Copyright Ó 2010 by the Genetics Society of America DOI: 10.1534/genetics.110.120691
RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity in Escherichia coli
Christian J. Rudolph, Akeel A. Mahdi, Amy L. Upton and Robert G. Lloyd1 Institute of Genetics, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom Manuscript received May 11, 2010 Accepted for publication July 16, 2010
ABSTRACT Replication of the Escherichia coli chromosome usually initiates at a single origin (oriC) under control of DnaA. Two forks are established and move away in opposite directions. Replication is completed when these meet in a broadly defined terminus area half way around the circular chromosome. RecG appears to consolidate this arrangement by unwinding D-loops and R-loops that PriA might otherwise exploit to initiate replication at other sites. It has been suggested that without RecG such replication generates 39 flaps as the additional forks collide and displace nascent leading strands, providing yet more potential targets for PriA. Here we show that, to stay alive, cells must have either RecG or a 39 single-stranded DNA (ssDNA) exonuclease, which can be exonuclease I, exonuclease VII, or SbcCD. Cells lacking all three nucleases are inviable without RecG. They also need RecA recombinase and a Holliday junction resolvase to survive rapid growth, but SOS induction, although elevated, is not required. Additional requirements for Rep and UvrD are identified and linked with defects in DNA mismatch repair and with the ability to cope with conflicts between replication and transcription, respectively. Eliminating PriA helicase activity removes the requirement for RecG. The data are consistent with RecG and ssDNA exonucleases acting to limit PriA-mediated re-replication of the chromosome and the consequent generation of linear DNA branches that provoke recombination and delay chromosome segregation.
EPLICATION of the Escherichia coli chromosome and R-loops, potentially enabling replication to initiate R initiates at a single origin (oriC) under the control wherever such structures arise (Marians 2000; Sandler of DnaA (Messer 2002). Two forks are established, and Marians 2000; Heller and Marians 2006b; which then move round the circular chromosome in Michel et al. 2007; Gabbai and Marians 2010). Kogoma opposite directions. Duplication of the chromosome is and co-workers identified a constitutive form of SDR achieved when they meet in a broadly defined terminus (cSDR), which they proposed to initiate at R-loops, and area flanked by polar sequences (ter) that when bound distinguished it from an inducible form (iSDR), which is by the Tus terminator protein allow forks to enter but triggered in cells exposed to genotoxic agents and not leave this area (Mulcair et al. 2006; Duggin et al. characterized by its dependence on RecBCD enzyme 2008). Thus, the chromosome is divided into two (Kogoma 1997). cSDR is elevated in the absence of replichores within each of which replication proceeds RecG or RNase HI (Asai and Kogoma 1994; Hong et al. in a polar fashion from oriC toward ter. However, the two 1995). These two proteins provide different ways of replisome complexes meeting within the terminus area eliminating R-loops. RecG is a double-stranded DNA may not be those assembled at oriC, but new complexes (dsDNA) translocase and dissociates the RNA from the assembled following the rescue of stalled or damaged structure by catalyzing branch migration whereas RNase forks (Gabbai and Marians 2010). HI digests the RNA from the RNA:DNA hybrid (Hong Studies by Kogoma and co-workers showed that this et al. 1995; Vincent et al. 1996; Fukuoh et al. 1997; highly evolved replichore arrangement is compromised McGlynn et al. 1997; Singleton et al. 2001). Strains when DnaA-independent stable DNA replication (SDR) lacking both proteins are inviable, indicating that is initiated via PriA-mediated DnaB loading and repli- excessive levels of SDR may be harmful (Hong et al. some assembly at sites other than oriC (Kogoma 1997). 1995). Following UV irradiation, which triggers iSDR, PriA facilitates DnaB loading at stalled forks, D-loops, DrecG cells show a very extended and PriA helicase- dependent delay in chromosome segregation and cell division (Rudolph et al. 2009a). The majority of the Supporting information is available online at http://www.genetics.org/ DNA synthesis detected during this period is DnaA cgi/content/full/genetics.110.120691/DC1. independent and associated with an increase in the 1Corresponding author: Institute of Genetics, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom. number of replication forks traversing the chromosome. E-mail: [email protected] It can lead to replication of both origin and terminus
Genetics 186: 473–492 (October 2010) 474 C. J. Rudolph et al. areas of the chromosome and of all regions in between. fork collisions and therefore of 39 flaps. In the absence of However, it can also lead to disproportionate amplifica- RecG, these flaps would also have a longer half-life, tion of some chromosomal areas and to the accumula- increasing the opportunity for their targeting by PriA or tion of branched DNA resistant to cleavage by a Holliday for the loading of RecA. Furthermore, without RecG, junction resolvase (Rudolph et al. 2009a,b). any D-loops established by subsequent recombination Although SDR disturbs the replichore arrangement, would be stabilized, further increasing the likelihood of it is not obvious why this should have such dramatic perpetuating cycles of fork collisions and re-replication. effects in the absence of RecG. We have suggested that According to this scenario, 39–59 ssDNA exonucleases by increasing the number of replication fork collisions, might be rather vital in the absence of RecG. In this SDR may trigger repeated cascades of chromosome re- article we show that the presence of exonuclease I replication and recombination and that by limiting SDR (ExoI), exonuclease VII (ExoVII), or the SbcCD nucle- and dissociating recombination intermediates RecG ase, all of which can digest ssDNA from 39 ends, is reduces the likelihood of such pathology (Rudolph needed to keep DrecG cells alive. This requirement can et al. 2009a). be overcome by eliminating the helicase activity of PriA, Exactly what happens when forks meet in the termi- consistent with the idea that a major function of RecG is nus area is not known, although it is generally assumed to curb pathological replication of the chromosome. that the replisome components dissociate as any remain- The presence of at least one of these three enzymes is ing gaps are filled in and the nascent strands are finally also needed to help keep rep and uvrD cells alive, but for sealed by DNA ligase (Figure 1A, i–iii). However, the different reasons consistent with ssDNA exonucleases priming of SDR on either strand at sites other than oriC having multiple roles in DNA replication and repair. means some forks will now meet outside of the normal termination zone (Figure 1B, i) (Kogoma 1997). Studies of DNA replication in vitro raised the possibility that MATERIALS AND METHODS without Tus to arrest forks at ter, the replisome of one Strains: Bacterial strains are listed in supporting informa- fork might sometimes displace the 39 end of the nascent tion, Table S1. All constructs used for synthetic lethality assays leading strand of the fork coming in the other direction are based on E. coli K-12 MG1655 DlacIZYA strains carrying (Hiasa and Marians 1994). If such displacement derivatives of pRC7 (Bernhardt and De Boer 2004). The were to occur in vivo, it would generate a 39 flap (Figure quiescent rusA gene was activated to express the RusA Holliday 1B, ii). Unless removed, the exposed single-stranded junction resolvase using constructs carrying rus-2,anIS10 insertion upstream of the coding sequence (Mahdi et al. DNA (ssDNA) might provide a template on which 1996). Chromosomal genes were inactivated using Tn10 or kan the RecFOR proteins could establish a RecA filament, insertions, conferring resistance to tetracycline (Tcr) and thus provoking recombination (Umezu et al. 1993; kanamycin (Kmr), respectively, or with deletions tagged with Morimatsu and Kowalczykowski 2003). Alternatively, sequences encoding resistance to chloramphenicol (Cmr; cat), r r the branch point might provide a substrate that PriA kanamycin (kan), trimethoprim (Tm ; dhfr), apramycin (Apra ; apra), or spectinomycin (Spcr; spc)(Mahdi et al. 2006; Zhang could exploit with the aid of its helicase activity to load et al. 2010). New deletion alleles of xseA (DxseATdhfr; DnaB and initiate re-replication of the DNA, with DxseATcat), recA (DrecATspc; DrecATcat; DrecATkan), recQ leading strand synthesis primed perhaps via DnaB– (DrecQTapra), uvrD (DuvrDTcat), and sbcCD (DsbcCDTspc) DnaG interactions (Figure 1C, i–iii) (Heller and were made using the one-step gene inactivation method of atsenko anner Marians 2006b). Depending on which fork had its D and W (2000). The DuvrD, DrecA, and DxseA alleles remove all but 39, 45, and 48 bp, respectively, from the leading strand displaced, the new fork would move 59 and 39 ends of the coding sequence. For the sbcCD deletion, either toward oriC or toward ter, generating DNA 50 bp at the beginning of sbcD and 100 bp at the end of sbcC branches with duplex ends that provoke RecBCD- were retained. mediated recombination, thus establishing yet more Plasmids: pRC7 is a low-copy-number, mini-F derivative of 1 ernhardt e oer new forks (Figure 1C, iv and v). Pathological cascades of the lac construct pFZY1 (B and D B 2004). pJJ100 (recG1), pAM375 (recB1), pAM390 (ruvABC1), and this nature may explain the over-replication of DNA pAM409 (recG1 ruvABC1) are derivatives of pRC7 encoding observed in vivo in the absence of Tus/ter control the wild-type genes indicated. Their construction has been (Krabbe et al. 1997; Markovitz 2005). described elsewhere (Mahdi et al. 2006; Zhang et al. 2010). A39 flap might be eliminated in wild-type cells by 39–59 pAM401 (sbcCD1) and pAM490 (rnhA1) are also derivatives of ssDNA exonucleases or converted to a 59 flap by branch pRC7. In each case, the indicated wild-type coding sequence was PCR amplified from MG1655 using 59 and 39 primers migration and then eliminated by 59–39 ssDNA exonu- incorporating ApaI sites, and the product was cloned into the cleases (Figure 1, D and E). RecG is well suited to carry ApaI site within the lacIq gene of pRC7. The inserts are out the conversion as it has a particularly high affinity for transcribed in the same orientation as the disrupted lacIq. 39 flap structures and very efficiently unwinds the strand Media and general methods: LB broth and 56/2 minimal ending 59 at the branch point (McGlynn and Lloyd salts media and methods for monitoring cell growth and for anaka asai strain construction by P1vir-mediated transduction have been 2001; T and M 2006). Without RecG, the cited (Al-Deib et al. 1996; McGlynn and Lloyd 2000; initial number of SDR events initiated would be in- Trautinger et al. 2005). The incidence of spontaneous creased, leading to even higher levels of unscheduled resistance to rifampicin (rpoB mutants) was determined by Exonuclease-Deficient DrecG Cells 475
Figure 1.—Models depicting possible outcomes of replication fork collision [adapted from Rudolph et al (2009b)]. (A) Fork merging and nascent strand ligation. (B) Pathological replication resulting from unscheduled replication fork collisions or during normal termination: (B, i) Schematic of the E. coli chromosome showing normal replication from oriC and the presence of several additional replication forks initiated as a result of SDR induction. The opposed arrowheads indicate the positions of unscheduled fork collisions outside of the normal termination zone bounded by Tus-ter (for simplicity, only two ter sites are depicted). (B, ii) Nascent strand displacement following unscheduled collisions triggered by SDR or, at a lower frequency, in the absence of SDR. (C, i–v) Pathological, PriA helicase-dependent replication in the absence of RecG generates a dsDNA branch that can provoke recombination. (D) Termination achieved via a 59 ssDNA exonuclease after RecG converts a 39 flap to a 59 flap. (E) Termination achieved via a 39 ssDNA exonuclease. (F) Pathological, PriA helicase-independent replication in the presence of RecG. spreading 100-ml samples of broth cultures grown to 2 3 109 SOS induction: SOS induction was analyzed using sulA cells/ml on LB agar plates supplemented with rifampicin at a (sfiA)TlacZ fusion strains. Cultures were grown in broth to an final concentration of 50 mg/ml, which were then incubated A650 of 0.3 and split in two before adding mitomycin C to one overnight. Wild-type strain MG1655 typically yields ,10 re- half to a final concentration of 1 mg/ml. Incubation was then sistant colonies under these conditions whereas derivatives continued for 1 hr, and samples were assayed for b-galactosidase lacking a functional MutHLS system usually yield several activity as described (Miller 1972). hundred (mutator phenotype). Synthetic lethality assays: The rationale for synthetic lethal- Measuring sensitivity to DNA damage: Sensitivity to UV ity assays has been described (Bernhardt and De Boer 2004; light and ionizing radiation was measured using exponential- Mahdi et al. 2006). Essentially, a wild-type gene of interest is 8 1 phase cells grown to an A650 of 0.4 (1–2 3 10 cells/ml for cloned in pRC7, a lac mini-F plasmid that is rapidly lost, and strain MG1655). Samples of appropriate dilutions were used to cover a null mutation in the chromosome in a Dlac irradiated on the surface of LB agar plates and survivors were background. A mutation in another gene of interest is then scored after 18–24 hr incubation. Survival data are means from introduced into the chromosome. If the double mutant is at least two, and usually three to six, independent experi- viable, the plasmid-free cells segregated during culture will ments. Errors (SE) range between 5% and 15% of the mean. form LacÀ colonies on agar plates. If synthetically lethal, they Sensitivity to mitomycin C was determined by growing cultures will fail to grow and only Lac1 colonies formed by cells to an A650 of 0.4 and spotting 10 ml of serial 10-fold dilutions retaining the plasmid will be observed. When viability is from 10À1 to 10À5 on LB agar with or without mitomycin C at a reduced but not eliminated, the colonies formed by cells final concentration of 0.5 mg/ml and incubating at 37°. Plates retaining the plasmid are notably larger than those formed by were photographed after 24 hr incubation, unless stated plasmid-free cells. To record the phenotype, cultures of strains otherwise. Sensitivity to 2-aminopurine (2-AP; Sigma) was carrying the relevant pRC7 derivatives were grown overnight in determined by the same method, using LB agar containing LB broth containing ampicillin to maintain plasmid selection, 2-AP at a final concentration of 300 mg/ml. diluted 80-fold in LB broth, and grown without ampicillin 476 C. J. Rudolph et al.
1 selection to an A650 of 0.4 before spreading dilutions on LB pRC7, a lac mini-F plasmid that is rapidly lost. The agar or 56/2 glucose minimal salts agar supplemented with X- plasmid was used to cover DrecG in a strain also deleted gal and IPTG. Plates were photographed and scored after 48 hr for the lac operon and carrying additional mutations ° (LB agar) or 72 hr (56/2 agar) at 37 , unless stated otherwise. inactivating one or more of the enzymes with known Plasmid-free cells forming small white colonies were re- streaked to see if they could be subcultured, and the streaked ssDNA exonuclease activity. We tested ExoI (encoded by ehman ussbaum plates were photographed after incubation at 37° for 24–48 hr xonA), which attacks 39 ends (L and N (LB agar) or 48–72 hr (56/2 glucose salts agar), as indicated. In 1964); RecJ, which attacks 59 ends (Lovett and Kolodner certain specified cases where plasmid-free segregants (LacÀ 1989); and ExoVII (encoded by xseA), which can target clones) form healthy colonies on 56/2 agar, but fail to appear either end (Chase and Richardson 1974). We also on LB agar, sample colonies from the 56/2 agar plates were tested the SbcCD enzyme, which has multiple nuclease grown in 56/2 glucose minimal salts medium to an A650 of 0.4 activities, including the ability to remove 39 overhangs and further tested to quantify the effect. Each culture was diluted in 10-fold steps from 10À1 to 10À5, and 10-ml aliquots from partial duplexes and to cut hairpin structures halker onnelly were spotted on both LB and 56/2 glucose minimal agar. (C et al. 1988; C et al. 1998, 1999; Colony-forming ability was recorded by photographing the Eykelenboom et al. 2008). Synthetic lethality between plates after incubation for 24 hr (LB agar) or 48 hr (56/2 agar), the covered and the uncovered mutations is revealed if unless specified otherwise. the construct fails to show growth of plasmid-free LacÀ Identification of 2-AP-resistant suppressors: Samples from clones (white colonies and white sectors within blue seven independent cultures of strain N7037, which is deleted colonies) on agar plates supplemented with X-gal and for xonA, xseA, and sbcCD and therefore sensitive to 2-AP and ahdi mitomycin C, were spread on LB agar plates containing 2- IPTG (M et al. 2006). A reduction in viability is aminopurine at a final concentration of 300 mg/ml. A few indicated when the colonies formed by plasmid-free colonies of 2-AP-resistant derivatives were visible on each plate cells are smaller than the blue/sectored colonies after 24 hr at 37°. Seven resistant clones, one from each of the formed by those cells that retained the plasmid at the original cultures, were purified for further analysis. Four time of plating. In such cases, viability can be evaluated exhibited a strong mutator phenotype but remained as sensitive to mitomycin C as the parent. The other three were further by streaking samples of the colonies on the not mutators but exhibited increased resistance to mitomycin relevant agar media to see if they can be subcultured. A C. In one of these three, strain N7050, the mutation re- failure to subculture indicates that the colonies formed sponsible was identified as an allele of rpoC in the following were the result of abortive growth following plasmid loss D T way: The sbcCD kan allele in N7050 was first replaced with a and dilution of the relevant plasmid-encoded gene deletion tagged with resistance to spectinomycin (DsbcCDTspc), and the resulting construct (N7683) was transduced with P1 product. The emergence of large colony variants in- phage grown on pools of cells carrying random kan insertions dicates a viability defect that can be overcome by the in the chromosome generated in strain MG1655 using the EZ- acquisition of suppressors. Tn5 ,kan-2. Tnp Transposome system (Epicentre Technol- 39 ssDNA exonuclease activity is vital for cells lacking r ogies). The Km transductants were screened for those that RecG: The assays conducted revealed that DrecG cells were also sensitive to mitomycin C and 2-AP on the basis that such a clone would carry a kan insertion linked to the wild-type lacking ExoI, ExoVII, and SbcCD are inviable on LB allele of the suppressor locus. One candidate was identified agar, showing no ability to form visible colonies without a (N7704) and shown by PCR sequencing to carry an insertion in covering recG1 plasmid (Figure 2A, i and ii; Figure S1, yijC at minute 89.64 of the genetic map. P1 phage from this xiv). Some colonies of plasmid-free cells are detected on r clone was used to transduce N7683 to Km . Fifty-three percent minimal salts agar, but these are tiny and tend to of the transductants tested proved sensitive to mitomycin C and to 2-AP; i.e., they had lost the suppressor. P1 grown on a accumulate suppressors, as evident from the appearance transductant retaining the suppressor phenotype (N7711) was of large colony variants (Figure 2A, iii and iv). Otherwise, used to transduce strain N7427, which is deleted for xonA, xseA, DrecG cells lacking any one or two of ExoI, ExoVII, or and sbcCD. In this case, 37% of the Kmr transductants selec- SbcCD form colonies on both LB and minimal salts agar ted acquired resistance to mitomycin C and to 2-AP; i.e., they and can be subcultured on both types of media without had inherited the suppressor. These proved as resistant as the original isolate, N7050, from which we concluded that the acquisition of suppressors (Tables 1 and 2; Figure S1; mutation linked to yijC was the sole factor responsible for the data not shown). These observations reveal that DrecG suppression in that isolate. Further genetic analyses sug- cells need a 39 exonuclease to stay alive and that this gested a mutation in the vicinity of the rpoBC operon. PCR requirement can be satisfied by any one of ExoI, ExoVII, sequencing revealed a G-to-A transition at bp 2755 in rpoC, or SbcCD. However, SbcCD alone is able to do so encoding an R919H substitution in RpoC, the b9-subunit of RNA polymerase. efficiently only if RecJ is present. Without RecJ, the cells form small colonies on LB agar that take nearly 24 hr to become visible to the naked eye (Figure 2A, v; Figure 2C, strain N7317). ExoI and ExoVII show no such limitation RESULTS (Figure S1, xv and xvi). The scenario outlined in Figure 1 predicts that ssDNA The only nuclease activity reported for ExoI is the exonucleases might be vital in the absence of RecG. To ability to digest unpaired ssDNA from a 39 end (Lehman investigate whether this is the case, we exploited a and Nussbaum 1964). Therefore, the fact that this synthetic lethality assay based on a recG1 derivative of enzyme suffices to keep DrecG cells alive, and robustly Exonuclease-Deficient DrecG Cells 477
Figure 2.—Maintenance of cell viability by the combined actions of DNA helicases and ssDNA exonucleases. (A) Effect of RecG. (B and C) Effect of RecJ. (D) Effects of RecQ, HelD, DinG, Rep, and UvrD. (A, B, and D) Synthetic lethality assays. These, and similar assays reported in subsequent figures, are described in detail in materials and methods. The relevant genotype of the construct used is shown above each photograph, with the strain number in parentheses. The fraction of white colonies is shown below with the number of white colonies/total colonies analyzed in parentheses. The spot assays in C are of cultures of the strains indicated as serially diluted in 10-fold steps from 10À1 to 10À5 before spotting 10-ml samples on the media indicated, as described in materials and methods. so (Table 1; Figure S1, x), is highly informative. It shows However, the ability to digest 39 ssDNA is vital only in the that the viability of these cells can be maintained absence of RecG. With RecG present, cells lacking ExoI, provided unpaired 39 ssDNA can be degraded, indicat- ExoVII, and SbcCD remain viable (Figure 2A, i), which ing that the accumulation of such strands might have implies that ssDNA species with exposed 39 termini toxic consequences. ExoVII and SbcCD appear to pro- either do not accumulate or can be dealt with by other vide the only other nucleases capable of eliminating this means. This observation also argues against the idea that threat. None of the several other E. coli enzymes ExoI is needed to keep some essential protein complex reported to attack ssDNA from 39 ends, such as exo- intact. For example, ExoI is known to bind the ssDNA nuclease III, exonuclease IX, and exonuclease X binding protein SSB, which itself interacts with a (ExoX) (Viswanathan and Lovett 1999; Lombardo number of other proteins associated with genome repli- et al. 2003; Centore et al. 2008), appears up to the task. cation and maintenance (Shereda et al. 2008). 478 C. J. Rudolph et al.
TABLE 1 Viability of exonuclease-deficient cells lacking RecG, RuvABC, RecA, Rep, UvrD, or RNaseHI
Colony formation by plasmid-free segregants of synthetic lethality constructsa pAM401 pJJ100 pAM390 pAM490 Other chromosomal mutation(s)b sbcCD1/c recG1/DrecG ruv1/Druv rnhA1/DrnhA None, or any 1 or 2 of xonA, xseA, sbcCD, recJ 1111 xonA xseA recJ 11 * À xonA sbcCD recJ 1111 xseA sbcCD recJ 1111 xonA xseA sbcCD 1 ÀÀÀ rep or uvrD plus any 1 or 2 of xonA, xseA, sbcCD 1 xonA xseA sbcCD recQ 1 xonA xseA sbcCD helD 1 xonA xseA sbcCD dinG 1 xonA xseA sbcCD recF * priA300 1111 xonA xseA sbcCD priA300 111d Àe a As determined by using a synthetic lethality assay (materials and methods). 1, plasmid-free segregants form well-developed colonies on LB agar equal to or approaching in size those formed by cells retaining the plasmid (unless indicated otherwise in the text). They also account for 25–75% of the total colonies observed and can be subcultured without difficulty. À, colonies of plasmid-free segregants not detected on either LB or 56/2 minimal salts agar, except as indicated in the text. *, plasmid-free segregants account for .20% of the total colonies observed on LB agar but establish colonies that are much smaller than those formed by cells retaining the plasmid; they establish much stronger colonies on 56/2 minimal salts agar and can be subcultured without difficulty under these conditions. See text for additional details. b The mutations identified were deletions or in some cases (recA and recJ )Tn10 insertions (see Table S1), except for rus-2, which is an orf-56TIS10 insertion activating rusA, and priA300, which is a base substitution encoding helicase-defective PriAK230R. c The chromosome carries sbcCD1, except as indicated in column 1. d The colonies formed on LB agar are smaller than those established by cells retaining the plasmid, but these two types of col- onies are about equal in size on 56/2 minimal salts agar. e Small colonies of plasmid-free segregants detected on 56/2 minimal salts agar, but could not be subcultured.
Assays with constructs based on an sbcCD1 derivative of colonies, but only on LB agar (Figure 2D, vi and vii). pRC7 revealed that ExoIÀ ExoVIIÀ SbcCDÀ cells grow As with cells lacking RecG, the presence of any one of very slowly on LB agar if RecJ is eliminated. However, ExoI, ExoVII, or SbcCD suffices to maintain robust provided RecG is available, they form colonies on viability on LB agar (Table 1 and data not shown). These minimal salts agar that can be subcultured without observations indicate that ExoIÀ ExoVIIÀ SbcCDÀ cells difficulty (Figure 2B, i–iv; Figure 2C, strain N7074). From are likely to have multiples defects in DNA macromo- these data, and those in Table 1 and Figure S1, it seems lecular metabolism. that, whereas DrecG cells have a specific requirement for a ExoIÀ ExoVIIÀ SbcCDÀ cells are sensitive to mito- 39 ssDNA exonuclease, recG1 cells can be maintained by mycin C and 2-aminopurine: The above data (Figure either a 39 ora59 activity. However, the 39 activity has to be 3C; Table 1) demonstrate that any combination of ExoI, ExoI, ExoVII, or SbcCD. No other nuclease present in ExoVII, and SbcCD can be eliminated from wild-type E. coli seems to be able to maintain viability. cells without obvious loss of viability, as reported Rep and UvrD promote viability in rich media: We (Dermic 2006). The mutants form healthy colonies on extended our studies to investigate whether DNA LB agar and are almost as resistant to UV light and helicases other than RecG are required to support mitomycin C as the wild type (Figure 3, A and C; Table 2), growth of ExoIÀ ExoVIIÀ SbcCDÀ cells. RecQ, HelD, with the exception of a construct lacking all three and DinG proved dispensable (Figure 2D, i–iii; Table 1). nucleases. Cells lacking ExoI, ExoVII, and SbcCD are However, Rep, a 39–59 DNA helicase considered to be fairly resistant to UV light but proved sensitive to involved with DNA replication (Yarranton and Gefter ionizing radiation and mitomycin C, although not as 1979), proved essential for colony formation on LB agar, sensitive as a recB strain defective in DNA double-strand although dispensable on minimal salts agar (Figure 2D, break repair (Figure 3, B and C; data not shown). They iv and v), which contrasts with the requirement for RecG are also sensitive to the base analog 2-AP (Figure 3C). A under both conditions (cf. Figure 2D, iv and v, with strain lacking ExoX in addition to ExoI, ExoVII, and Figure 2A, ii and iii). Likewise, UvrD, a 39–59 DNA SbcCD was made without difficulty (Table 2, strain helicase associated initially with DNA repair (Matson N7007), but did not appear different from an ExoIÀ 1986), is required specifically for good growth of ExoVIIÀ SbcCDÀ strain in terms of sensitivity to UV, Exonuclease-Deficient DrecG Cells 479
Figure 3.—Effect of DNA-damaging agents on strains depleted of 39 ssDNA exonucleases. (A) Sensitivity to UV light. (B) Sen- sitivity to ionizing radiation. (C) Sensitivity to mitomycin C and 2-AP. (D) Effect of MutS on the sensitivity of an ExoIÀ ExoVIIÀ SbcCDÀ strain to 2-AP and mitomycin C. (E) Synthetic lethality assays showing the effect of eliminating MutS on the viability of ExoIÀ ExoVIIÀ SbcCDÀ strains lacking RecG, RecJ, Rep, or UvrD. (F and G) mutS derivatives of ExoIÀ ExoVIIÀ SbcCDÀ cells lacking RecJ or Rep accumulate suppressors during growth on LB agar. 480 C. J. Rudolph et al.
TABLE 2 Effect of RecG on UV sensitivity of exonuclease-depleted strains
rec1 constructs DrecG constructs xonA, xseA, sbcCD, Exonuclease exoX, recJ genotype deficiency (polarity) Strain Survivala Strain Survivala Wild type None TB28 0.68 N5742 0.14 xonA ExoIÀ (39–59) N6946 0.67 N7293 0.21 xseA ExoVIIÀ (39–59 and 59–39) N6951 0.68 N7297 0.11 sbcCD SbcCDÀ (39–59) N5281 0.59 N7295 0.11 recJ RecJÀ (59–39) N4934 0.62 N7294 0.044 xonA xseA ExoIÀ ExoVIIÀ N6954 0.58 N7301 0.093 xonA sbcCD ExoIÀ SbcCDÀ N6945 0.44 N7299 0.059 xonA recJ ExoIÀ RecJÀ N7065 0.22 N7298 0.028 xseA sbcCD ExoVIIÀ SbcCDÀ N6952 0.65 N7303 0.13 xseA recJ ExoVIIÀ RecJÀ N7066 0.32 N7302 0.0028 sbcCD exoX SbcCDÀ ExoXÀ N7004 0.80 NCb sbcCD recJ SbcCDÀ RecJÀ N7056 0.63 N7300 0.0067 xonA xseA sbcCD ExoIÀ ExoVIIÀ SbcCDÀ N6953 0.33 Inviablec N7037 0.30 xonA xseA recJ ExoIÀ ExoVIIÀ RecJÀ N7036 0.0072 N7317d 0.001d xonA sbcCD exoX ExoIÀ SbcCDÀ ExoXÀ N7005 0.49 NCb xonA sbcCD recJ ExoIÀ SbcCDÀ RecJÀ N7063 0.17 N7312 0.011 xseA sbcCD exoX ExoVIIÀ SbcCDÀ ExoXÀ N7006 0.79 NCb xseA sbcCD recJ ExoVIIÀ SbcCDÀ RecJÀ N7064 0.21 N7311 0.00025 xonA xseA sbcCD exoX ExoIÀ ExoVIIÀ SbcCDÀ ExoXÀ N7007 0.23 NCb xonA xseA sbcCD recJ ExoIÀ ExoVIIÀ SbcCDÀ RecJÀ N7074d 0.001d inviablec a Survival was determined at a dose of 30 J/m2. Except where indicated otherwise, survival was determined by using cultures grown to exponential phase in LB broth and irradiated on the surface of LB agar plates. Values are means of three to seven experiments. b NC, not constructed. c As revealed by using synthetic lethality constructs carrying pJJ100 (recG1). d These strains cannot be subcultured on LB agar (N7074) or subculture poorly (N7317), but can be grown with no difficulty, and their UV survival determined, by using 56/2 glucose minimal salts media under otherwise identical conditions. mitomycin C, or 2-AP (Table 2 and data not shown). improves the ability of ExoIÀ ExoVIIÀ SbcCDÀ cells Reducing both 39 and 59 activities increases sensitivity to lacking UvrD to form colonies on LB agar (Figure 3E, i; UV, as reported (Viswanathan and Lovett 1998; Table 3). Thus, the poor growth seen with MutS present Dermic 2006), but especially if RecG is also absent may be attributed in part to some consequence of (Table 2; Figure 3A). abortive mismatch repair. Samples of ExoIÀ ExoVIIÀ SbcCDÀ cells spread on LB Eliminating MutS from ExoIÀ ExoVIIÀ SbcCDÀ cells agar supplemented with 2-AP at 300 mg/ml give rise to does not restore resistance to mitomycin C (Figure 3D), colonies of resistant derivatives within 24–48 hr. These nor does it eliminate the dependence on RecG, RecJ, suppressors appear at a frequency of 1/106 initial cells and Rep for growth on LB agar (Figure 3E, ii–iv; Table plated. We isolated seven independent isolates for 3). There is some improvement in the recovery of further analysis. Four of these proved strong mutators plasmid-free colonies of recJ and rep derivatives (Figure (see materials and methods), consistent with a defect 3E, iii and iv), but this is largely attributable to the in mismatch repair. A mutS construct confirmed that outgrowth of suppressors, which is not surprising, given resistance to 2-AP is restored by inactivating the the mutator phenotype of the mutS construct. The MutHLS-dependent and methyl-directed mismatch re- outgrowth of suppressors is very evident when cells pair system (Figure 3D). These data indicate that subcultured in minimal salts medium are plated on LB mismatch repair is compromised in ExoIÀ ExoVIIÀ agar (recJ derivative; Figure 3F) or when white colonies SbcCDÀ cells and leads to inviability when mismatches initially detected on minimal salts indicator plates are increased by exposure to 2-AP. They fit with previ- are streaked directly on LB agar (rep derivative; Figure ous studies demonstrating the involvement of ExoI 3G). and ExoVII in mismatch repair (Harris et al. 1998; One of the three suppressor strains that did not Viswanathan and Lovett 1998; Burdett et al. 2001; display a mutator phenotype proved strongly resistant Viswanathan et al. 2001) and suggest that SbcCD may to both 2-AP and mitomycin C (Figure 4A, strain also be engaged in this process. Eliminating MutS also N7050). This isolate carries an rpoC mutation encoding Exonuclease-Deficient DrecG Cells 481
TABLE 3 Suppression of the reduced viability of exonuclease-deficient cells lacking recombination/repair activities
Effect of mutS, rpoC[R919H], and priA300 mutations on colony formation by plasmid-free segregants of pAM401 sbcCD1/DsbcCD DxonA DxseA constructsa Other chromosomal mutation(s)b mut1 rpo1 pri1 mutS rpoC[R919H] priA300 None 1111 DrecG ÀÀb 1 recJ284 **,c 1 Àd Drep Àd *,c,d 1 DuvrD *,c 111 DruvABC **,c *,c 1 DruvABC rus-2 1 DrecA or recA269 Àd Àb,d *,e a 1, À, and * are defined in Table 1, footnote a. b Plasmid-free segregants for pin-prick colonies that cannot be subcultured. c The colonies observed develop outgrowths of suppressors that form large colonies on subculture. d Plasmid-free segregants establish robust colonies on 56/2 minimal salts agar where they account for .35% of the total and can be subcultured without difficulty using 56/2 salts media. e Plasmid-free segregants can be subcultured on LB agar.
an R919H substitution in the b9-subunit of RNA poly- and Lloyd 2000; Trautinger and Lloyd 2002). These merase. Genetic reconstructions established that this rpo* mutations also promote growth of uvrD rep double mutation alone is responsible for the suppression mutants in rich media (Guy et al. 2009). They are (materials and methods). In other work, a particular thought to act by destabilizing RNAP transcription class of stringent RNA polymerase (RNAP) mutations complexes, thereby reducing the likelihood of patho- (rpo*) was shown to act as partial suppressors of the DNA logical consequences following collisions with replica- repair-deficient phenotype of ruv mutants (McGlynn tion forks (Trautinger et al. 2005; Rudolph et al. 2007).
Figure 4.—Mutations in subunits of RNAP modulate the phenotype of ExoIÀ ExoVIIÀ SbcCDÀ strains. (A) Effect of RpoB and RpoC mutations on sensitivity to mitomycin C and 2-AP. (B) Synthetic lethality assays showing the effect of RpoCR919H on the viability of derivatives lacking RecG, RecJ, Rep, or UvrD. (B, ii, b) Abortive growth of a plasmid-free colony from B, ii, a. 482 C. J. Rudolph et al.
We examined three rpoB alleles in a strain lacking ExoI, Any one of ExoI, ExoVII, or SbcCD is sufficient to ExoVII, and SbcCD and found that each conferred maintain robust viability in cells lacking RuvABC (Figure resistance to mitomycin C and, to a lesser extent, to 2-AP 5A, i; Table 1). But, as we found with DrecG cells, RecJ is (Figure 4A). Thus, the sensitivity of the ExoIÀ ExoVIIÀ redundant, except when both ExoI and ExoVII are SbcCDÀ strain may reflect in part a reduced ability to missing, in which case the cells grow very slowly, even resolve conflicts between DNA replication and tran- more slowly than their DrecG counterparts (Figure 5B, i scription, which are elevated when the template DNA is and ii). Thus, it seems that, under conditions promoting corrupted. rapid growth, recombination is provoked specifically in However, none of the RNAP mutations tested elimi- cells lacking the 39 ssDNA exonuclease activities of ExoI, nates the requirement for RecG to maintain viability. ExoVII, and SbcCD and for reasons that have little to do The synthetic lethality assays exploited revealed that with any defect in mismatch repair or with any conflict some white colonies do appear on LB agar, but are tiny between replication and transcription. and cannot be subcultured without acquisition of Recombination is necessary in ExoIÀ ExoVIIÀ suppressors (Figure 4B, i and ii; Table 3; data not SbcCDÀ cells: We investigated recA derivatives of ExoIÀ shown). Therefore, although conflicts between replica- ExoVIIÀ SbcCDÀ cells to see if the recombination tion and transcription most probably contribute to the detected using ruv constructs is needed to maintain sensitivity of ExoIÀ ExoVIIÀ SbcCDÀ cells to mitomycin C viability. Synthetic lethality assays revealed that these and 2-AP, we suspect that such conflicts are not the cells do need RecA to form colonies on LB agar, primary reason for the inviability observed when the although not on minimal salts agar (Figure 6, A and B; cells are also depleted of RecG. However, they might be Table 3), which is consistent with the absence of any the reason why the cells need Rep and UvrD to grow well need for RuvABC under these conditions. A construct on LB agar as the rpoC suppressor abolishes the re- made using recATTn10 yields plasmid-free colonies on quirement (Figure 4B, iii and iv; Table 3). They may also LB agar, but these appear at a reduced frequency, grow help to explain why the cells need RecJ, although such very slowly, and reveal large colony variants on sub- conflicts cannot be the only reason as the recJ rpoC culture (Figure 6A, iii and iv). These variants proved to construct still shows a growth defect relative to the recJ1 be recA1 derivatives resulting from excision of Tn10 control (Figure 4B, cf. i and v). (data not shown). Their accumulation under these 39 ssDNA exonucleases limit recombination: Early conditions emphasizes the need for RecA to maintain genetic studies indicated that ExoI and SbcCD eliminate viability. The rpoC[R919H] suppressor does little to substrates that RecA protein might otherwise exploit to eliminate this requirement. Plasmid-free colonies are initiate homologous DNA pairing and strand exchange more frequent, but still very small, and the emergence of (Kushner et al. 1971; Lloyd and Buckman 1985; large colony variants remains a major feature (Figure Kowalczykowski 2000). To determine whether such 6C, i and ii; Table 3). substrates accumulate in ExoIÀ ExoVIIÀ SbcCDÀ cells, We made a lexA3 construct to investigate whether RecA we examined a DruvABC derivative on the premise that might be needed to induce the SOS repair response. SOS the initiation of recombination might be toxic without is induced when RecA is assembled on ssDNA exposed an efficient system for resolving Holliday junctions. during replication of damaged DNA or following DNA Synthetic lethality assays revealed that a DruvABC de- breakage and triggers autocleavage of LexA protein, the rivative of an ExoIÀ ExoVIIÀ SbcCDÀ strain grows very SOS repressor (Sassanfar and Roberts 1990). The poorly on LB agar, forming very small colonies without a lexA3 allele encodes a mutant repressor resistant to covering plasmid, colonies that become overgrown with autocleavage. Consequently, lexA3 cells, like recA cells, suppressors (Figure 5A, i and ii; Table 1; data not cannot induce SOS (Sassanfar and Roberts 1990). The shown). Activation of the RusA Holliday junction construct made revealed that lexA3 does not prevent resolvase (rus-2 insertion) largely abolishes this defect ExoIÀ ExoVIIÀ SbcCDÀ cells from growing on LB agar. (Figure 5A, iii; Table 3), demonstrating that the poor Indeed, they form fairly robust colonies (Figure 6C, iii). growth is indeed most likely due to an accumulation of Furthermore, they still need RecG to do so (Figure 6C, iv) Holliday junctions. However, the ruv cells have no as well as RecA, Rep, and UvrD (data not shown). Some difficulty growing on minimal salts agar (Figure 5A, small white colonies are seen with the construct lacking iv), indicating that the accumulation of Holliday junc- RecG, but these are full of suppressors (Figure 6C, v). tions is a problem that arises during conditions permit- From these data we conclude that excessive SOS ting rapid growth. The rpoC[R919H] suppressor expression is not the main reason why the viability of improves colony growth on LB agar, but the effect is ExoIÀ ExoVIIÀ SbcCDÀ cells is so reduced in the absence marginal (Figure 5A, v; Table 3). Eliminating MutS also of RecG, Rep, or UvrD. The data also enable us to increases colony growth (Figure 5A, vi; Table 3), but conclude that these cells rely on the recombinase much of this increase can be attributed to the accumu- activity of RecA to survive growth in rich media rather lation of suppressors, triggered no doubt by the mutator than on its ability to promote SOS induction. However, phenotype (data not shown). the recombinase activity requires assembly of a RecA Exonuclease-Deficient DrecG Cells 483
Figure 5.—ExoIÀ ExoVIIÀ SbcCDÀ cells need a Holliday junction resolvase to maintain rapid growth. (A) Synthetic lethality assays revealing the extremely poor growth on LB agar compared with minimal agar of ExoIÀ ExoVIIÀ SbcCDÀ cells lacking RuvABC. Robust growth on LB agar is restored by activating the RusA resolvase (rus-2 mutation) but not by eliminating MutS, nor by expressing RpoCR919H. (B) Synthetic lethality assays demonstrating how the ability of SbcCD to maintain the viability of DruvABC cells depends on RecJ.
filament on ssDNA, in which case it might be expected normally such DNA would be vulnerable to exonuclease to cause some increase in the expression of SOS genes. digestion. We investigated this possibility and found that the basal It is therefore significant that even after 48 hr level of expression is increased quite substantially. This incubation, ExoIÀ ExoVIIÀ SbcCDÀ cells lacking RecF is clear from the approximately fivefold higher levels of form small and sickly colonies on LB agar, although they b-galactosidase seen in ExoIÀ ExoVIIÀ SbcCDÀ cells grow well enough on minimal salts agar (Figure 6D, i carrying the lacZ1 gene fused to the LexA-regulated sulA and ii; Table 1), whereas those lacking RecB are quite (sfiA) promoter (Table 4). It is also clear that the viable, forming colonies on both types of media as presence of any one of ExoI, ExoVII, or SbcCD curbs efficiently as the recB1 parent. Unlike the recF derivative, this increase, whereas the rpoC[R919] suppressor does the colonies formed on LB agar are well developed not. The SOS response is induced strongly in every case after only 24 hr incubation (Figure 6E; data not shown). following exposure to mitomycin C. The relevant recB construct was made both with and These data demonstrate that ssDNA must be exposed without the aid of a covering plasmid, but any covering and in a form that not only is vulnerable to attack by plasmid used is eliminated with such a high frequency ExoI, ExoVII, or SbcCD but also is amenable to RecA under nonselective conditions that it prohibits use of loading. Two pathways have been identified by which a our standard assay for synthetic lethality. However, this stable RecA filament can be assembled on ssDNA: one itself emphasizes the viability of the plasmid-free cells mediated by RecBCD enzyme and the other by the (Table S1, strains N7783 and N7789). We conclude RecFOR proteins. RecBCD unwinds and resects duplex that at least some of the ssDNA on which RecA is DNA ends and loads RecA at a 39 ssDNA overhang loaded to initiate recombination is generated by means created after the enzyme activity has been modulated other than RecBCD-mediated digestion of duplex DNA at a x-sequence. The 39 end of the exposed strand may ends. be held within the RecBCD complex, protecting it RecG limits recombination during normal growth: A from other nucleases, while RecB’s helicase activity central tenet of the model in Figure 1 is that recombi- may strip away any SSB protein (Kowalczykowski nogenic 39 flaps are generated more frequently in cells 2000; Amundsen and Smith 2003; Singleton et al. lacking RecG because of the increased incidence of 2004). The RecFOR proteins normally load RecA at unscheduled fork collisions. Accordingly, and on the ssDNA gaps. They enable RecA to displace SSB protein, basis that a ruv mutant would have no means to resolve which has a higher affinity for ssDNA (Umezu et al. 1993; Holliday junctions efficiently by junction cleavage Morimatsu and Kowalczykowski 2003; Cox 2007). (Zhang et al. 2010), a Druv DrecG cell should be more However, RecFOR can also assemble RecA at ssDNA sensitive to a reduction in 39 exonuclease activity than a ends generated independently of RecBCD, although ruv single mutant. We used a ruv1 recG1 derivative of 484 C. J. Rudolph et al.
Figure 6.—The recombinase activity of RecA is needed to maintain rapid growth of ExoIÀ ExoVIIÀ SbcCDÀ cells. (A and B) Synthetic lethality and spot dilution assays showing how ExoIÀ ExoVIIÀ SbcCDÀ cells lacking RecA grow well on minimal agar but are inviable on LB agar. (C–E) Synthetic lethality and spot dilution assays showing the effects of RpoCR919H, lexA3, RecF, and RecB on the viability of ExoIÀ ExoVIIÀ SbcCDÀ cells.
pRC7 to test this possibility and examined the effect of Robust growth is maintained in this case if RecG is present eliminating any two of ExoI, ExoVII, and SbcCD. or RecA eliminated (Figure 7B, ii and iii). Removing all three would be uninformative as we have Taken together, these data indicate that potentially shown this to compromise the viability of both Druv and recombinogenic substrates do arise more frequently in DrecG strains. With all three nucleases present, DruvABC the absence of RecG but can be eliminated efficiently via DrecG cells show no major reduction in viability in that the action of 39 ssDNA exonucleases, particularly by they form good colonies without the covering plasmid ExoI. However, the inability of ExoIÀ ExoVIIÀ SbcCDÀ (Figure 7A, i and ii). If recombinogenic 39 flaps do arise cells lacking either RuvABC or RecA to establish good more frequently in the absence of RecG, then it seems colonies on LB agar indicates that recombinogenic that these can be removed efficiently by exonuclease substrates are generated under conditions promoting digestion. rapid growth even with RecG present and that the cells ExoI proved sufficient to maintain robust viability must engage in and complete recombination if they are (Figure 7A, iii). ExoVII also keeps the cells alive, but the to stay alive. If these recombinogenic substrates are 39 slightly smaller size of the plasmid-free colonies observed flaps, or dsDNA branches arising from subsequent PriA- with the relevant construct suggests that it might be a little mediated replication, RecG is clearly unable to convert less effective (Figure 7A, iv). SbcCD proved rather in- all 39 flaps to 59 flaps, or if it can, then RecJ cannot effective, with the cells forming small and rather sickly eliminate all the 59 flaps formed. The fact that elimi- colonies without the covering plasmid (Figure 7B, i). nating RecA enables SbcCD to maintain robust viability Exonuclease-Deficient DrecG Cells 485
TABLE 4 Effect of ssDNA exonuclease deficiency on SOS expression
b-Galactosidase activitya Exonuclease deficiency (additional genotype) Strain no. No. of experiments ÀMMC 1MMC None N7843 10 77 6 8 1474 6 70 ExoIÀ N7844 7 94 6 4 1591 6 96 ExoVIIÀ N7859 4 83 6 12 1343 6 60 SbcCDÀ N7845 4 65 6 13 1384 6 61 ExoIÀ ExoVIIÀ N7860 4 136 6 6 1542 6 95 ExoIÀ SbcCDÀ N7846 4 102 6 5 1354 6 82 ExoVIIÀ SbcCDÀ N7847 4 109 6 7 1309 6 47 ExoIÀ ExoVIIÀ SbcCDÀ N7837 7 398 6 28 1962 6 52 None (rpoC[R919H]) N7888 4 135 6 7 2520 6 42 ExoIÀ ExoVIIÀ SbcCDÀ (rpoC[R919H]) N7889 4 476 6 40 2365 6 75 a Measured in DlacIZYA strains carrying a sulAT lacZ1 fusion as described in materials and methods and expressed as Miller Units. Values are means 6 SE. MMC = mitomycin C. in the absence of RecG is significant (Figure 7B, iii). It is reducing or eliminating the helicase activity of PriA (Al- consistent with the formation of dsDNA branches that Deib et al. 1996; Jaktaji and Lloyd 2003; Rudolph et al. provoke RecBCD-mediated recombination (Figure 1C, 2009a; Zhang et al. 2010). We exploited priA300, which iii and iv). Without RecA to load on the 39 ssDNA encodes helicase-defective PriAK230R, to investigate exposed after an encounter with x, RecBCD recombi- whether the same holds true for the inviability caused nase activity is aborted and its dsDNA exonuclease in ExoIÀ ExoVIIÀ SbcCDÀ cells. Synthetic lethality activity (ExoV) becomes rampant (Dabert et al. 1992; constructs revealed that priA300 restores robust viability Kuzminov and Stahl 1997). SbcCD removes the (Figure 8A, i–iv; Table 3). The priA300 allele also ssDNA, enabling the DNA to be targeted and further improves the ability of ExoIÀ ExoVIIÀ SbcCDÀ cells digested by another RecBCD molecule (Zahradka et al. lacking RuvABC or RecA to form colonies on LB agar, 2009). This combination of nuclease activities facilitates although viability is still compromised in each case, as complete removal of the DNA branch, thus possibly evident from the more robust growth of cells retaining explaining the restoration of robust viability. the covering plasmid (Figure 8A, v and vi; Table 3). PriA activates recombination in cells lacking RecG: However, priA300 does not confer viability in the Previous studies revealed that most, if not all, aspects of absence of both RecG and RecA (Figure 8, vii). It also the recG mutant phenotype are suppressed by mutations does not suppress the sensitivity of ExoIÀ ExoVIIÀ
Figure 7.—RecG limits the requirement for RuvABC to maintain rapid growth of ExoIÀ ExoVIIÀ SbcCDÀ cells. (A and B) Syn- thetic lethality assays showing the effect of 39 ssDNA exonucleases on the viability of a strain lacking RuvABC and RecG. 486 C. J. Rudolph et al.
Figure 8.—Effect of priA300 on ExoIÀ ExoVIIÀ SbcCDÀ cells depleted of RecA, RecG, RuvABC, or UvrD. (A) Synthetic lethality assays. (B) Spot dilution assays showing sensitivity to mitomycin C and 2-AP.
SbcCDÀ cells to mitomycin C and 2-AP (Figure 8B). have reduced viability (Sandler 2000; Mahdi et al. These observations, together with the fact that priA300 2006). is not otherwise a suppressor of ruv or recA (Jaktaji and RNase HI-deficient cells require ssDNA exonu- Lloyd 2003), demonstrate that it is PriA, and in cleases to stay alive: The data presented above fit with particular some activity that depends on its ability to the idea that the inviability of ExoIÀ ExoVIIÀ SbcCDÀ unwind DNA, that is responsible for the recombination cells lacking RecG is a consequence of SDR initiated via provoked in cells lacking ExoI, ExoVII, and SbcCD and PriA helicase activity. However, we were conscious that for making these cells dependent on RecG for their RecG is needed to facilitate recovery of recombinants in viability. However, it is also clear from the weak growth of crosses with ruv strains, a fact previously interpreted cells lacking RuvABC or RecA (Figure 8A, v and vi) and as evidence that RecG and RuvABC provide alterna- the inviability of those lacking both RecG and RecA that tive ways for processing recombination intermediates eliminating PriA helicase activity does not prevent (Lloyd 1991). To investigate whether it is SDR that is recombination altogether. responsible rather than some recombination defect, We also examined priA300 cells lacking ExoI, ExoVII, we analyzed exonuclease-deficient constructs lacking SbcCD, and UvrD and found that they, too, form robust RNase HI (DrnhA). This enzyme degrades RNA from colonies on LB agar (Figure 8A, viii; Table 3). Their RNA:DNA duplexes, including from R-loops, and its vigorous growth contrasts sharply with the feeble absence is known to trigger a high level of cSDR, high colonies formed by the equivalent priA1 cells (Figure enough in fact to sustain viability in the absence of 2D, vi), which we have demonstrated to be due in large origin firing (Kogoma 1997). There is no evidence that measure to some consequence of abortive mismatch it has any direct role in recombination. repair as it can be alleviated by eliminating MutS (Figure We observed that removing RNase HI mimics the 3E, i). Therefore, we suspect that the abortive mismatch effect of removing RecG in that DrnhA cells are inviable repair in these cells creates substrates that PriA can if ExoI, ExoVII, and SbcCD are eliminated. The exploit via its helicase activity to load DnaB and thus synthetic lethality constructs that were exploited yielded initiate replication in a manner that reduces viabili- no plasmid-free colonies on either LB agar or minimal ty. We were unable to investigate the effect of priA300 salts agar (Figure 9, i–iii; Table 1; data not shown). This on ExoIÀ ExoVIIÀ SbcCDÀ cells lacking Rep, as we is consistent with SDR being the trigger for the in- were unable to make the relevant construct. Previous viability of exonuclease-depleted DrecG cells. However, studies established that priA300 rep double-mutant cells removing RNase HI differs in that priA300 does little to Exonuclease-Deficient DrecG Cells 487
Figure 9.—RNase HI promotes survival of cells depleted of ssDNA exonucleases, RuvABC, or RecG. (A and B) Synthetic le- thality assays. (C) Sensitivity to UV light. restore viability. The plasmid-free cells form tiny colo- the viability of ruv cells (Figure 9B, v and vi). The nies on minimal agar but fail to grow on LB agar (Figure differences observed may be explained by the possibility 9A, iv and v; Table 3). The cells are also inviable on LB that RNase HI may also help to process Okazaki agar if ExoI, ExoVII, and RecJ are missing but grow well fragments during replication (Ogawa and Okazaki on minimal agar (Figure 9B, i and ii). As with cells 1984; Kornberg and Baker 1992). Without it, gaps may lacking RecG, the presence of either ExoI or ExoVII is accumulate in the lagging strand, compounding any sufficient to maintain viability, even if both SbcCD and difficulties arising from the increase in SDR but also RecJ are missing (Figure 9B, iii and iv; Table 1; data not compromising viability even when SDR is reduced. shown). The two differences may be explained by the incomplete suppression of SDR by priA300 and the presence of RecG, which could convert some 39 flaps to DISCUSSION 59 flaps. Without RecJ and ExoVII, PriA might then Recent studies of how the integrity of the genome and initiate pathological re-replication by targeting these cell viability are maintained during the course of DNA flaps (Figure 1F). The PriAK230R protein would retain replication in bacteria have focused on describing what the ability to do so as the template for DnaB loading is happens to replication forks as they encounter blocking already single stranded, eliminating the need for heli- lesions in or on the DNA template or on dissecting the case activity. Removing RNase HI also mimics the effect of re- molecular mechanisms that enable cells to overcome eller moving RecG in that it increases the sensitivity of ruv such blocks and complete replication (H and arians ahdi ichel mutant cells to killing by UV light. However, the effect is M 2006b; M et al. 2006; M et al. 2007; not as severe (Figure 9C). On the other hand, even with Rudolph et al. 2007; Guy et al. 2009; Boubakri et al. a full complement of ssDNA exonucleases available, 2010; Gabbai and Marians 2010). By comparison, removing RNase HI makes DrecG cells inviable, as much less attention has been paid to what happens reported (Hong et al. 1995), whereas it only reduces when one fork runs into another, an event that usually 488 C. J. Rudolph et al.
Figure 10.—Models illustrating how DNA molecules containing single-strand flaps might be processed by RecG translocase or by ssDNA exonucleases or targeted by RecA or PriA to provoke recombination (see discussion for further details). happens only once at the termination of replication to DrecG cells only when these cells have suffered damage during the bacterial cell cycle, but which occurs many to their DNA. Thus, given that DrecG cells lacking ExoI, times along each chromosome during S-phase in eu- ExoVII, and SbcCD are inviable, it would seem that DNA karyotes. In this article, we have presented evidence must somehow become ‘‘damaged’’ in these cells even consistent with the idea that the termination of DNA without application of external genotoxic agents, i.e.,as synthesis does not always run smoothly, at least in E. coli, a result of events that occur during normal growth. and that, consequently, replication fork encounters We considered the possibility that this ‘‘damage’’ need to be limited. might reflect abortive repair of DNA base-pair mis- The data presented demonstrate that DrecG cells need matches generated during chromosome replication a39 ssDNA exonuclease to stay alive and that this (Iyer et al. 2006). Both ExoI and ExoVII are implicated requirement can be satisfied by any one of ExoI, ExoVII, in nascent strand removal following initiation of mis- or SbcCD or eliminated by inactivating the helicase match repair by the MutHLS proteins (Burdett et al. activity of PriA. The data are consistent with a model in 2001; Viswanathan et al. 2001). Inactivating these two which unscheduled initiation of replication via PriA- enzymes, and any possible substitute, might therefore mediated replisome assembly at sites remote from oriC leave recombinogenic nicks or gaps in the nascent (SDR) compromises the highly evolved replichore strands, which might then account for the fact that arrangement that orchestrates duplication and trans- homologous recombination and means to resolve Holli- mission of the E. coli chromosome and that normally day junctions are essential for the survival of cells limits fork collisions to a single event during each cycle lacking ExoI, ExoVII, and SbcCD (Figures 5A and 6A). of cell growth and division. The data fit with the idea that Indeed, we discovered that cells lacking ExoI, ExoVII, a major role of RecG is to limit such replication (Figure and SbcCD are sensitive to 2-AP, one of the hallmarks of 1). Taken together, the data suggest that the ability of a DNA mismatch repair defect downstream of the PriA to secure chromosome duplication in times of initiation step (Glickman and Radman 1980). These replicative stress comes at a price (Rudolph et al. cells also need UvrD to grow well on LB agar, a DNA 2009a,b). helicase required for displacement of the nascent stand Previous studies demonstrated that mutations reduc- containing the mismatched base (Iyer et al. 2006). ing or eliminating PriA helicase activity suppress the Eliminating MutS restores resistance and reduces the sensitivity of recG mutants to mitomycin C (Al-Deib et al. need for UvrD, establishing that the cells do indeed 1996; Gregg et al. 2002; Jaktaji and Lloyd 2003). They suffer from abortive mismatch repair. However, elimi- also eliminate most of the delay in division observed nating MutS does not remove the requirement for RecG following irradiation of DrecG cells with UV light and RuvABC, indicating that the cells must have at least (Rudolph et al. 2009a). However, it is significant that one additional defect. DrecG cells are quite healthy, with a doubling time close A reduced ability to resolve conflicts between DNA to that of wild type. This implies that PriA becomes toxic replication and transcription is indicated by the fact Exonuclease-Deficient DrecG Cells 489 that, in addition to UvrD protein, ExoIÀ ExoVIIÀ cuses subsequent degradation on the strand ending 59, SbcCDÀ cells need Rep to grow on LB agar, but not on leaving a 39 tail on which RecBCD then loads RecA to minimal salts agar. Previous studies revealed that rep initiate recombination (Smith 1990; Kowalczykowski uvrD double mutants have the same growth constraint 2000; Singleton et al. 2004; Amundsen et al. 2007; and that this can be alleviated by mutations shown to Dillingham and Kowalczykowski 2008). However, destabilize RNAP transcription complexes (Guy et al. although RecBCD is crucial for the repair of DNA 2009). We suggest that, in the absence of ExoI, ExoVII, breaks, recBC single mutants are viable, as are recA and SbcCD to remodel a fork stalled at a ternary mutants, which would suggest that chromosome break- transcription complex, UvrD might have greater diffi- age is rather rare under normal growth conditions. culty compensating for the absence of Rep and vice Breaks are detectable in recBC cells (Seigneur et al. versa. Consistent with this, we discovered that the 1998), and viability is reduced (Capaldo-Kimball and requirement for UvrD and Rep is alleviated in the Barbour 1971), but some of the breaks may be presence of an rpoC mutation encoding an R919H pathological in origin, being a consequence of RecBCD substitution in the b9-subunit of RNAP. This mutation inactivation. ExoV is thought to be particularly instru- and other previously described rpoB alleles also allevi- mental in eliminating the Holliday junction structure ates the sensitivity of ExoIÀ ExoVIIÀ SbcCDÀ cells to 2-AP formed at reversed forks, thus limiting fork breakage via and mitomycin C. However, they do not eliminate the RuvABC (Seigneur et al. 1998). It is therefore signifi- need for RecG or for RecA, indicating that there is yet cant that cells lacking ExoI, ExoVII, and SbcCD do not another defect in these cells. require RecBCD to stay alive. Furthermore, the viability Studies with strains carrying sulATlacZ fusions re- of recB recG and recB ruv cellsisnodifferentfromthatof vealed that the SOS response is constitutively elevated in recBC single mutants (Lloyd et al. 1987; Lloyd and ExoIÀ ExoVIIÀ SbcCDÀ cells and that ExoI, ExoVII, and Buckman 1991). Taken together, these observations SbcCD all contribute to keeping its expression at a low indicate that increased chromosome breakage is not the level during normal growth in LB broth. Furthermore, primary reason for the failure of cells lacking ExoI, SOS expression remains high in the presence of the ExoVII, and SbcCD to form colonies when RecG, RuvABC, rpoC[R919H] suppressor mutation, indicating that the or RecA is inactivated and that something other than SOS-inducing signal is generated at the same high level the processing of duplex DNA ends is responsible for even when conflicts between DNA replication and the initial generation of potentially toxic 39 ssDNA. transcription might be reduced. Hiasa and Marians (1994) presented evidence in- The high level of SOS expression, coupled with the dicating that a 39 flap might be generated when the fact that cells lacking ExoI, ExoVII, and SbcCD become replisome of one replication fork displaces the 39 end of inviable if RecA, RecG, or RuvABC is eliminated is the nascent leading strand of the fork coming in the significant. It indicates (1) that ssDNA species with free other direction. This led us to propose that 39 flaps 39 ends accessible to these nucleases are generated with might be generated in this way quite frequently in a or without RecG present; (2) that these strands enable DrecG strain as a result of the increase in SDR, which RecA to initiate recombination with high efficiency; and leads to additional and unscheduled replication fork (3) that this recombination is essential, providing the encounters outside of the normal termination area only means apart from exonuclease digestion of dealing (Rudolph et al. 2009a,b, 2010). Flaps of this nature with the exposed strands. So, how do these recombino- would be expected to be particularly problematic for genic 39 strands arise? And why are they particularly cells lacking RecG and depleted of 39 ssDNA exonu- problematic in the absence of RecG? There is clearly a cleases. As outlined in Figure 10, they could provide deficiency in mismatch repair and most probably a templates for RecA loading and strand exchange. The reduced ability to resolve conflicts between DNA repli- loading of RecA might also provoke recombination. cation and transcription. However, neither defect is Otherwise, PriA could target the branch point to initiate sufficient to explain why ExoIÀ ExoVIIÀ SbcCDÀ cells further replication, which would convert the flap to a need RecA, RecG, and RuvABC to stay alive. dsDNA branch, thus providing an alternative route to Chromosome breakage would most certainly expose a recombination via RecBCD enzyme. Such exchanges need for RecA. Breakages occur when forks encounter would generate a network of partially replicated chro- nicks or gaps in the template strands or when they stall mosomes with numerous DNA branches (Figure 1C, iv and reverse to establish a Holliday junction structure and v), as observed (Rudolph et al. 2009b). thatcouldbetargetedandcleavedbyRuvABC(Kuzminov The suggestion that cells lacking RecG and depleted 1995; Seigneur et al. 1998). RecBCD enzyme would of 39 ssDNA exonucleases suffer damage to their DNA as normally be expected to unwind the duplex DNA end a result of unscheduled replication fork collisions is exposed in both cases and to degrade both strands via consistent with the fact that the problem largely its ExoV activity until it encounters a x-sequence in the disappears when the helicase activity of PriA is elimi- strand ending 39, whereupon its activity is modified in nated (Figure 8), especially as PriA helicase mutants some manner that is not fully understood but that fo- reduce SDR (Tanaka et al. 2003). Given that DrecG and 490 C. J. Rudolph et al.
DrnhA cells have in common a high level of cSDR 2000; Gari et al. 2008; Sun et al. 2008). However, initiated at R-loops (Kogoma 1997), it is also consistent although RecG might be targeted to replication forks with the fact that eliminating RNase HI mimics the via its interaction with SSB (Lecointe et al. 2007), direct effect of eliminating RecG (Figure 9). However, priA300 evidence that it promotes replication fork reversal in vivo does little to eliminate the problem in this case. is distinctly lacking. Furthermore, it exhibits a strong Furthermore, RecJ is also needed to maintain viability. preference in vitro for unwinding forks that mimic These two observations are exactly what we would a39 flap structure of the type depicted in Figure 1 predict if the RecG present in these cells were able to (McGlynn and Lloyd 2001). convert a 39 flap to a 59 flap (Figure 10). PriA might then We thank Carol Buckman and Lynda Harris for excellent technical be able to initiate replisome assembly without need of its help and colleagues identified in Table S1 for plasmids and strains. helicase activity as there would be no lagging strand to This work was supported by a program grant to R.G.L. from the UK get in the way of DnaB loading, but alternatively, PriC Medical Research Council and by an early career fellowship to C.J.R. might do so (Heller and Marians 2005, 2006a,b). from the Leverhulme Trust. However, cells lacking RNase HI not only are less effective in eliminating R-loops but also may be de- fective in Okazaki fragment processing (Ogawa and LITERATURE CITED Okazaki 1984), which, if true, would create at least two Al-Deib, A. A., A. A. Mahdi and R. G. Lloyd, 1996 Modulation of different problems for chromosome replication and recombination and DNA repair by the RecG and PriA helicases of thus make it difficult to draw definitive conclusions Escherichia coli K-12. J. Bacteriol. 178: 6782–6789. Amundsen,S.K.,andG.R.Smith, 2003 Interchangeable parts of À À À from rnhA derivatives of ExoI ExoVII SbcCD cells. the Escherichia coli recombination machinery. Cell 112: 741– By reducing fork collisions to a single event per cell 744. mundsen aylor eddy mith cycle and restricting termination to a defined area, the A , S. K., A. F. T ,M.R and G. R. S , 2007 Intersubunit signaling in RecBCD enzyme, a complex replichore arrangement of the chromosome enables protein machine regulated by Chi hot spots. Genes Dev. 21: complete replication of the chromosome to be achieved 3296–3307. with minimum conflict with transcription and in a way Asai, T., and T. Kogoma, 1994 Roles of ruvA, ruvC and recG gene functions in normal and DNA damage-inducible replication of that allows the 8-bp KOPS (FtsK orienting polar sequen- the Escherichia coli chromosome. Genetics 137: 895–902. ces) DNA elements to be arranged symmetrically in each Bernhardt, T. G., and P. A. de Boer, 2004 Screening for synthetic chromosome arm to direct efficient FtsK-mediated lethal mutants in Escherichia coli and identification of EnvC (YibP) chromosome segregation during cell division (Bigot as a periplasmic septal ring factor with murein hydrolase activity. Mol. Microbiol. 52: 1255–1269. et al. 2005; Reyes-Lamothe et al. 2008). With RecG Bigot, S., O. Saleh,C.Lesterlin,C.Pages,M.El Karoul et al., present to unwind R-loops, D-loops, and 39 ssDNA flaps, 2005 KOPS: DNA motifs that control E. coli chromosome segre- and a full complement of exonucleases with the ability to gation by orienting the FtsK translocase. EMBO J. 24: 3770–3780. Boubakri, H., A. L. de Septenville,E.Viguera and B. Michel, digest any ssDNA flaps and dsDNA branches that may 2010 The helicases DinG, Rep and UvrD cooperate to promote arise, wild-type cells are able to maintain the advantages replication across transcription units in vivo. EMBO J. 29: 145–157. conferred. Burdett, V., C. Baitinger,M.Viswanathan,S.T.Lovett and P. Modrich, 2001 In vivo requirement for RecJ, ExoVII, ExoI, However, we cannot exclude other interpretations of and ExoX in methyl-directed mismatch repair. Proc. Natl. Acad. the data presented, especially because RecG, PriA, Sci. USA 98: 6765–6770. SbcCD, and ExoVII act on a variety of substrates Capaldo-Kimball, F., and S. D. Barbour, 1971 Involvement of recombination genes in growth and viability of Escherichia coli in vitro. Thus, cells lacking multiple exonucleases may K-12. J. Bacteriol. 106: 204–212. accumulate so much ssDNA that they simply die when Centore, R. C., R. Lestini and S. J. Sandler, 2008 XthA (exonu- DNA macromolecular metabolism is further compro- clease III) regulates loading of RecA onto DNA substrates in mised by the elimination of RecG. We did not test the log phase Escherichia coli cells. Mol. Microbiol. 67: 88–101. Chalker, A. F., D. R. F. Leach and R. G. Lloyd, 1988 Escherichia coli viability of DrecG cells lacking every possible combina- sbcC mutants permit stable propagation of DNA replicons con- tion of ssDNA exonucleases to eliminate this possibility. taining a long DNA palindrome. Gene 71: 201–205. Furthermore, the RecBCD enzyme has a potent ssDNA Chase, J. W., and C. C. Richardson, 1974 Exonuclease VII of Escher- ichia coli: mechanism of action. J. Biol. Chem. 249: 4553–4561. exonuclease activity that might be able to eliminate a 39 Connelly, J. C., L. A. Kirkham and D. R. Leach, 1998 The SbcCD flap. Thus, the lesion responsible for the inviability of nuclease of Escherichia coli is a structural maintenance of chromo- exonuclease-depleted DrecG cells, rather than being a 39 somes (SMC) family protein that cleaves hairpin DNA. Proc. Natl. Acad. Sci. USA 95: 7969–7974. ssDNA flap generated during unscheduled fork colli- Connelly, J. C., E. S. de Leau and D. R. Leach, 1999 DNA cleavage sions as we suggest, may be some downstream conse- and degradation by the SbcCD protein complex from Escherichia quence of replication fork blockage resulting from an coli. Nucleic Acids Res. 27: 1039–1046. ox inability to process DNA strands by nuclease digestion. C , M. M., 2007 Regulation of bacterial RecA protein function. Crit. Rev. Biochem. Mol. Biol. 42: 41–63. RecG itself has been shown to drive replication fork Dabert, P., S. D. Ehrlich and A. Gruss, 1992 x sequence protects reversal in vitro, a key feature of models for promoting against RecBCD degradation of DNA in vivo. Proc. Natl. Acad. replication restart in both bacteria and eukaryotes that Sci. USA 89: 12073–12077. Datsenko, K. A., and B. L. Wanner, 2000 One-step inactivation of incorporate the need to process the nascent DNA chromosomal genes in Escherichia coli K-12 using PCR products. strands (Seigneur et al. 1998; McGlynn and Lloyd Proc. Natl. Acad. Sci. USA 97: 6640–6645. Exonuclease-Deficient DrecG Cells 491
Dermic, D., 2006 Functions of multiple exonucleases are essential Lecointe, F., C. Serena,M.Velten,A.Costes,S.McGovern et al., for cell viability, DNA repair and homologous recombination 2007 Anticipating chromosomal replication fork arrest: SSB tar- in recD mutants of Escherichia coli. Genetics 172: 2057–2069. gets repair DNA helicases to active forks. EMBO J. 26: 4239–4251. Dillingham, M. S., and S. C. Kowalczykowski, 2008 RecBCD en- Lehman, I. R., and R. Nussbaum, 1964 The deoxyribonucleases of zyme and the repair of double-stranded DNA breaks. Microbiol. Escherichia coli. V. On the specificity of exonuclease I. J. Biol. Mol. Biol. Rev. 72: 642–671. Chem. 239: 2628–2636. Duggin, I. G., R. G. Wake,S.D.Bell and T. M. Hill, 2008 The rep- Lloyd,R.G.,1991 Conjugationalrecombination in resolvase-deficient lication fork trap and termination of chromosome replication. ruvC mutants of Escherichia coli K-12 depends on recG. J. Bacteriol. Mol. Microbiol. 70: 1323–1333. 173: 5414–5418. Eykelenboom, J. K., J. K. Blackwood,E.Okely and D. R. Leach, Lloyd, R. G., and C. Buckman, 1985 Identification and genetic 2008 SbcCD causes a double-strand break at a DNA palindrome analysis of sbcC mutations in commonly used recBC sbcB strains in the Escherichia coli chromosome. Mol. Cell 29: 644–651. of Escherichia coli K-12. J. Bacteriol. 164: 836–844. Fukuoh, A., H. Iwasaki,K.Ishioka and H. Shinagawa, 1997 ATP- Lloyd, R. G., and C. Buckman, 1991 Genetic analysis of the recG lo- dependent resolution of R-loops at the ColE1 replication origin cus of Escherichia coli K-12 and of its role in recombination and by Escherichia coli RecG protein, a Holliday junction-specific heli- DNA repair. J. Bacteriol. 173: 1004–1011. case. EMBO J. 16: 203–209. Lloyd, R. G., C. Buckman and F. E. Benson, 1987 Genetic analysis Gabbai, C. B., and K. J. Marians, 2010 Recruitment to stalled rep- of conjugational recombination in Escherichia coli K-12 strains de- lication forks of the PriA DNA helicase and replisome-loading ac- ficient in RecBCD enzyme. J. Gen. Microbiol. 133: 2531–2538. tivities is essential for survival. DNA Repair (Amst.) 9: 202–209. Lombardo, M. J., I. Aponyi,M.P.Ray,M.Sandigursky,W.A. Gari, K., C. Decaillet,M.Delannoy,L.Wu and A. Constantinou, Franklin et al., 2003 xni-deficient Escherichia coli are proficient 2008 Remodeling of DNA replication structures by the branch for recombination and multiple pathways of repair. DNA Repair point translocase FANCM. Proc. Natl. Acad. Sci. USA 105: 16107– (Amst.) 2: 1175–1183. 16112. Lovett, S. T., and R. D. Kolodner, 1989 Identification and purifi- Glickman, B. W., and M. Radman, 1980 Escherichia coli mutator mu- cation of a single-stranded-DNA-specific exonuclease encoded by tants deficient in methylation-instructed DNA mismatch correc- the recJ gene of Escherichia coli. Proc. Natl. Acad. Sci. USA 86: tion. Proc. Natl. Acad. Sci. USA 77: 1063–1067. 2627–2631. Gregg, A. V., P. McGlynn,R.P.Jaktaji and R. G. Lloyd, Mahdi,A.A.,G.J.Sharples,T.N.Mandal andR.G.Lloyd, 2002 Direct rescue of stalled DNA replication forks via the com- 1996 Holliday junction resolvases encoded by homologous rusA bined action of PriA and RecG helicase activities. Mol. Cell 9: genes in Escherichia coli K-12 and phage 82. J. Mol. Biol. 257: 561–573. 241–251. Mahdi, A. A., C. Buckman,L.Harris and R. G. Lloyd, 2006 Rep Guy, C. P., J. Atkinson,M.K.Gupta,A.A.Mahdi,E.J.Gwynn et al., and PriA helicase activities prevent RecA from provoking unnec- 2009 Rep provides a second motor at the replisome to promote essary recombination during replication fork repair. Genes Dev. duplication of protein-bound DNA. Mol. Cell 36: 654–666. 20: 2135–2147. Harris, R. S., K. J. Ross,M.J.Lombardo andS.M.Rosenberg, Marians, K. J., 2000 Replication and recombination intersect. Curr. 1998 Mismatch repair in Escherichia coli cells lacking single-strand Opin. Genet. Dev. 10: 151–156. exonucleases ExoI, ExoVII, and RecJ. J. Bacteriol. 180: 989–993. Markovitz, A., 2005 A new in vivo termination function for DNA Heller, R. C., and K. J. Marians, 2005 The disposition of nascent polymerase I of Escherichia coli K12. Mol. Microbiol. 55: 1867– strands at stalled replication forks dictates the pathway of repli- 1882. some loading during restart. Mol. Cell 17: 733–743. Matson, S. W., 1986 Escherichia coli helicase II (uvrD gene product) Heller, R. C., and K. J. Marians, 2006a Replication fork reactiva- translocates unidirectionally in a 39 to 59 direction. J. Biol. Chem. tion downstream of a blocked nascent leading strand. Nature 261: 10169–10175. 439: 557–562. McGlynn, P., and R. G. Lloyd, 2000 Modulation of RNA polymer- Heller, R. C., and K. J. Marians, 2006b Replisome assembly and ase by (p)ppGpp reveals a RecG-dependent mechanism for rep- the direct restart of stalled replication forks. Nat. Rev. Mol. Cell lication fork progression. Cell 101: 35–45. Biol. 7: 932–943. McGlynn, P., and R. G. Lloyd, 2001 Rescue of stalled replication Hiasa, H., and K. J. Marians, 1994 Tus prevents overreplication of forks by RecG: simultaneous translocation on the leading and oriC plasmid DNA. J. Biol. Chem. 269: 26959–26968. lagging strand templates supports an active DNA unwinding Hong, X., G. W. Cadell and T. Kogoma, 1995 Escherichia coli RecG model of fork reversal and Holliday junction formation. Proc. and RecA proteins in R-loop formation. EMBO J. 14: 2385–2392. Natl. Acad. Sci. USA 98: 8227–8234. Iyer, R. R., A. Pluciennik,V.Burdett and P. L. Modrich, McGlynn, P., A. A. Al-Deib,J.Liu,K.J.Marians and R. G. Lloyd, 2006 DNA mismatch repair: functions and mechanisms. Chem. 1997 The DNA replication protein PriA and the recombination Rev. 106: 302–323. protein RecG bind D-loops. J. Mol. Biol. 270: 212–221. Jaktaji, R. P., and R. G. L loyd, 2003 PriA supports two distinct Messer, W., 2002 The bacterial replication initiator DnaA. DnaA pathways for replication restart in UV-irradiated Escherichia coli and oriC, the bacterial mode to initiate DNA replication. FEMS cells. Mol. Microbiol. 47: 1091–1100. Microbiol. Rev. 26: 355–374. Kogoma, T., 1997 Stable DNA replication: interplay between DNA Michel, B., H. Boubakri,Z.Baharoglu,M.LeMasson and R. replication, homologous recombination, and transcription. Lestini, 2007 Recombination proteins and rescue of arrested Microbiol. Mol. Biol. Rev. 61: 212–238. replication forks. DNA Repair (Amst.) 6: 967–980. Kornberg, A., and T. A. Baker, 1992 DNA polymerase I of E. coli, Miller, J. H., 1972 Experiments in Molecular Genetics. Cold Spring pp. 113–164 in DNA Replication, Ed. 2. W. H. Freeman, New York. Harbor Laboratory Press, Cold Spring Harbor, NY. Kowalczykowski, S. C., 2000 Initiation of genetic recombination Morimatsu, K., and S. C. Kowalczykowski, 2003 RecFOR pro- and recombination-dependent replication. Trends Biochem. teins load RecA protein onto gapped DNA to accelerate DNA Sci. 25: 156–165. strand exchange: a universal step of recombinational repair. Krabbe, M., J. Zabielski,R.Bernander and K. Nordstrom, Mol. Cell 11: 1337–1347. 1997 Inactivation of the replication-termination system affects Mulcair, M., P. Schaeffer,A.Oakley,H.Cross,C.Neylon et al., the replication mode and causes unstable maintenance of plas- 2006 A molecular mousetrap determines polarity of termina- mid R1. Mol. Microbiol. 24: 723–735. tion of DNA replication in E. coli. Cell 125: 1309–1319. Kushner, S. R., H. Nagaishi,A.Templin and A. J. Clark, Ogawa, T., and T. Okazaki, 1984 Function of RNase H in DNA rep- 1971 Genetic recombination in Escherichia coli: the role of exo- lication revealed by RNase H defective mutants of Escherichia coli. nuclease I. Proc. Natl. Acad. Sci. USA 68: 824–827. Mol. Gen. Genet. 193: 231–237. Kuzminov, A., 1995 Collapse and repair of replication forks in Reyes-Lamothe, R., X. Wang and D. Sherratt, 2008 Escherichia coli Escherichia coli. Mol. Microbiol. 16: 373–384. and its chromosome. Trends Microbiol. 16: 238–245. Kuzminov, A., and F. W. Stahl, 1997 Stability of linear DNA in recA Rudolph, C. J., P. Dhillon,T.Moore and R. G. Lloyd, mutant Escherichia coli cells reflects ongoing chromosomal DNA 2007 Avoiding and resolving conflicts between DNA replication degradation. J. Bacteriol. 179: 880–888. and transcription. DNA Repair (Amst.) 6: 981–993. 492 C. J. Rudolph et al.
Rudolph, C. J., A. L. Upton,L.Harris and R. G. Lloyd, recombination-dependent DNA replication. Genes Cells 8: 2009a Pathological replication in cells lacking RecG DNA trans- 251–261. locase. Mol. Microbiol. 73: 352–366. Trautinger, B. W., and R. G. Lloyd, 2002 Modulation of DNA re- Rudolph, C. J., A. L. Upton and R. G. Lloyd, 2009b Replication fork pair by mutations flanking the DNA channel through RNA poly- collisions cause pathological chromosomal amplification in cells merase. EMBO J. 21: 6944–6953. lacking RecG DNA translocase. Mol. Microbiol. 74: 940–955. Trautinger, B. W., R. P. Jaktaji,E.Rusakova and R. G. Lloyd, Rudolph, C. J., A. L. Upton,G.S.Briggs and R. G. Lloyd, 2010 Is 2005 RNA polymerase modulators and DNA repair activities re- RecG a general guardian of the bacterial genome? DNA Repair solve conflicts between DNA replication and transcription. Mol. (Amst.) 9: 210–223. Cell 19: 247–258. Sandler, S. J., 2000 Multiple genetic pathways for restarting Umezu, K., N. Chi and R. D. Kolodner, 1993 Biochemical interac- DNA replication forks in Escherichia coli K-12. Genetics 155: tion of the Escherichia coli RecF, RecO, and RecR proteins with 487–497. RecA protein and single-stranded DNA binding protein. Proc. Sandler, S. J., and K. J. Marians, 2000 Role of PriA in replication Natl. Acad. Sci. USA 90: 3875–3879. fork reactivation in Escherichia coli. J. Bacteriol. 182: 9–13. Vincent, S. D., A. A. Mahdi and R. G. Lloyd, 1996 The RecG Sassanfar,M.,andJ.W.Roberts, 1990 Nature of the SOS-inducing branch migration protein of Escherichia coli dissociates R-loops. signal in Escherichia coli. The involvement of DNA replication. J. Mol. Biol. 264: 713–721. J. Mol. Biol. 212: 79–96. Viswanathan, M., and S. T. Lovett, 1998 Single-strand DNA-specific Seigneur, M., V. Bidnenko,S.D.Ehrlich and B. Michel, exonucleases in Escherichia coli: roles in repair and mutation 1998 RuvAB acts at arrested replication forks. Cell 95: 419–430. avoidance. Genetics 149: 7–16. Shereda, R. D., A. G. Kozlov,T.M.Lohman,M.M.Cox and J. L. Viswanathan, M., and S. T. Lovett, 1999 Exonuclease X of Escher- Keck, 2008 SSB as an organizer/mobilizer of genome mainte- ichia coli. A novel 39-59 DNase and DnaQ superfamily member in- nance complexes. Crit. Rev. Biochem. Mol. Biol. 43: 289–318. volved in DNA repair. J. Biol. Chem. 274: 30094–30100. Singleton, M. R., S. Scaife and D. B. Wigley, 2001 Structural anal- Viswanathan, M., V. Burdett,C.Baitinger,P.Modrich and S. T. ysis of DNA replication fork reversal by RecG. Cell 107: 79–89. Lovett, 2001 Redundant exonuclease involvement in Escheri- Singleton, M. R., M. S. Dillingham,M.Gaudier,S.C.Kowalczy- chia coli methyl-directed mismatch repair. J. Biol. Chem. 276: kowski and D. B. Wigley, 2004 Crystal structure of RecBCD 31053–31058. enzyme reveals a machine for processing DNA breaks. Nature Yarranton, G. T., and M. L. Gefter, 1979 Enzyme-catalyzed DNA 432: 187–193. unwinding: studies on Escherichia coli rep protein. Proc. Natl. Smith, G. R., 1990 RecBCD enzyme, pp. 78–98 in Nucleic Acids and Acad. Sci. USA 76: 1658–1662. Molecular Biology, edited by F. Eckstein and D. M. J. Lilley. Zahradka,K.,M.Buljubasic,M.Petranovic and D. Zahradka, Springer-Verlag, Berlin. 2009 Roles of ExoI and SbcCD nucleases in ‘‘reckless’’ DNA degra- Sun, W., S. Nandi,F.Osman,J.S.Ahn,J.Jakovleska et al., dation in recA mutants of Escherichia coli. J. Bacteriol. 191: 1677–1687. 2008 The FANCM ortholog Fml1 promotes recombination at Zhang, J., A. A. Mahdi,G.S.Briggs and R. G. Lloyd, stalled replication forks and limits crossing over during DNA 2010 Promoting and avoiding recombination: contrasting activ- double-strand break repair. Mol. Cell 32: 118–128. ities of the Escherichia coli RuvABC Holliday junction resolvase Tanaka, T., and H. Masai, 2006 Stabilization of a stalled replication and RecG DNA translocase. Genetics 185: 23–37. fork by concerted actions of two helicases. J. Biol. Chem. 281: 3484–3493. Tanaka, T., C. Taniyama,K.Arai and H. Masai, 2003 ATPase/heli- case motif mutants of Escherichia coli PriA protein essential for Communicating editor: G. R. Smith GENETICS
Supporting Information http://www.genetics.org/cgi/content/full/genetics.110.120691/DC1
RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity in Escherichia coli
Christian J. Rudolph, Akeel A. Mahdi, Amy L. Upton and Robert G. Lloyd
Copyright Ó 2010 by the Genetics Society of America DOI: 10.1534/genetics.110.120691 2 SI C. J. Rudolph et al.
FIGURE S1.—Synthetic lethality assays showing how depletion of one or more of ExoI, ExoVII, SbcCD or RecJ affects the viability of cells lacking RecG. C. J. Rudolph et al. 3 SI
TABLE S1
Escherichia coli K-12 strains
Strain Relevant Genotypea Sourceb
(a) General P1 donors
BW2513 xseA758::kan (BABA et al. 2006)
DL729 sbcCD::kan David Leach
E15 dam::cat mutS::spc/str M. Radman
JC12334 tnaA::Tn10 recF143 A. J. Clark
JIG486 xonA::apra (GROVE et al. 2008)
JJC735 rep::cat (BIDNENKO et al. 1999)
JJC1382 sulA::MudAplacMuB::Tn9 (SANDLER 1996)
N3072 recA269::Tn10 (LLOYD et al. 1987)
N3793 recG263::kan (AL-DEIB et al. 1996)
N4452 recG265::cat (JAKTAJI and LLOYD 2003)
N4700 rnhA::cat R. Crouch (BACHMANN 1996)
STL4534 exoX1::npt Susan Lovett
SWM1001 helD::cat (MENDONCA et al. 1995)
TRM308 recB268::Tn10 (MAHDI et al. 2006)
(c) MG1655 and derivatives
MG1655 F– rph-1
AM1655 recG::apra This work
AM1750 pAM409 (lac+ recG+ ruvABC+) / lacIZYA TB28 pAM409 to Apr
AM1581 lacIZYA recB268::Tn10 (MAHDI et al. 2006)
AM1874 xseA::dhfr (GROVE et al. 2008)
AM1955 ruvABC::apra This work
AM1986 recA::spc This work
AM1994 sbcCD::spc This work
AM1999 pAM490 (lac+ rnhA+) / lacIZYA rnhA::cat N7287 pAM490 to Apr
AM2014 mutS::kan This work
AM2069 lacIZYA recG::apra zjf-920Tn10 priB202 rpoB[S1332L] RGL & A. A. Mahdi, unpublished work
AM2037 recA::cat This work
AM2073 lacIZYA recG::apra zjf-920Tn10 priB202 rpoB[G1260D] RGL & A. A. Mahdi, unpublished work
AM2155 lacIZYA argE86::Tn10 TB28 x P1.N4837 to Tcr
AM2156 lacIZYA argE86::Tn10 rpoB[A1714T] Selection for resistance to 50 μg/ml
4 SI C. J. Rudolph et al.
rifampicin. The rpoB mutation encodes and I572F substitution in RpoB.
AM2265 uvrD::cat This work
AU1006 pJJ100 (lac+ recG+) / lacIZYA recG::apra rnhA::cat JJ1119 P1.N4704 to Cmr
AU1178 pAM390 (lac+ ruvABC+) / lacIZYA rnhA::cat N6254 P1.N4704 to Cmr
AU1179 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::apra N6254 P1.AM1955 to Aprar
AU1181 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::apra rnhA::cat AU1179 P1.N4704 to Cmr
AU1190 lacIZYA rnhA::cat Plasmid free derivative of AU1178
AU1191 lacIZYA ruvABC::apra Plasmid free derivative of AU1179
AU1192 lacIZYA ruvABC::apra rnhA::cat Plasmid free derivative of AU1181
HB169 lacIZYA<>frt dinG::kan Peter McGlynn. dinG::kan is from the
KEIO collection (BABA et al. 2006).
JJ1017 pJJ100 (lac+ recG+) / lacIZYA recG265::cat N5742 pJJ100 to Apr
JJ1119 pJJ100 (lac+ recG+) / lacIZYA recG::apra JJ1017 P1.AM1655 to Aprar (Cms)
N4256 recG263::kan (JAKTAJI and LLOYD 2003)
N4278 recB268::Tn10 (MEDDOWS et al. 2004)
N4560 recG265::cat (MAHDI et al. 2006)
N4704 rnhA::cat MG1655 P1.N4700 to Cmr
N4837 argE86::Tn10 (JAKTAJI and LLOYD 2003)
N4884 rpoB*35 ruvABC::cat (MAHDI et al. 2006)
N4934 recJ284::Tn10 (MAHDI et al. 2006)
N4971 recG263::kan ruvABC::cat (JAKTAJI and LLOYD 2003)
N5123 malE::Tn10 lexA3 (TRAUTINGER et al. 2005)
N5281 sbcCD::kan MG1655 x P1.DL729 to Kmr
N5288 exoX1::npt MG1655 x P1.STL4534 to Kmr
N5305 phoR79::Tn10 sbcC201 proC29 (GROVE et al. 2008)
N5500 priA300 (JAKTAJI and LLOYD 2003)
N5602 recQ::kan (MAHDI et al. 2006)
N5742 lacIZYA recG265::cat TB28 P1.N4452 to Cmr
N5917 priA300 lacIZYA<>aph (Kmr) N5500 P1.TB12 to Kmr
N5924 priA300 lacIZYA<>aph (Kmr) pCP20 N5917 pCP20 to Apr at 30°C
N5926 priA300 lacIZYA<>frt Plasmid free, frt (Kms) N5924 selected at
42°C (DATSENKO and WANNER 2000)
N6254 pAM390 (lac+ ruvABC+) / lacIZYA TB28 pAM390 to Apr
N6268 lacIZYA ruvABC::cat (MAHDI et al. 2006)
N6269 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat N6268 pAM390 to Apr
N6283 pJJ100 (lac+ recG+) / lacIZYA TB28 pJJ100 to Apr
C. J. Rudolph et al. 5 SI
N6310 lacIZYA ruvABC::cat rus-2 (orf-56::IS10) (MAHDI et al. 2006)
N6329 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat rus-2 (orf-56::IS10) N6310 x pAM390 to Apr
N6627 pAM409 (lac+ recG+ ruvABC+) / lacIZYA recG::apra AM1750 P1.AM1655 to Aprar
N6628 pAM409 (lac+ recG+ ruvABC+) / lacIZYA recG::apra ruvABC::cat N6627 P1.N4884 to Cmr
N6666 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat AM1750 P1.N4884 to Cmr
N6945 sbcCD::kan xonA::apra N5281 x P1.JIG486 to Aprar
N6946 xonA::apra MG1655 P1.JIG486 to Aprar
N6949 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat xonA::apra N6269 P1.JIG486 to Aprar
N6951 xseA::dhfr MG1655 P1.AM1874 to Tmr
N6952 sbcCD::kan xseA::dhfr N5281 x P1.AM1874 to Tmr
N6953 sbcCD::kan xonA::apra xseA::dhfr N6945 x P1.AM1874 to Tmr
N6954 xonA::apra xseA::dhfr N6946 P1.AM1874 to Tmr
N6954 xonA::apra N6946 x P1.AM1874 to Tmr
N6955 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat xseA::dhfr N6269 P1.AM1874 to Tmr
N6975 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat xonA::apra N6949 P1.AM1874 to Tmr xseA::dhfr
N6978 lacIZYA sbcCD::kan RGL unpublished work
N7000 phoR79::Tn10 sbcC201 N5281 x P1.N5305 to Tcr (Kms)
N7001 xonA::apra phoR79::Tn10 sbcC201 N6945 x P1.N5305 to Tcr (Kms)
N7002 xseA::dhfr phoR79::Tn10 sbcC201 N6952 x P1.N5305 to Tcr (Kms)
N7003 xonA::apra xseA::dhfr phoR79::Tn10 sbcC201 N6953 x P1.N5305 to Tcr
N7004 phoR79::Tn10 sbcC201 exoX1::npt N7000 x P1.N5288 to Kmr
N7005 xonA::apra phoR79::Tn10 sbcC201 exoX1::npt N7001 x P1.N5288 to Kmr
N7006 xseA::dhfr phoR79::Tn10 sbcC201 exoX1::npt N7002 x P1.N5288 to Kmr
N7007 xonA::apra xseA::dhfr phoR79::Tn10 sbcC201exoX1::npt N7003 x P1.N5288 to Kmr
N7008 lacIZYA ruvABC::cat xonA::apra xseA::dhfr Plasmid free derivative of N6975
N7009 pAM401 (lac+ sbcCD+) / lacIZYA ruvABC::cat xonA::apra N7008 x pAM401 to Apr xseA::dhfr
N7010 lacIZYA ruvABC::cat xonA::apra Plasmid free derivative of N6949
N7013 lacIZYA ruvABC::cat xseA::dhfr Plasmid free derivative of N6955
N7031 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat rus-2 (orf-56::IS10) N6329 x P1.JIG486 to Aprar xonA::apra
N7036 xonA::apra xseA::dhfr recJ284::Tn10 N6954 P1.N4934 to Tcr
N7037 lacIZYA xonA::apra xseA::dhfr sbcCD::kan N6954 P1.N6978 to Kmr
N7042 pAM401 (lac+ sbcCD+) / lacIZYA xonA::apra xseA::dhfr N7037 pAM401 to Apr sbcCD::kan
N7043 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat rus-2 (orf-56::IS10) N7031 x P1.AM1874 to Tmr
6 SI C. J. Rudolph et al.
xonA::apra xseA::dhfr
N7045 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat rus-2 (orf-56::IS10) N7043 x P1.N6978 to Kmr xonA::apra xseA::dhfr sbcCD::kan
N7050 lacIZYA xonA::apra xseA::dhfr sbcCD::kan rpoC[R919H] Selection on N7037 for resistance to 2- AP
N7055 pAM401 (lac+ sbcCD+) / lacIZYA xonA::apra xseA::dhfr N7042 x P1. N4934 to Tcr sbcCD::kan recJ284::Tn10
N7056 sbcCD::kan recJ284::Tn10 N5281 x P1.N4934 to Tcr
N7057 sbcCD::kan xonA::apra xseA::dhfr mutS::spc N6953 x P1.E15 to Spcr
N7060 pAM401 (lac+ sbcCD+) / lacIZYA xonA::apra xseA::dhfr N7042 x P1.N4884 Cmr sbcCD::kan ruvABC::cat
N7063 sbcCD::kan xonA::apra recJ284::Tn10 N6945 x P1.N4934 to Tcr
N7064 sbcCD::kan xseA::dhfr recJ284::Tn10 N6952 x P1.N4934 to Tcr
N7065 xonA::apra recJ284::Tn10 N6946 x P1.N4934 to Tcr
N7066 xseA::dhfr recJ284::Tn10 N6951 P1. N4934 to Tcr
N7071 lacIZYA ruvABC::cat rus-2 (orf-56::IS10) xonA::apra xseA::dhfr Plasmid free derivative of N7045 sbcCD::kan
N7074 lacIZYA xonA::apra xseA::dhfr sbcCD::kan recJ284::Tn10 Plasmid free derivative of N7055 (56/2)
N7266 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xonA::apra JJ1017 P1.JIG486 to Aprar
N7267 pJJ100 (lac+ recG+) / lacIZYA recG265::cat recJ284::Tn10 JJ1017 P1.N4934 to Tcr
N7268 pJJ100 (lac+ recG+) / lacIZYA recG265::cat sbcCD::kan JJ1017 P1.N6978 to Kmr
N7274 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xseA::dhfr JJ1017 P1.AM1874 to Tmr
N7280 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xonA::apra N7266 P1.N4934 to Tcr recJ284::Tn10
N7281 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xonA::apra sbcCD::kan N7266 P1.N6978 to Kmr
N7282 pJJ100 (lac+ recG+) / lacIZYA recG265::cat sbcCD::kan N7268 P1.N4934 to Tcr recJ284::Tn10
N7284 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xseA::dhfr xonA::apra N7274 P1.JIG486 to Aprar
N7285 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xseA::dhfr N7274 P1.N4934 to Tcr recJ284::Tn10
N7286 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xseA::dhfr sbcCD::kan N7274 P1.N6978 to Kmr
N7289 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xseA::dhfr xonA::apra N7284 P1.N4934 to Tcr recJ284::Tn10
N7290 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xseA::dhfr xonA::apra N7284 P1.N6978 to Kmr sbcCD::kan
N7291 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xseA::dhfr sbcCD::kan N7286 P1.N4934 to Tcr recJ284::Tn10
N7292 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xonA::apra sbcCD::kan N7281 P1.N4934 to Tcr recJ284::Tn10
C. J. Rudolph et al. 7 SI
N7293 lacIZYA recG265::cat xonA::apra Plasmid free derivative of N7266
N7294 lacIZYA recG265::cat recJ284::Tn10 Plasmid free derivative of N7267
N7295 lacIZYA recG265::cat sbcCD::kan Plasmid free derivative of N7268
N7297 lacIZYA recG265::cat xseA::dhfr Plasmid free derivative of N7274
N7298 lacIZYA recG265::cat xonA::apra recJ284::Tn10 Plasmid free derivative of N7280
N7299 lacIZYA recG265::cat xonA::apra sbcCD::kan Plasmid free derivative of N7281
N7300 lacIZYA recG265::cat sbcCD::kan recJ284::Tn10 Plasmid free derivative of N7282
N7301 lacIZYA recG265::cat xseA::dhfr xonA::apra Plasmid free derivative of N7284
N7302 lacIZYA recG265::cat xseA::dhfr recJ284::Tn10 Plasmid free derivative of N7285
N7303 lacIZYA recG265::cat xseA::dhfr sbcCD::kan Plasmid free derivative of N7286
N7308 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat xonA::apra N6975 x P1.N4934 to Tcr xseA::dhfr recJ284::Tn10
N7311 lacIZYA recG265::cat xseA::dhfr sbcCD::kan recJ284::Tn10 Plasmid free derivative of N7291
N7312 lacIZYA recG265::cat xonA::apra sbcCD::kan recJ284::Tn10 Plasmid free derivative of N7292 (56/2)
N7314 pAM401 (lac+ sbcCD+) / lacIZYA recG265::cat xonA::apra N7312 x pAM401 to Apr sbcCD::kan recJ284::Tn10
N7316 pAM401 (lac+ sbcCD+) / lacIZYA recG265::cat xonA::apra N7314 x P1.AM1874 to Tmr sbcCD::kan recJ284::Tn10 xseA::dhfr
N7317 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xseA::dhfr xonA::apra Plasmid free derivative of N7289 (56/2) recJ284::Tn10
N7318 pAM401 (lac+ sbcCD+) / lacIZYA xonA::apra xseA::dhfr N7042 x P1.N3072 to Tcr sbcCD::kan recA269::Tn10
N7325 lacIZYA ruvABC::cat xonA::apra xseA::dhfr Plasmid free derivative of N6975
N7334 lacIZYA xonA::apra TB28 P1.JIG486 to Aprar
N7336 lacIZYA xonA::apra xseA::dhfr N7334 P1.AM1874 to Tmr
N7337 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xonA::apra N7325 pAM409 to Apr xseA::dhfr
N7338 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xonA::apra N7337 N3793 to Kmr (Apr) xseA::dhfr recG263::kan
N7356 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xseA::dhfr xonA::apra N7290 P1.E15 to Spcr sbcCD::kan mutS::spc/str
N7357 pAM401 (lac+ sbcCD+) / lacIZYA xonA::apra xseA::dhfr N7060 x P1.E15 to Spcr sbcCD::kan ruvABC::cat mutS::spc
N7382 lacIZYA sbcCD::spc TB28 P1.AM1994 to Spcr
N7395 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xonA::apra N7338 x P1.AM1986 to Spcr xseA::dhfr recG263::kan
N7417 lacIZYA sbcCD::spc xonA::apra N7382 P1.JIG486 to Aprar
N7418 lacIZYA sbcCD::spc xseA::dhfr N7382 P1.AM1874 to Tmr
8 SI C. J. Rudolph et al.
N7424 lacIZYA xonA::apra xseA::dhfr recJ284::Tn10 N7336 P1.N4934 to Tcr
N7427 lacIZYA sbcCD::spc xseA::dhfr xonA::apra N7418 P1.JIG486 to Aprar
N7429 pAM490 (lac+ rnhA+) / lacIZYA xonA::apra xseA::dhfr N7336 pAM490 to Apr
N7438 lacIZYA sbcCD::spc xonA::apra recJ284::Tn10 N7417 P1.N4934 to Tcr
N7439 lacIZYA sbcCD::spc xseA::dhfr recJ284::Tn10 N7418 P1.N4934 to Tcr
N7441 pAM490 (lac+ rnhA+) / lacIZYA xonA::apra xseA::dhfr rnhA::cat N7429 P1.N4704 to Cmr (Apr)
N7447 pAM490 (lac+ rnhA+) / lacIZYA xonA::apra xseA::dhfr N7424 pAM490 to Apr recJ284::Tn10
N7452 pAM490 (lac+ rnhA+) / lacIZYA xonA::apra xseA::dhfr rnhA::cat N7441 x P1.N6978 to Kmr sbcCD::kan
N7457 pAM490 (lac+ rnhA+) / lacIZYA sbcCD::spc xonA::apra N7438 pAM490 to Apr recJ284::Tn10
N7458 pAM490 (lac+ rnhA+) / lacIZYA sbcCD::spc xseA::dhfr N7439 pAM490 to Apr recJ284::Tn10
N7481 pAM490 (lac+ rnhA+) / lacIZYA xonA::apra xseA::dhfr N7447 P1.N4704 to Cmr (Apr) recJ284::Tn10 rnhA::cat
N7482 pAM490 (lac+ rnhA+) / lacIZYA sbcCD::spc xonA::apra N7457 P1.N4704 to Cmr (Apr) recJ284::Tn10 rnhA::cat
N7483 pAM490 (lac+ rnhA+) / lacIZYA sbcCD::spc xseA::dhfr N7458 P1.N4704 to Tcr (Apr) recJ284::Tn10 rnhA::cat
N7504 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xonA::apra N7010 pAM409 to Apr
N7505 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xseA::dhfr N7013 pAM409 to Apr
N7510 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat N6628 P1.N3793 to Kmr (Apras) recG263::kan
N7517 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xonA::apra N7504 P1.N3793 to Kmr recG263::kan
N7518 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xseA::dhfr N7505 P1.N3793 to Kmr recG263::kan
N7545 lacIZYA sbcCD::spc xseA::dhfr xonA::apra mutS::kan N7427 x P1.AM2014 to Kmr
N7561 pJJ100 (lac+ recG+) / lacIZYA sbcCD::spc xseA::dhfr xonA::apra N7427 pJJ100 to Apr
N7570 pJJ100 (lac+ recG+) / lacIZYA sbcCD::spc xseA::dhfr xonA::apra N7561 x P1.N3793 to Kmr recG263::kan
N7573 lacIZYA priA300 xonA::apra N5926 P1.JIG486 to Aprar
N7574 lacIZYA priA300 xonA::apra xseA::dhfr N7573 P1.AM1874 to Tmr
N7576 pAM490 (lac+ rnhA+) / lacIZYA priA300 xonA::apra xseA::dhfr N7574 pAM490 to Apr
N7584 pAM490 (lac+ rnhA+) / lacIZYA priA300 xonA::apra xseA::dhfr N7576 P1.N4704 to Cmr rnhA::cat
N7591 pAM490 (lac+ rnhA+) / lacIZYA priA300 xonA::apra xseA::dhfr N7584 P1.N6978 to Kmr rnhA::cat sbcCD::kan
C. J. Rudolph et al. 9 SI
N7611 pAM401 (lac+ sbcCD+) / lacIZYA priA300 xonA::apra xseA::dhfr N7574 x pAM401 to Apr
N7615 pAM401 (lac+ sbcCD+) / lacIZYA priA300 xonA::apra xseA::dhfr N7611 x P1.N6978 to Kmr sbcCD::kan
N7623 pAM401 (lac+ sbcCD+) / lacIZYA priA300 xonA::apra xseA::dhfr N7615 x P1.N3072 to Tcr sbcCD::kan recA269::Tn10
N7625 pAM401 (lac+ sbcCD+) / lacIZYA priA300 xonA::apra xseA::dhfr N7615 x P1.N4560 to Cmr sbcCD::kan recG265::cat
N7626 pAM401 (lac+ sbcCD+) / lacIZYA priA300 xonA::apra xseA::dhfr N7615 x P1.N4884 to Cmr sbcCD::kan ruvABC::cat
N7627 lacIZYA priA300 xonA::apra xseA::dhfr plasmid free derivative of N7611
N7628 lacIZYA priA300 xonA::apra xseA::dhfr sbcCD::kan N7627 x P1.N6978 to Kmr
N7671 pAM401 (lac+ sbcCD+) / lacIZYA priA300 xonA::apra xseA::dhfr N7625 x P1.N3072 to Tcr sbcCD::kan recG265::cat recA269::Tn10
N7676 pAM401 (lac+ sbcCD+) / lacIZYA xonA::apra xseA::dhfr N7042 x P1.JC12334 to Tcr sbcCD::kan tna::Tn10 recF143
N7679 lacIZYA xonA::apra xseA::dhfr sbcCD::kan recA269::Tn10 plasmid free derivative of N7318 (56/2)
N7683 lacIZYA xonA::apra xseA::dhfr sbcCD::spc rpoC[R919H] N7050 x P1.N7382 to Spcr (and Kms)
N7684 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7427 x pAM401 to Apr xonA::apra
N7687 lacIZYA priA300 xonA::apra xseA::dhfr sbcCD::spc N7628 x P1.N7382 to Spcr
N7691 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7684 x P1.N5602 to Kmr xonA::apra recQ::kan
N7692 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7684 x P1.N4934 to Tcr xonA::apra recJ284::Tn10
N7694 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xonA::apra N7517 x P1.N7382 to Spcr recG263::kan sbcCD::spc
N7695 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xseA::dhfr N7518 x P1.N7382 to Spcr recG263::kan sbcCD::spc
N7696 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7545 x pAM401 to Apr xonA::apra mutS::kan
N7699 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7692 x P1.AM2014 to Kmr xonA::apra recJ284::Tn10 mutS::kan
N7704 lacIZYA xonA::apra xseA::dhfr sbcCD::spc yijC::kan N7683 x P1.(Kmr pool) to Kmr Rpo+
N7707 lacIZYA sbcCD::spc xseA::dhfr xonA::apra recJ284::Tn10 Plasmid free derivative of N7699 mutS::kan identified and grown on 56/2 salts agard
N7711 lacIZYA xonA::apra xseA::dhfr sbcCD::spc yijC::kan rpoC[R919H] N7683 x P1.N7704 to Kmr
N7713 lacIZYA sbcCD::spc xseA::dhfr xonA::apra yijC::kan rpoC[R919H] N7427 x P1.N7711 to Kmr (and MCr)
N7714 lacIZYA xonA::apra xseA::dhfr sbcCD::spc argE86::Tn10 N7711 x P1.N4837 to Tcr
N7715 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7713 x pAM401 to Apr
10 SI C. J. Rudolph et al.
xonA::apra yijC::kan rpoC[R919H]
N7716 lacIZYA xonA::apra xseA::dhfr sbcCD::spc rpoB[S1332L] N7714 x P1.AM2069 to Arg+
N7717 lacIZYA xonA::apra xseA::dhfr sbcCD::spc rpoB[G1260D] N7714 x P1.AM2073 to Arg+
N7719 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7715 x P1.N3072 to Tcr xonA::apra yijC::kan rpoC[R919H] recA::Tn10
N7720 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7715 x P1.N4934 to Tcr xonA::apra yijC::kan rpoC[R919H] recJ284::Tn10
N7721 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7715 x P1.N4560 to Cmr xonA::apra yijC::kan rpoC[R919H] recG265::cat
N7722 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7715 x P1.N4884 to Cmr xonA::apra yijC::kan rpoC[R919H] ruvABC::cat
N7723 lacIZYA xonA::apra xseA::dhfr sbcCD::spc rpoB*35 N7714 x P1.N4884 to Arg+
N7745 lacIZYA rpoC[R919H] AM2156 x P1.N7050 to Arg+ Rifs (rpoB+)
N7765 pAM401 (lac+ sbcCD+) / lacIZYA ruvABC::cat rus-2 (orf-56::IS10) N7071 x pAM401 to Apr xonA::apra xseA::dhfr sbcCD::kan
N7783c lacIZYA sbcCD::spc xseA::dhfr xonA::apra recB268::Tn10 N7684 x P1.TRM308 to Tcr
N7787 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7684 x P1.SWM101 to Cmr xonA::apra helD::cat
N7789 lacIZYA sbcCD::spc xseA::dhfr xonA::apra recB268::Tn10 N7427 x P1.TRM308 to Tcr
N7794 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7684 x P1.N5123 to Tcr xonA::apra malE::Tn10 lexA3
N7800 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7794 x P1.N3793 to Kmr xonA::apra malE::Tn10 lexA3 recG263::kan
N7806 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA758::kan N7684 x P1.BW25113 to Kmr (Tms) xonA::apra
N7814 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7684 x P1.HB169 to Kmr xonA::apra dinG::kan
N7816 lacIZYA xseA::dhfr TB28 x P1.AM1874 to Tmr
N7817 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7691 x P1.JJC735 to Cmr (Kms, recQ+) xonA::apra rep::cat
N7823 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7806 x P1.N4560 to Cmr xonA::apra recG265::cat
N7835 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7684 x P1.AM2037 to Cmr xonA::apra recA::cat
N7837 lacIZYA sbcCD::spc xseA::dhfr xonA::apra N7427 x P1.JJC1382 to Cmr (Apr) sulA::MudAplacMuB::Tn9
N7841 lacIZYA sbcCD::spc xseA::dhfr xonA::apra argE86::Tn10 N7427 x P1.N4837 to Tcr
N7842 pAM401 (lac+ sbcCD+) / lacIZYA priA300 xonA::apra xseA::dhfr N7615 x P1.AM2265 to Cmr
C. J. Rudolph et al. 11 SI
sbcCD::kan uvrD::cat
N7843 lacIZYA sulA::MudAplacMuB::Tn9 TB28 x P1.JJC1382 to Cmr (Apr)
N7844 lacIZYA xonA::apra sulA::MudAplacMuB::Tn9 N7334 x P1.JJC1382 to Cmr (Apr)
N7845 lacIZYA sbcCD::spc sulA::MudAplacMuB::Tn9 N7382 x P1.JJC1382 to Cmr (Apr)
N7846 lacIZYA sbcCD::spc xonA::apra sulA::MudAplacMuB::Tn9 N7417 x P1.JJC1382 to Cmr (Apr)
N7847 lacIZYA sbcCD::spc xseA::dhfr sulA::MudAplacMuB::Tn9 N7418 x P1.JJC1382 to Cmr (Apr)
N7854 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7684 x P1.AM2265 to Cmr xonA::apra uvrD::cat
N7855 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7696 x P1.AM2265 to Cmr xonA::apra mutS::kan uvrD::cat
N7857 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7696 x P1.JJC735 to Cmr xonA::apra mutS::kan rep::cat
N7859 lacIZYA xseA::dhfr sulA::MudAplacMuB::Tn9 N7816 x P1.JJC1382 to Cmr (Apr)
N7860 lacIZYA xonA::apra xseA::dhfr sulA::MudAplacMuB::Tn9 N7336 x P1.JJC1382 to Cmr (Apr)
N7861 lacIZYA sbcCD::spc xseA::dhfr xonA::apra rpoC[R919H] N7841 x P1.N7050 to Arg+ (MCr, 2- APr)
N7867 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7715 x P1.JJC735 to Cmr xonA::apra yijC::kan rpoC[R919H] rep::cat
N7868 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA::dhfr N7715 x P1.AM2265 to Cmr xonA::apra yijC::kan rpoC[R919H] uvrD::cat
N7888 lacIZYA rpoC[R919H] sulA::MudAplacMuB::Tn9 N7745 x P1.JJC1382 to Cmr (Apr)
N7889 lacIZYA sbcCD::spc xseA::dhfr xonA::apra rpoC[R919H] N7861 x P1.JJC1382 to Cmr (Apr) sulA::MudAplacMuB::Tn9
TB12 lacIZYA<>aph (Kmr) (BERNHARDT and DE BOER 2003)
TB28 lacIZYA<>frtd (BERNHARDT and DE BOER 2003) aOnly the relevant additional genotype of the MG1655 derivatives is shown b During transduction of recipients carrying derivatives of the Apr construct, pRC7, selection was imposed for both the donor marker and for resistance to ampicillin. Plasmid-free derivatives were identified as white colonies on LB supplemented with X- Gal and IPTG or, when the plasmid free cells do not grow well on LB agar, on similarly supplemented 56/2 glucose minimal salts agar, as indicated. c During construction of this strain it transpired that the Tcr transductants had great difficulty retaining pAM401. Hence, although the transductants could be purified on LB agar supplemented with tetracycline, they could not be purified on plates supplemented with both tetracycline and ampicillin, despite having been selected on such plates initially (we assume the - lactamase encoded by the plasmid-carrying cells reduces the local concentration of ampicillin, allowing outgrowth of the selected Tcr, plasmid-free cells). N7783 is one such plasmid-free transductant. To confirm that a sbcCD::spc xseA::dhfr xonA::apra recB268::Tn10 strain is viable, recB268::Tn10 was introduced by PI transduction into a plasmid-free strain N7427. Transduction was efficient and the Tcr colonies purified without any sign of having acquired a suppressor. The resulting construct (N7789) proved to have a phenotype identical to that of N7783. dAbbreviated to lacIZYA in derivatives.
12 SI C. J. Rudolph et al.
SUPPORTING REFERENCES
AL-DEIB, A. A., A. A. MAHDI and R. G. LLOYD, 1996 Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12. J. Bacteriol. 178: 6782-6789. BABA, T., T. ARA, M. HASEGAWA, Y. TAKAI, Y. OKUMURA et al., 2006 Construction of Escherichia coli K-12 in-frame, single- gene knockout mutants: the Keio collection. Mol Syst Biol 2: 1-11. BACHMANN, B. J., 1996 Derivations and genotypes of some mutant derivatives of Escherichia coli K-12, pp. 2460-2488 in Escherichia coli and Salmonella Cellular and Molecular Biology, (Second Edition), edited by F. C. NEIDHARDT, R. CURTISS III, J. L. INGRAHAM, E. C. C. LIN, K. B. LOW et al. ASM Press, Washington, D.C. BERNHARDT, T. G., and P. A. DE BOER, 2003 The Escherichia coli amidase AmiC is a periplasmic septal ring component exported via the twin-arginine transport pathway. Mol Microbiol 48: 1171-1182. BIDNENKO, V., M. SEIGNEUR, M. PENEL-COLIN, M. F. BOUTON, S. DUSKO EHRLICH et al., 1999 sbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants. Mol Microbiol 33: 846-857. DATSENKO, K. A., and B. L. WANNER, 2000 One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97: 6640-6645. GROVE, J. I., L. HARRIS, C. BUCKMAN and R. G. LLOYD, 2008 DNA double strand break repair and crossing over mediated by RuvABC resolvase and RecG translocase. DNA Repair (Amst) 7: 1517-1530. JAKTAJI, R. P., and R. G. LLOYD, 2003 PriA supports two distinct pathways for replication restart in UV-irradiated Escherichia coli cells. Mol. Microbiol. 47: 1091-1100. LLOYD, R. G., C. BUCKMAN and F. E. BENSON, 1987 Genetic analysis of conjugational recombination in Escherichia coli K-12 strains deficient in RecBCD enzyme. J. Gen. Microbiol. 133: 2531-2538. MAHDI, A. A., C. BUCKMAN, L. HARRIS and R. G. LLOYD, 2006 Rep and PriA helicase activities prevent RecA from provoking unnecessary recombination during replication fork repair. Genes Dev. 20: 2135-2147. MEDDOWS, T. R., A. P. SAVORY and R. G. LLOYD, 2004 RecG helicase promotes DNA double-strand break repair. Mol Microbiol 52: 119-132. MENDONCA, V. M., H. KLEPIN and S. A. MATSON, 1995 DNA helicases in recombination and repair: construction of a uvrD helD recQ mutant deficient in recombination and repair. J Bacteriol 177: 1326-1335. SANDLER, S. J., 1996 Overlapping functions for recF and priA in cell viability and UV-inducible SOS expression are distinguished by dnaC809 in Escherichia coli K-12. Mol Microbiol 19: 871-880. TRAUTINGER, B. W., R. P. JAKTAJI, E. RUSAKOVA and R. G. LLOYD, 2005 RNA polymerase modulators and DNA repair activities resolve conflicts between DNA replication and transcription. Mol. Cell 19: 247-258.