extensive studies in the past years and replication fork arrest is now recognized as an important source of DNA rearrangements in all organisms (Michel, 2000; Carr, 2002; Kolodner et al, 2002). A growing number of proteins known to participate in homologous recombination, DNA repair or replication restart have been proposed to act at blocked forks in vivo, and some of them directly bind Y-shaped DNA molecules that mimic fork structures in vitro (Jones and Nakai, 2000; Sandler and Marians, 2000; McGlynn and Lloyd, 2002; Hishida et al, 2004; Flores et al, 2005; Heller and Marians, 2005a). The rules that govern the action of different fork-binding proteins at inactivated replication forks in vivo presumably depend on the conditions of fork arrest and are still largely unknown. Replication fork arrest leads in several Escherichia coli replication mutants to a reaction called replication fork reversal (RFR; Seigneur et al, 1998; reviewed in Michel et al, 2004). This reaction involves the annealing of leading- and lagging-strand ends, to form a Holliday junction (HJ) adjacent to a DNA double-strand end (Figure 1A). In E. coli, DNA double-strand ends are processed by the recombinase-exonuclease RecBCD and HJs by the resolvase RuvABC (Kuzminov, 1999). In the absence of RecBCD, resolution of the RFR-made HJ by RuvABC leads to fork breakage (Figure 1E). The RFR model was based on direct quantitative analysis of fork breakage and supported by genetic data. To date, the reaction was proposed to occur in: (i) two helicase mutants, the rep mutant defective for an accessory replicative helicase and dnaBts cells, in which the main replicative helicase is inactivated at high temperature (Seigneur et al, 1998); (ii) three Pol III mutants, dnaEts, dnaNts and holDQ10am, respectively, affected for the catalytic Pol III subunit, the processivity clamp and one of the subunits of the clamp loader (Flores et al, 2001; Grompone et al, 2002) and (iii) finally in the priA mutant defective for the main replication restart pathway (Grompone et al, 2004a). 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. Download figure Download PowerPoint Although the processing of reversed forks by recombination proteins is well understood (Figure 1B–D), less is known about the mechanism of their formation. In vitro, both the homologous recombination protein RecA and the helicase RecG can reverse a particular fork structure (McGlynn and Lloyd, 2000; Robu et al, 2001, 2004). In vivo, RecA is required for RFR in the dnaBts mutant affected for the replicative helicase (Seigneur et al, 2000), but not in other replication mutants (Seigneur et al, 2000; Flores et al, 2001; Grompone et al, 2002, 2004a). RecG has been proposed to promote RFR at replication forks blocked by a UV lesion (McGlynn and Lloyd, 2002), a reaction which is now controversial (Donaldson et al, 2004; Wang, 2005). To date, in most conditions of replication inactivation, the mechanism by which a blocked replication fork is converted into an HJ remains unknown. RuvA, RuvB and RuvC proteins form two complexes: a RuvAB complex with helicase and branch migration activities and a RuvABC complex that resolves HJs (reviewed in West, 1997). Forks are not broken when the RuvABC complex is inactive, because there is no other HJ resolvase in wild-type E. coli. This requirement for RuvABC for HJ resolution prevented us from testing whether the RuvAB complex could be able to bind blocked replication forks in vivo and to convert them into HJs. To address this question, we took advantage of the RusA protein (Mandal et al, 1993; Mahdi et al, 1996). RusA is a resolvase encoded by a cryptic E. coli prophage; it specifically resolves HJs in vitro (Sharples et al, 1994). In vivo, RusA is able to resolve HJs in E. coli as well as in heterologous organisms such as yeast and mammalian cells (Doe et al, 2000; Saintigny et al, 2002). The rusA gene is not expressed in wild-type E. coli due to the lack of a functional promoter, but mutations such as rus-1 activate the expression of a functional RusA protein and suppress the homologous recombination defect of ruvAB or ruvABC mutants (Mandal et al, 1993; Mahdi et al, 1996). In addition to HJs made by homologous recombination, RusA cleaves reversed forks in the helicase mutant dnaBts, in which RFR requires RecA (Seigneur et al, 2000). In this work, we used the RusA HJ resolvase to test in different replication mutants whether RFR occurs in the absence of RuvAB. We first compared in detail two Pol III mutants, dnaNts affected for the processivity clamp and dnaEts affected for the polymerase subunit. RusA cleaves forks in dnaNts ruvABC mutant cells, indicating that reversal of dnaNts-blocked forks does not require RuvAB. In contrast, RusA is not able to cleave blocked forks in dnaEts ruvAB mutants, indicating that the RuvAB helicase is required for the conversion of dnaEts-blocked forks into HJs. The analysis of the other replication mutants in which RFR occurs confirms that RuvAB is required for fork reversal in some but not all of them. Results RFR occurs in dnaNts ruvABC Replication fork breakage is catalyzed in Pol IIIts mutants by the action of the HJ resolvase RuvABC on reversed forks (Grompone et al, 2002). Quantification of the amount of chromosomal DNA able to enter pulse-field gels is used to measure fork breakage (Seigneur et al, 1998; see Materials and methods). As only fully linear DNA is able to enter pulse-field gels, breakage of both replication forks in a circular chromosome is needed for a measurable linearization. Since the presence of RecB and RecC proteins prevents forks cleavage (Figure 1; Michel et al (2004) and references therein), RuvABC-dependent breakage can only be measured in cells in which either recB or recC is inactivated (see Supplementary Table S1 for strain constructions). In the dnaNts recB mutant, a high level of fork breakage occurs at 37°C (62.3%), which is lower when DnaN is fully inactivated at 42°C (36.7%) and is largely abolished by ruvABC inactivation (8.1% at 37°C; Figure 2A and Supplementary Table S2). The lower level of fork breakage in the dnaNts recB mutant at 42°C compared to 37°C has been previously described and is specific for this mutant (Grompone et al, 2002). Reasons for this observation are presently unknown (a partial DnaN activity may be required for RFR, or the DnaN mutant protein may block access to the replication fork at 42°C; Grompone et al, 2002). 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). Download figure Download PowerPoint In order to test whether the helicase action of the RuvAB complex is required for the conversion of inactivated replication forks into HJs, we used ruvABC rus-1 mutant cells, which lack RuvABC and express the RusA resolvase. dnaNts recB (RuvABC active), dnaNts recB ruvABC (no active resolvase) and dnaNts recB ruvABC rus-1 cells (RusA active) were compared. A high level of DNA breakage was also observed at 37°C in the ruvABC rus-1 mutant context, that is, when RusA is active (59.9%), indicating that HJs are formed at dnaNts-blocked forks in the absence of RuvAB (Figure 2A and Supplementary Table S2). Similar results were obtained when the RecBC complex is inactivated at high temperature by recBts and recCts mutations (called here recBCts) rather than a recB null mutation (Supplementary Table S2). It should be noted that 38% linear DNA forms in the recBCts ruvABC rus-1 mutant in which replication is not affected by a mutation (JJC2712; Figure 2B and Supplementary Table S2). The origin of spontaneous chromosome breakage is unknown. It may result from spontaneous replication arrest and resolution by RusA of Ruv-independent RFR occurring at these arrested replication forks. In addition, the level of linear DNA in replication-proficient cells is higher in ruvABC mutants when RusA is expressed (38.1% of linear DNA in recBCts ruvABC rus-1 cells at 42°C versus 20% in recB ruvABC mutant; Seigneur et al, 1998), presumably because RusA resolves recombination intermediates that would otherwise prevent some broken chromosomes from entering pulse-field gels. Nevertheless, the use of the rus-1 allele and the comparison of DnaN+ and dnaNts cells allow us to conclude that at dnaNts-blocked forks RFR does not require RuvABC. The occurrence of RFR in the absence of RuvAB suggests alternative pathways of RFR, but it does not exclude the involvement of RuvAB in RFR in the dnaNts mutant. RFR does not occur in dnaEts ruvABC The rus-1 allele was similarly used to test whether HJs are still formed at blocked forks in the dnaEts mutant when RuvABC proteins are absent. In contrast with the high level of fork breakage in the dnaEts recBCts mutant (64%; Figure 2B and Supplementary Table S3), the level of chromosome breakage in dnaEts recBCts ruvABC rus-1 was not significantly different from that in a DnaE+ recBCts ruvABC rus-1 mutant (37.2 versus 38%; Figure 2B and Supplementary Table S3). Similar results were observed when RecBCD was inactivated by a recB null mutation (recB268∷Tn10, JJC2647; Supplementary Table S3). Consequently, the comparison of DnaE+ and dnaEts cells indicates that DnaE inactivation does not cause fork breakage when RusA is active and RuvABC absent. Similar results were obtained when only RuvA and RuvB were absent (ruvA60∷Tn10 mutation, J1JC2725; Supplementary Table S3). The absence of RusA-dependent fork breakage in the dnaEts recBCts ruvAB(C) rus-1 mutant did not result from a peculiar inhibition of the RusA protein in this mutant, as the rus-1 allele fully suppressed the sensitivity to UV irradiation caused by ruvAB and ruvABC mutations in this background as in all mutants (data not shown). In order to ascertain that the lack of dnaEts-induced fork breakage in a ruvABC rus-1 context results from the absence of RuvABC, a plasmid carrying functional ruvABC genes was introduced in the ruvABC mutant (the low copy plasmid routinely used for complementation experiments, pGB-ruvABC, has no deleterious effect in contrast with high copy plasmids carrying ruvABC genes; Seigneur et al, 1998; Grompone et al, 2002; Lopez et al, 2005). Expression of RuvABC from pGB-ruvABC in the dnaEts recBCts ruvABC rus-1 mutant restored more than 60% of linear DNA, whereas the plasmid vector had no effect (Figure 2B and Supplementary Table S3). This result confirms that the inactivation of ruvABC is responsible for the lower level of fork breakage dnaEts recBCts ruvABC rus-1 cells compared to dnaEts recBCts. The plasmid pGB-ruvAB, which expresses only RuvA and RuvB (Seigneur et al, 1998), was also tested. It was previously reported that cells that express RuvAB and RusA but not RuvC are unable to resolve HJ, presumably because, as shown in vitro, RuvAB prevents RusA action by masking the HJ (Chan et al, 1997). Accordingly, when pGB-ruvAB was introduced in dnaEts recBCts ruvABC rus-1 cells, it prevented homologous recombination at 30°C (rendering cells UV sensitive) and did not restore fork breakage (measured by pulse-field gel quantification, data not shown). In conclusion, the lack of dnaEts-induced fork breakage when RusA is active and RuvAB inactive indicates that RFR requires a functional RuvAB complex in the dnaEts mutant, in contrast with the dnaNts mutant. RuvAB is required for RFR at dnaEts-blocked forks in recA or recFOR mutants Previous results indicate that in both dnaEts and dnaNts mutants a futile reaction takes place at blocked forks prior to RFR: RecA binds to the fork with the help of the presynaptic proteins RecQ, RecJ and RecFOR, and the resulting recombination intermediate is then undone by the UvrD helicase (Flores et al, 2005). In order to determine whether the requirement for RuvAB for RFR is dependent on this ‘RecA-binding/RecA-removal’ reaction, RusA-catalyzed fork breakage was tested in recF, recO and recA backgrounds (Figure 3, and Supplementary Tables S4 and S5). As in the RecAFOR+ context, the level of DNA breakage measured in a dnaEts recBCts ruvABC rus-1 context (RusA active and ruvABC inactive) was significantly lower (31–37%) than in the respective RuvABC+ isogenic strain (55–65%), and not significantly different from the DnaE+ recBCts ruvABC rus-1 recA (or recO) mutant (32.5–41%; Figure 3A, and Supplementary Tables S4 and S5). Expression of RuvABC from a plasmid in dnaEts recBCts ruvABC rus-1 recF (recO) cells restored a high level of fork breakage, indicating that the low level of breakage results from the absence of RuvABC (Figure 3B and Supplementary Table S5). These results show that RuvAB is required for the occurrence of RusA-catalyzed fork breakage, and hence for the formation of reversed forks in the dnaEts mutant, regardless of the presence of RecFOR or RecA. Similarly, breakage in a dnaEts recBCts recF uvrD mutant (60.5%) was decreased to 42% by the combination of ruvABC rus-1 mutations, a level similar to that of the DnaE+ recBCts recO uvrD ruvABC rus-1 mutant (JJC2722; Supplementary Table S5). This observation shows that, in a uvrD recFOR context also, RusA does not act at forks inactivated by the dnaEts mutation when the RuvAB complex is absent. We conclude that RuvAB is required for RFR in the dnaEts mutant regardless of the ‘RecA-binding/RecA-removal’ reaction. 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). Download figure Download PowerPoint Preventing RFR is not lethal in dnaEts at 37°C dnaEts cells can propagate at 37°C, but with a reduced ability to form colonies (Figure 4A; Grompone et al, 2002). dnaEts cells are chronically induced for the SOS response and their viability is strongly improved by mutations that abolish or decrease SOS induction, such as lexAind, recA, recFOR or recQ mutations (Flores et al, 2005; Figure 4). The dnaEts ruvABC mutant is not viable at 37°C (Figure 4A) and its lethality is suppressed by the inactivation of either recA or recFOR (Figure 4). However, the lethality of the dnaEts ruvABC mutant is not suppressed by the sole inactivation of the SOS induction, since dnaEts lexAind ruvABC and dnaEts recQ ruvABC mutants are killed upon shift to 37°C (data not shown). As RecFOR and RecA are required for recombinational gap repair as well as SOS induction, we propose that their inactivation alleviates the need for RuvABC by preventing recombinational gap repair (allowing, e.g., gap filling by a polymerase). Indeed, gaps may form during chromosome replication in the dnaEts mutant as a consequence of defects in lagging-strand synthesis, and recombine in a RecA- and RecFOR-dependent way, forming HJs that render RuvABC essential for viability. If RFR were required for the viability of the dnaEts mutant at 37°C, dnaEts ruvABC recF and dnaEts ruvABC recA strains would not be viable because of the lack of RFR. Since these mutants can propagate at 37°C, we conclude that the dnaEts mutant does not require RFR for growth at this temperature. 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). Download figure Download PowerPoint We tested whether the inactivation of ruvABC, which suppresses fork breakage, suppresses the requirement for RecBC for growth of dnaEts cells at 37°C. To avoid the lethal effect of gap repair in dnaEts cells that lack RuvABC, the question was addressed in a recF background. The inactivation of ruvABC allowed a slow growth of the dnaEts recF recB mutant at 37°C in liquid medium (Figure 4B) and allowed colony formation (data not shown). This result supports the idea that RuvAB is responsible for the requirement for RecB upon replication impairment. RecA, RecG and UvrD are not required for RFR in dnaNts As RFR is RecA-dependent in the dnaBts mutant and RuvAB-dependent in dnaEts mutant, we tested a possible redundant role of RuvAB and RecA for the catalysis of RFR in the dnaNts mutant by measuring chromosome breakage in a dnaNts mutant that lacks both. As RecFOR can be required for RecA binding to block forks, the recO mutation was also tested. A high level of RusA-catalyzed fork breakage was observed in dnaNts recB ruvABC rus-1 recA (or recO) mutants (66–71%; Table I), indicating that dnaNts-blocked forks are reversed in the absence of both RuvAB and RecA (or both RuvABC and RecFOR). Table 1. RecA, RecG and UvrD are not required for RFR in dnaNts Percentage linear chromosome Strain Relevant genotype 30°C 37°C 42°C N JJC1319a dnaNts recB recA 41±6 65.3±5.8 41.7±6.3 3/3/4 JJC1420a dnaNts recB recA ruvABC 33.6±3 22.2±4.6 24.3±3.7 2/4/3 JJC2665 dnaNts recB recA ruvABC rus-1 ND 66.4±1.2 44.8±4.5 2/2 JJC2681 dnaNts recBCts recA ruvABC rus-1 ND 68±2.5 53.6±4.2 4/3 JJC2339a dnaNts recBCts recO 31.8 66.7±6.4 46.5±4.5 1/4/3 JJC2766 dnaNts recBCts recO ruvABC 9.6 12.8±4.1 12.7±1.3 1/3/3 JJC2680 dnaNts recBCts recO ruvABC rus-1 ND 71.3±3.6 45.4±0.6 4/2 JJC2021 (pBR-Gam) dnaNts recA (pBR-Gam+) 25.1 67.5±5 47.7±3.9 1/7/3 JJC2966/JJC2975 (pBR-Gam) dnaNts recA recG ruvABC rus-1 (pBR-Gam+) 29.7±0.3 58.7±2.7 42.1±1.4 2/8/5 JJC2978 (pBR-Gam) dnaNts recA recG ruvABC (pBR-Gam+) 23.4 18.5±3.7 12.3±0.5 1/3/3 JJC2981 (pBR-Gam) recA recG ruvABC rus-1 (pBR-Gam+) 16.5 29.3±0.8 23.9±1.8 1/3/3 JJC2354/2374a dnaNts recBCts recO uvrD 23.3±3.7 67.1±4.2 49.7±5.3 2/13/7 JJC2412a dnaNts recBCts recO uvrD ruvABC 12.3±3 6.7±4.2 10±5.4 2/2/2 JJC2741/JJC2729 dnaNts recBCts recO uvrD ruvABC rus-1 24.3±0.8 67.9±5.1 53.2±4.1 2/4/4 aResults are the average of values previously published (Grompone et al, 2002; Flores et al, 2004, 2005) and values obtained in this work. New values did not differ from previous ones. ND=not defined. RuvAB and RecG share the property of being able to migrate HJs in vitro and in vivo (Lloyd and Sharples, 1993a). RecG has also been shown to reverse fork-like structures in vitro, and possibly in vivo (McGlynn and Lloyd, 2000; Robu et al, 2004). We tested whether RecG could be the helicase that reverses dnaNts-blocked forks in the ruvAB recA context, but we observed that RusA-mediated fork breakage occurs in dnaNts cells that lack RecA, RuvAB and RecG (58%; Table I). This indicates that forks are reversed by yet another activity. Finally, RusA-catalyzed fork cleavage occurs in the dnaNts mutant when RecFOR, UvrD and RuvABC are all inactive (JJC2741/JJC2729; Table I). Therefore, these experiments did not allow identification of the RFR pathway that operates at dnaNts-blocked forks. RuvAB is required for RFR in holDQ10am and rep mutants, but not in the priA mutant Based on the occurrence of RuvABC-dependent fork breakage in a recBC mutant context, RFR was proposed to occur in several replication mutants. The holDQ10am mutant carries an amber mutation in the gene encoding the HolD protein, one of the polypeptides of the Pol III clamp loader. This mutant was isolated in a background in which the mutation is poorly suppressed (Flores et al, 2001). In the AB1157 background used here, the amber mutation is only partially suppressed also since the holDQ10am mutant is lethal when RecBC is inactivated and the holDQ10am recBCts mutant suffers fork breakage at 42°C (Figure 5 and Supplementary Table S6). RFR also occurs in the priA mutant, in which spontaneously arrested replication forks persist because of the inactivation of the main replication restart pathway (Grompone et al, 2004a) and in the rep mutant (Seigneur et al, 1998). The role of the Rep helicase in vivo is not entirely elucidated; it is proposed to remove obstacles in front of replication forks and to participate in replication restart (Seigneur et al, 1998; Sandler, 2000; Heller and Marians, 2005b). In holDQ10am, priA and rep mutants, RFR was shown to be RecA-independent and the molecular mechanism of the reaction is still unknown (Seigneur et al, 2000; Flores et al, 2001; Grompone et al, 2004a). To test whether RuvAB is required for RFR in these three mutants, holDQ10am, rep and priA mutations were combined with the recBCts ruvABC rus-1 mutations. As these combinations of mutations affect growth, strain constructions were made in the presence of a complementing plasmid with conditional replication origin (see Materials and methods). Therefore, mutants were constructed in HolD+, PriA+ or Rep+ backgrounds, respectively, and holDQ10am-, priA- or rep-deficient cells were isolated after segregation of the plasmid (in addition, these mutants were propagated on minimal medium to further prevent the appearance of suppressor mutations). 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). Download figure Download PowerPoint Measures of fork breakage by pulse-field gels showed that RusA did not cause breakage in the holDQ10am recBCts ruvABC rus-1 mutant (28.9 versus 51.5% in RuvABC+ cells; Figure 5 and Supplementary Table S6). Therefore, similarly to dnaEts-blocked forks, holDQ10am-blocked forks are not converted to HJs in the absence of RuvAB. In contrast, the level of DNA breakage was similar in priA recB and priA recB ruvABC rus-1 cells, indicating that HJs form at priA-inactivated forks in the absence of RuvAB (Figure 5 and Supplementary Table S6). Finally, the level of fork breakage in rep recBCts ruvABC rus-1 cells (47.6%) was lower than in rep recBCts cells (67.7%), indicating that RuvAB is invol
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