The RdgC protein of Escherichia coli binds DNA and counters a toxic effect of RecFOR in strains lacking the replication restart protein PriA
2003; Springer Nature; Volume: 22; Issue: 3 Linguagem: Inglês
10.1093/emboj/cdg048
ISSN1460-2075
Autores Tópico(s)Cancer therapeutics and mechanisms
ResumoArticle3 February 2003free access The RdgC protein of Escherichia coli binds DNA and counters a toxic effect of RecFOR in strains lacking the replication restart protein PriA Timothy Moore Timothy Moore Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH UK Search for more papers by this author Peter McGlynn Peter McGlynn Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH UK Search for more papers by this author Hien-Ping Ngo Hien-Ping Ngo Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH UK Search for more papers by this author Gary J. Sharples Gary J. Sharples Present address: Centre for Infectious Diseases, University of Durham, Wolfson Research Institute, Queen's Campus, Stockton-on-Tees, TS17 6BH UK Search for more papers by this author Robert G. Lloyd Corresponding Author Robert G. Lloyd Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH UK Search for more papers by this author Timothy Moore Timothy Moore Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH UK Search for more papers by this author Peter McGlynn Peter McGlynn Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH UK Search for more papers by this author Hien-Ping Ngo Hien-Ping Ngo Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH UK Search for more papers by this author Gary J. Sharples Gary J. Sharples Present address: Centre for Infectious Diseases, University of Durham, Wolfson Research Institute, Queen's Campus, Stockton-on-Tees, TS17 6BH UK Search for more papers by this author Robert G. Lloyd Corresponding Author Robert G. Lloyd Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH UK Search for more papers by this author Author Information Timothy Moore1, Peter McGlynn1, Hien-Ping Ngo1, Gary J. Sharples2 and Robert G. Lloyd 1 1Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH UK 2Present address: Centre for Infectious Diseases, University of Durham, Wolfson Research Institute, Queen's Campus, Stockton-on-Tees, TS17 6BH UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:735-745https://doi.org/10.1093/emboj/cdg048 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info PriA protein provides a means to load the DnaB replicative helicase at DNA replication fork and D loop structures, and is therefore a key factor in the rescue of stalled or broken forks and subsequent replication restart. We show that the nucleoid-associated RdgC protein binds non-specifically to single-stranded (ss) DNA and double-stranded DNA. It is also essential for growth of a strain lacking PriA, indicating that it might affect replication fork progression or fork rescue. dnaC suppressors of priA overcome this inviability, especially when RecF, RecO or RecR is inactivated, indicating that RdgC avoids or counters a toxic effect of these proteins. Mutations modifying ssDNA-binding (SSB) protein also negate this toxic effect, suggesting that the toxicity reflects inappropriate loading of RecA on SSB-coated ssDNA, leading to excessive or untimely RecA activity. We suggest that binding of RdgC to DNA limits RecA loading, avoiding problems at replication forks that would otherwise require PriA to promote replication restart. Mutations in RNA polymerase also reduce the toxic effect of RecFOR, providing a further link between DNA replication, transcription and repair. Introduction The rdgC gene of Escherichia coli encodes a 34 kDa protein associated with the nucleoid (Ryder et al., 1996; Murphy et al., 1999). Its deletion has no obvious effect except in a nuclease-deficient recBC sbcBC mutant background where it confers a recombination dependent growth phenotype (hence rdg). Thus, a ΔrdgC recBC sbcBC strain is only viable provided the RecA and RecF proteins necessary for recombination in this background are functional (Ryder et al., 1996). Given recombination underpins genome replication (Cox et al., 2000), this raises the possibility that RdgC may normally aid replication fork progression. Replication forks assembled at oriC are inherently processive, but the current view is that their progress is often blocked by lesions in or on the template DNA, with estimates ranging from 10 to 50% of cells requiring some form of fork reactivation (Cox et al., 2000; McGlynn and Lloyd, 2002b). The problem becomes acute in cells exposed to a DNA-damaging agent such as UV light and is complicated by the stalling of transcription complexes at non-pairing lesions in the template strand, creating further obstacles to fork progression (Hanawalt et al., 1994; McGlynn and Lloyd, 2000; van den Boom et al., 2002). Recent studies in E.coli have suggested that stalled forks might unwind so that the parental strands reanneal and the nascent daughter strands anneal to form a Holliday junction (Seigneur et al., 1998). This may occur spontaneously via release of positive supercoiling ahead of the fork, but is more likely catalysed either by RecG helicase or via the strand exchange activity of RecA, aided perhaps by the RecF, RecO and RecR proteins (Robu et al., 2001; Singleton et al., 2001; McGlynn and Lloyd, 2002a, b). Once formed, the Holliday junction may be driven further from the lesion by the RuvAB branch migration complex (Seigneur et al., 1998). Such reactions would require the replisome complex to have dissociated, which raises the question of how DNA synthesis might subsequently resume. Furthermore, the offending lesion has to be repaired or bypassed for this renewed synthesis to continue without mishap. Fork reversal and Holliday junction formation provide possible solutions to these problems. Backing away from the block may create room for repair, although the timing of repair is still unknown and may depend on how replication is resumed. Creating a Holliday junction provides a substrate that recombination enzymes can exploit to promote restart. At least two general models have been proposed. Both rely on the primosome assembly factor PriA to load the DnaB replicative helicase (Liu and Marians, 1999; Liu et al., 1999; Marians, 2000; Sandler and Marians, 2000), but employ two different DNA structures for this purpose. The first relies on direct restoration of a (corrected) fork structure, the second on recombination to first form a D loop, which is then converted to a fork (Seigneur et al., 1998; Gregg et al., 2002; McGlynn and Lloyd, 2002a, b). The latter may sometimes involve fork breakage, possibly via RuvABC-mediated cleavage of a Holliday junction formed during fork reversal. However, Courcelle and Hanawalt have proposed alternative models for direct fork restoration that do not involve formation of a Holliday junction (Courcelle and Hanawalt, 1999, 2001). Both direct and indirect models of fork rescue emphasize the critical role of PriA. This is consistent with the fact that inactivation of PriA results in reduced cell viability, defective cell division, sensitivity to DNA damage, chronic SOS induction and recombination deficiency (Nurse et al., 1991; Kogoma et al., 1996). PriA initiates assembly of a primosome at replication fork and D loop structures via a series of defined protein—protein interactions involving PriB, DnaT and possibly PriC, culminating in transfer of DnaB from a DnaB—DnaC complex to the PriA—DNA complex and subsequent binding of DnaG primase (McGlynn et al., 1997; Liu and Marians, 1999; Liu et al., 1999). DNA polymerase III holoenzyme can then be loaded to form a fully functional replisome capable of coupled leading and lagging strand synthesis. The interaction of PriA with the DnaB—DnaC complex is normally crucial for DnaB loading at replication fork and at D loop structures. However, certain amino acid substitutions in DnaC can circumvent this requirement, enabling DnaB to be loaded without PriA and suppressing the phenotype of priA null strains (Sandler et al., 1996; Xu and Marians, 2000; Gregg et al., 2002). Such dnaC suppressor mutations accumulate rapidly in cultures of priA null strains because of the very substantial improvement in cell viability (Sandler et al., 1996; Gregg et al., 2002). In this paper, we show that RdgC is a DNA-binding protein that forms stable complexes with both single-stranded (ss) DNA and double-stranded (ds) DNA, and that it is required for growth of priA null strains and for the ability of dnaC suppressor mutations to improve their viability. We also show that the RecF, RecO and RecR proteins are responsible for the low viability of priA dnaC rdgC mutant strains and that this toxic effect can be eliminated by changes to the ssDNA-binding protein, SSB. The results presented provide new insight into the interactions between DNA replication, recombination and repair, and raise the possibility that nucleoid organization is important in maintaining replication fork progression in rapidly dividing cells. Results Distribution of rdgC genes in bacteria BLAST searches identified rdgC genes in the Beta (e.g. Neisseria sp.) and Gamma (e.g. E.coli) subdivisions of the Proteobacteria, but in no other species (Figure 1). Thus, rdgC appears to have arisen in the lineage leading to the Beta and Gamma Proteobacteria. It is present in all fourteen species of these groups for which a complete genome sequence is available. The encoded protein sequences show a high degree of conservation (alignments not shown), but provide no insight as to their function. Figure 1.Evolutionary distribution of rdgC. The tree was generated by alignment of 23S rRNA sequences using Clustal_X. An rdgC-like gene is present in the lineage and species highlighted in bold. Download figure Download PowerPoint Purification and physical properties of RdgC The 34 kDa E.coli wild-type RdgC protein was expressed from pGS853 and purified to homogeneity as described in Materials and methods (Figure 2A). Gel filtration indicated that the protein might form a dimer in solution (Figure 2B). Glutaraldehyde cross-linking of the purified protein followed by SDS—PAGE analysis revealed a single cross-linked species with an apparent molecular mass of nearly 100 kDa (Figure 2C). This would be consistent with a trimer of RdgC. However, the absence of any intermediate bands resulting from the cross-linking of only two monomers argues against this possibility. We therefore conclude that native RdgC is a dimer with a molecular mass of 68 kDa, although we cannot exclude the possibility of a trimeric state. Figure 2.Purification and physical properties of RdgC. (A) SDS—PAGE gel summarizing the recovery of RdgC at different stages of the purification protocol. Lane (a) molecular weight markers, (b) induced cell lysate, (c) 50—70% ammonium sulfate cut, (d—g) peak fractions from heparin, gel filtration, butyl—Sepharose and Q Sepharose columns, respectively. (B) Elution profile of RdgC during gel filtration. The elution volumes of molecular weight standards are indicated by arrowheads. (C) Glutaraldehyde cross-linking of RdgC. Reactions contained 10 μM RdgC and glutaraldehyde at 0 (a), 2 (b), 8 (c), 32 (d), 128 (e) and 512 μM (f), and were analysed by SDS—PAGE. Molecular markers are in (g). Monomeric (i) and cross-linked (ii) RdgC species are identified. Download figure Download PowerPoint RdgC binds DNA Two pieces of evidence suggest RdgC might be a DNA-binding protein. First, it is released from E.coli nucleoids by digestion with DNase I (Murphy et al., 1999). Secondly, during attempts at purification, RdgC was found to bind heparin and dsDNA cellulose columns (data not shown). Ryder et al. (1996) suggested RdgC might be an exonuclease, but no nuclease activity of any kind could be detected with the purified protein using a variety of linear and circular DNA substrates under a range of conditions (data not shown). However, band-shift assays confirmed that RdgC binds DNA. Well-defined complexes were detected with linear ssDNA and dsDNA substrates and with a variety of partial duplex and branched molecules (Figure 3A, panels i—vi). Two complexes are formed with a 61 nucleotide single strand (panel i) and four with a 61 bp linear duplex (panel ii). Although, we do not know the precise stoichiometry of these complexes, the results are consistent with the binding of two and four dimers of RdgC, respectively. Complexes formed with the branched substrates vary in number with length of ssDNA and dsDNA available (panels iii—vi). With linear duplexes, the number of complexes increased in proportion to DNA length, indicating that RdgC binds at any point along the DNA and not just to DNA ends (data not shown). The five distinct complexes detected with the Holliday junction substrate, J12, supports this conclusion. A distributive mode of binding is also supported by the fact that RdgC binds circular plasmid DNA and uniformly protects linear duplex DNA from attack by hydroxyl radicals (data not shown). Figure 3.DNA-binding activity of RdgC. (A) Gel assays showing binding of RdgC to linear and branched DNA structures. Binding reactions contained 0.1 nM DNA species, (i) 61 nucleotide ssDNA, (ii) 61 bp dsDNA, (iii) flayed duplex, (iv) three-strand junction, (v) Y-DNA, (vi) Holliday junction J12, and RdgC at 0, 0.5, 5, 50 and 500 nM in lanes a—e, f—j and k—o, respectively. (B) Effect of RecG and RdgC on cleavage of χ DNA by RuvC. Reactions contained 0.05 nM χ junction, 5 nM RuvC monomers (lanes b—i), 1, 10 and 100 nM RecG (lanes c—e), and 1, 10, 100 and 1000 nM RdgC dimers (lanes f—i), as indicated. The χ junction is labelled on all four arms and therefore all four possible products of junction resolution are detected (McGlynn and Lloyd, 2000). Download figure Download PowerPoint The binding data suggested that RdgC does not bind with higher affinity to branch points in DNA as opposed to linear duplex DNA (Figure 3A; data not shown). To confirm this finding, we investigated whether RdgC could interfere with the cleavage by RuvC resolvase of a Holliday junction structure in which the branch point is flanked by long duplex arms (Figure 3B, lane b). RecG protein, which binds with a high affinity to the branch point, clearly interferes with junction resolution (Figure 3B, lanes c—e). RdgC does not, even when in 400-fold molar excess over RuvC (Figure 3B, lanes f—i), indicating that it does not bind the branch point specifically. RdgC has a higher apparent binding affinity for dsDNA than for ssDNA (Figure 4A). This is particularly noticeable with short substrates. RdgC binds a 25 nucleotide single strand (Figure 4B, lane c), but the complex is unstable and dissociates on electrophoresis. It has a much higher affinity for a 25 bp linear duplex, forming a single, sharply-defined complex (Figure 4B, lane c). Competition binding studies confirmed that RdgC forms an unstable complex with the 25 nucleotide single strand. Hardly any retarded complex could be detected following the addition of poly[dIdC] (Figure 4B, lanes d—j and C). They also showed that the complexes formed with a 25 bp duplex and a 61 nucleotide single strand were much more stable (Figure 4B, lanes d—j and C). Complexes formed with the 61 bp linear duplex proved very stable, with no detectable dissociation during 2 h on ice. Figure 4.Affinity of RdgC for ssDNA and dsDNA. (A) Binding isotherm showing relative affinity of RdgC for a 61 bp linear duplex DNA and a 61 nucleotide single strand. Binding reactions contained 0.1 nM labelled DNA and RdgC at 0.025—410 nM. Data are means of two experiments. (B) Competition binding assays. Reactions contained 0.1 nM of the labelled DNA indicated. RdgC was present at 250 nM (25 nucleotide ssDNA), 25 nM (25 bp dsDNA), 20 nM (61 nucleotide ssDNA) and 2 nM (61 bp dsDNA). The RdgC concentration used in each case was the amount needed to achieve a significant bandshift without formation of a substantial fraction of higher order complexes. After 10 min on ice, 50 ng unlabelled poly[dIdC] competitor DNA was added as indicated and the reactions were kept on ice for a further 0, 5, 10, 30, 60, 90 or 120 min (lanes d—j) before electrophoresis, as described in Materials and methods. Horizontal and vertical lines identify substrate DNA and RdgC—DNA complexes, respectively. (C) Relative stability of RdgC complexes with ssDNA and dsDNA. The data from gels of the type shown in (B) were quantified and the complexes detected in the presence of poly[dIdC] expressed as the percentage of the DNA bound in the absence of competitor (B, lane c in each case). Data are averages of two or more experiments. Download figure Download PowerPoint RdgC is expressed at a high level in dividing cells Previous studies indicated that RdgC might be a fairly abundant, nucleoid-associated protein (Murphy et al., 1999). Western blots revealed that RdgC can be detected in extracts of wild-type E.coli cells (Figure 5A, lane b). The polyclonal antibodies used are highly specific for RdgC as no signal is detected in extracts from a ΔrdgC strain (Figure 5A, lane c). This enabled us to accurately measure RdgC in cell extracts at different phases of growth (Figure 5B). RdgC was at its highest level during exponential phase, reaching at its maximum ∼1000 dimers per cell. Its level decreased sharply to ∼50 dimers per cell in stationary phase (Figure 5C). This profile suggests RdgC might function during the period of DNA replication. Figure 5.Immunodetection of RdgC and effect of growth phase on expression. (A) Western blots of purified RdgC (6 ng, lane a) and cells extracts from wild-type strain MG1655 (lane b) and ΔrdgC strain N4586 (lane c) probed with anti-RdgC polyclonal antibodies. (B and C) Growth phase expression of RdgC. Strain MG1655 was grown in LB broth for 8 h from an initial A650 of 0.02. Samples were taken at intervals and assayed by western blotting for RdgC levels in total cell extracts and by microscopy for total cell numbers. (B) Western blots showing immunodetection of RdgC at the intervals shown. (C) Growth-dependent expression of RdgC. The data from blots of the type shown in (B) were quantified by reference to standard concentrations of pure RdgC. Squares indicate the amount of RdgC (dimers) per cell and triangles the total cell number. Data are averages of two experiments. Download figure Download PowerPoint Inviability of ΔrdgC priA and its suppression by dnaC mutations The high expression of RdgC during the period of very active DNA synthesis and its ability to bind DNA are consistent with the previous suggestion by Ryder et al. (1996) that RdgC may have a role in promoting chromosome replication. To investigate whether RdgC promotes replication fork progression, we tried to introduce the priA2 null mutation into a ΔrdgC strain. P1 phage grown on the priA2::Km strain, AG181, was used to transduce strain AB1157 and its ΔrdgC derivative, DIM037, selecting resistance to kanamycin. Although a high number of transductants were obtained with AB1157, none were obtained with DIM037, suggesting that a strain lacking both PriA and RdgC is inviable. Attempts to combine priA2 and ΔrdgC in the MG1655 background also failed. Given that mutations in dnaC such as dnaC212 or dnaC810 suppress priA (Sandler et al., 1996; Gregg et al., 2002), we tried to transduce ΔrdgC::Tm from strain N4586 to the priA2 dnaC212 strain AG181 and the priA2 dnaC810 strain DIM215, this time selecting resistance to trimethoprim. Transductant colonies were obtained in both cases, although they took several days to appear. However, they could be subcultured, indicating that a priA dnaC ΔrdgC construct is viable. One such clone, DIM063, was kept for further analysis. Strain DIM063 grows slowly in LB broth. The cells are highly filamentous and have grossly distorted nucleoids (Figure 6E), and only 10—20% are able to form colonies on LB agar, suggesting they have a major problem with chromosome replication and segregation. Filamentation is more extensive than in a priA2 single mutant (Figure 6C). By comparison cells of the priA dnaC212 parent, AG181, and of the ΔrdgC strain DIM037 have close to 100% viability. They generally resemble wild-type cells, although ΔrdgC cells appear a little more elongated and some form short filaments (Figure 6A, B and D). Thus, elimination of RdgC reverses the ability of dnaC212 to suppress the low viability of priA2 cells. Indeed, it exacerbates defects in cell division, which may explain the inability to construct a priA2 ΔrdgC strain. However, it does not restore sensitivity to DNA-damaging agents. Strain DIM063 is hardly more sensitive to UV than the priA2 dnaC212 parent and is certainly much more resistant than a priA2 single mutant (Figure 7C). It grows very weakly on LB agar containing mitomycin C (MC) at 0.5 μg/ml, but this can be attributed to slow growth and reduced cell viability (data not shown). Figure 6.Filamentous cell morphology of a ΔrdgC priA2 dnaC212 strain and suppression of filamentation by fgv mutations. Phase-contrast and DAPI images are merged to show nucleoid organization within the cell. The strains shown are (A) AB1157 (priA+ dnaC+ rdgC+), (B) DIM037 (ΔrdgC), (C) DIM070 (priA2), (D) AG181 (priA2 dnaC212), (E) DIM063 (priA2 dnaC212 ΔrdgC), (F) DIM061 (priA2 dnaC212 ΔrdgC rpoB), (G) DIM060 (priA2 dnaC212 ΔrdgC ssb) and (H) DIM064 (priA2 dnaC212 ΔrdgC recO). Details of the rpoB, ssb and recO suppressor mutations are in Table I. Download figure Download PowerPoint Figure 7.Suppressors of the slow growth and poor viability of a ΔrdgC priA dnaC strain. (A) Photograph of an LB agar plate streaked with ΔrdgC priA dnaC strain DIM063 and incubated for 60 h at 37°C. Colonies of fast-growing variants are readily visible against the background of small colonies of the parent strain. (B) Colony morphology of purified fast-growing variants of DIM063 incubated for 48 h on LB agar. The strains shown (all ΔrdgC priA2 dnaC212, except AG181) are (i) DIM063 (ΔrdgC priA dnaC parent strain), (ii) DIM057 (unidentified fgv), (iii) DIM060 (ssb), (iv) DIM061 (rpoB), (v) DIM064 (recO), (vi) AG181 (priA dnaC control strain), (vii) DIM089 (recF), (viii) DIM122 (recF143) and (ix) DIM123 (lexA3). Details of the ssb, rpoB, recF and recO suppressor mutations are in Table I. (C) Effect of priA, dnaC and rdgC mutations on sensitivity to UV light. The strains are AB1157 (wt), DIM037 (rdgC), AG181 (priA dnaC), DIM063 (priA dnaC rdgC) and DIM070 (priA). (D) UV sensitivity of derivates of priA dnaC rdgC strain DIM063 carrying suppressors of the slow-growth phenotype. The strains are HP126 (rpoB), DIM062 (ssb), DIM057 (unidentified fgv), HP125 (recO) and DIM089 (recF). Details of the rpoB, ssb, recO and recF mutations are in Table I. Download figure Download PowerPoint Previous studies revealed that the SOS response is chronically induced in priA2 strains (Nurse et al., 1991), and that this phenotype is alleviated by dnaC suppressors (Sandler, 1996). The highly filamentous morphology of DIM063 cells is indicative of chronic SOS induction, consistent with ΔrdgC reversing the effect of dnaC212 in a priA null background. To investigate this directly, we made constructs carrying lacZ fused to the SOS-inducible sfiA gene and tested SOS expression by measuring β-galactosidase activity. In cultures grown in LB broth to an A650 of 0.2, we detected 333 ± 24 units of enzyme activity in the priA2 construct, DIM173, compared with only 39 ± 1 units in the wild type, N5170. Activity was reduced to 83 ± 7 units in a priA2 dnaC212 construct, DIM175, consistent with the suppression of priA, but adding ΔrdgC partially reversed this effect, increasing activity in strain DIM177 to 164 ± 8 units. However, it is significant that this level is <50% of the activity in the priA2 construct. Taken together, these data indicate that chronic SOS induction is a feature of the priA2 dnaC212 ΔrdgC strain DIM063. However, this chronic induction may not be the only factor responsible for the severe growth defects. Cultures of a priA2 dnaC212 ΔrdgC strain accumulate fast-growing variants Strain DIM063 forms small colonies on LB agar and these are quickly overtaken by faster-growing variants. After 3 days incubation, these variants stand out against a background of small colonies (Figure 7A). They are stable and retain their fast-growth phenotype on subculture (Figure 7B). Such variants can also be selected directly by plating DIM063 on LB agar containing MC at 0.5 μg/ml, or rifampicin at 20 μg/ml. We isolated 39 independent isolates of these fast-growth variants (fgv). The gross filamentous phenotype of the parent (Figure 6E) is suppressed in all cases. Typical examples are shown in Figure 6F—H. However, phenotypic and genetic analyses indicated that they fall into at least four different classes. Table I lists examples that we have studied in some detail. All retain the priA2, dnaC212 and ΔrdgC mutations (data not shown), indicating the presence in each case of an additional suppressor mutation. The high frequency with which these suppressors arise suggests that a single suppressor mutation is responsible. This is supported by our mapping of the fgv mutations, which in each case showed that the slow growth phenotype of the parental strain could be fully restored by introducing the wild-type allele for the mutated gene identified. Table 1. Isolation and characterization of fast-growth variants (fgv) of strain DIM063a Strain Selection fgv allele Gene mutation Protein alteration UV survivalb DIM063 0.064 DIM089 LB fgv-010 recFT451A RecFW151R 0.000087 DIM064 LB fgv-007 recOΔ173−362c RecOΔ58-end 0.00014 HP125 LB fgv-037 recOΔT332d RecOΔ110-end 0.00022 HP101 LB fgv-013 recRΔ59−150e RecRΔ19-end 0.00014 DIM060 LB + MC fgv-004 ssbC289T SSBR97C 0.1 DIM062 LB + MC fgv-006 ssbΔ345−434f SSBΔ115−144 0.16 DIM061 LB + MC fgv-005 rpoBC1565A RpoBS522Y 0.084 DIM104 LB + Rif fgv-011 rpoBC1578A RpoBH526Q 0.11 DIM105 LB + Rif fgv-012 rpoBA1547G RpoBD516G 0.039 HP118 LB fgv-030 rpoBC604T RpoBR202C 0.27 HP126 LB fgv-038 rpoBG2542A RpoBE848K 0.15 DIM057 LB + MC fgv-001 Unknown — 0.31 DIM058 LB + MC fgv-002 Unknown — 0.23 DIM059 LB + MC fgv-003 Unknown — 0.28 a Samples from independent cultures of DIM063 (priA dnaC ΔrdgC) grown from independent inocula were spread on the indicated plates (MC at 0.5 μg/ml, Rif at 20 μg/ml), and after 48—60 h at 37°C, colonies of fast-growing variants were purified. b Fraction surviving 30 J/m UV relative to unirradiated cells. Survival of wild-type strain AB1157 was 0.5. Data are means from at least two experiments. c Deletion of a sequence flanked by tctcttgc direct repeats and one of the repeats. d Deletion from a run of 7 T residues. e Deletion of a sequence flanked by ccgggcg direct repeats and one of the repeats. f Deletion of a sequence flanked by gggtgg direct repeats and one of the repeats. RecFOR are toxic to ΔrdgC priA dnaC strains Of the 39 fgv isolates analysed, 16 proved quite sensitive to UV, and remained sensitive to MC. Genetic analyses with 14 of these indicated the presence of an additional mutation in recF (four isolates), recO (two isolates) or recR (eight isolates). Four were sequenced, two in recO and one each in recF and recR. The changes found indicated that protein function is most likely inactivated in each case (Table I). This is consistent with the observed sensitivity to UV (Figure 7B; Table I; data not shown). The recO mutation in strain DIM064 was transferred to wild-type strain AB1157 by exploiting its linkage to pheA. The resulting construct had the same sensitivity to UV as a recO null strain (Mahdi and Lloyd, 1989; data not shown). We also constructed a priA2 dnaC212 ΔrdgC strain carrying the well-characterized recF143 allele. This construct has the same fast-growth, UV-sensitive and non-filamentous cell morphology phenotype as the recF isolate, DIM089 (Figure 7Bviii; data not shown). Taken together, these data demonstrate that the RecF, RecO and RecR proteins are toxic to priA2 dnaC212 ΔrdgC. Furthermore, all three proteins have to be active in order to achieve this effect, suggesting a common basis for the toxicity. Modifications to SSB protein improve growth of ΔrdgC priA dnaC strains The remaining mutants grow well on LB agar containing MC. Indeed, several were selected on this basis (Table I). Two were shown by sequencing to carry mutations in ssb, which encodes the SSB protein (Table I, strains DIM060 and DIM062). One has a single nucleotide change encoding an arginine to cysteine substitution at position 97. The other has an in-frame deletion of 90 bp, eliminating 30 amino acid residues from the C-terminal half of SSB. A further three strains were tentatively identified as carrying fgv mutations in ssb on the basis of linkage to malE and the phenotypic similarity to strains DIM060 and DIM062 (data not shown). Given SSB is an essential protein playing crucial roles in DNA replication and repair (Kuzminov, 1999), it is most unlikely that the ssb alleles in these five strains lead to a su
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