RecG Protein and Single-Strand DNA Exonucleases Avoid Cell ...pAM401 (sbcCD1) and pAM490 (rnhA1) are...

Copyright � 2010 by the Genetics Society of AmericaDOI: 10.1534/genetics.110.120691

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell LethalityAssociated 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, 2010Accepted for publication July 16, 2010

ABSTRACT

Replication of the Escherichia coli chromosome usually initiates at a single origin (oriC) under control ofDnaA. Two forks are established and move away in opposite directions. Replication is completed whenthese meet in a broadly defined terminus area half way around the circular chromosome. RecG appears toconsolidate this arrangement by unwinding D-loops and R-loops that PriA might otherwise exploit toinitiate 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 potentialtargets 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 threenucleases are inviable without RecG. They also need RecA recombinase and a Holliday junction resolvaseto survive rapid growth, but SOS induction, although elevated, is not required. Additional requirementsfor Rep and UvrD are identified and linked with defects in DNA mismatch repair and with the ability tocope with conflicts between replication and transcription, respectively. Eliminating PriA helicase activityremoves the requirement for RecG. The data are consistent with RecG and ssDNA exonucleases acting tolimit PriA-mediated re-replication of the chromosome and the consequent generation of linear DNAbranches that provoke recombination and delay chromosome segregation.

REPLICATION of the Escherichia coli chromosomeinitiates at a single origin (oriC) under the control

of DnaA (Messer 2002). Two forks are established,which then move round the circular chromosome inopposite directions. Duplication of the chromosome isachieved when they meet in a broadly defined terminusarea flanked by polar sequences (ter) that when boundby the Tus terminator protein allow forks to enter butnot leave this area (Mulcair et al. 2006; Duggin et al.2008). Thus, the chromosome is divided into tworeplichores within each of which replication proceedsin a polar fashion from oriC toward ter. However, the tworeplisome complexes meeting within the terminus areamay not be those assembled at oriC, but new complexesassembled following the rescue of stalled or damagedforks (Gabbai and Marians 2010).

Studies by Kogoma and co-workers showed that thishighly evolved replichore arrangement is compromisedwhen DnaA-independent stable DNA replication (SDR)is initiated via PriA-mediated DnaB loading and repli-some assembly at sites other than oriC (Kogoma 1997).PriA facilitates DnaB loading at stalled forks, D-loops,

and R-loops, potentially enabling replication to initiatewherever such structures arise (Marians 2000; Sandler

and Marians 2000; Heller and Marians 2006b;Michel et al. 2007; Gabbai and Marians 2010). Kogomaand co-workers identified a constitutive form of SDR(cSDR), which they proposed to initiate at R-loops, anddistinguished it from an inducible form (iSDR), which istriggered in cells exposed to genotoxic agents andcharacterized by its dependence on RecBCD enzyme(Kogoma 1997). cSDR is elevated in the absence ofRecG or RNase HI (Asai and Kogoma 1994; Hong et al.1995). These two proteins provide different ways ofeliminating R-loops. RecG is a double-stranded DNA(dsDNA) translocase and dissociates the RNA from thestructure by catalyzing branch migration whereas RNaseHI digests the RNA from the RNA:DNA hybrid (Hong

et al. 1995; Vincent et al. 1996; Fukuoh et al. 1997;McGlynn et al. 1997; Singleton et al. 2001). Strainslacking both proteins are inviable, indicating thatexcessive levels of SDR may be harmful (Hong et al.1995). Following UV irradiation, which triggers iSDR,DrecG cells show a very extended and PriA helicase-dependent delay in chromosome segregation and celldivision (Rudolph et al. 2009a). The majority of theDNA synthesis detected during this period is DnaAindependent and associated with an increase in thenumber of replication forks traversing the chromosome.It can lead to replication of both origin and terminus

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.110.120691/DC1.

1Corresponding author: Institute of Genetics, University of Nottingham,Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom.E-mail: [email protected]

Genetics 186: 473–492 (October 2010)

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areas of the chromosome and of all regions in between.However, it can also lead to disproportionate amplifica-tion of some chromosomal areas and to the accumula-tion of branched DNA resistant to cleavage by a Hollidayjunction resolvase (Rudolph et al. 2009a,b).

Although SDR disturbs the replichore arrangement,it is not obvious why this should have such dramaticeffects in the absence of RecG. We have suggested thatby increasing the number of replication fork collisions,SDR may trigger repeated cascades of chromosome re-replication and recombination and that by limiting SDRand dissociating recombination intermediates RecGreduces the likelihood of such pathology (Rudolph

et al. 2009a).Exactly what happens when forks meet in the termi-

nus area is not known, although it is generally assumedthat the replisome components dissociate as any remain-ing gaps are filled in and the nascent strands are finallysealed by DNA ligase (Figure 1A, i–iii). However, thepriming of SDR on either strand at sites other than oriCmeans some forks will now meet outside of the normaltermination zone (Figure 1B, i) (Kogoma 1997). Studiesof DNA replication in vitro raised the possibility thatwithout Tus to arrest forks at ter, the replisome of onefork might sometimes displace the 39 end of the nascentleading strand of the fork coming in the other direction(Hiasa and Marians 1994). If such displacementwere to occur in vivo, it would generate a 39 flap (Figure1B, ii). Unless removed, the exposed single-strandedDNA (ssDNA) might provide a template on whichthe RecFOR proteins could establish a RecA filament,thus provoking recombination (Umezu et al. 1993;Morimatsu and Kowalczykowski 2003). Alternatively,the branch point might provide a substrate that PriAcould exploit with the aid of its helicase activity to loadDnaB and initiate re-replication of the DNA, withleading strand synthesis primed perhaps via DnaB–DnaG interactions (Figure 1C, i–iii) (Heller andMarians 2006b). Depending on which fork had itsleading strand displaced, the new fork would moveeither toward oriC or toward ter, generating DNAbranches with duplex ends that provoke RecBCD-mediated recombination, thus establishing yet morenew forks (Figure 1C, iv and v). Pathological cascades ofthis nature may explain the over-replication of DNAobserved in vivo in the absence of Tus/ter control(Krabbe et al. 1997; Markovitz 2005).

A 39 flap might be eliminated in wild-type cells by 39–59

ssDNA exonucleases or converted to a 59 flap by branchmigration and then eliminated by 59–39 ssDNA exonu-cleases (Figure 1, D and E). RecG is well suited to carryout the conversion as it has a particularly high affinity for39 flap structures and very efficiently unwinds the strandending 59 at the branch point (McGlynn and Lloyd

2001; Tanaka and Masai 2006). Without RecG, theinitial number of SDR events initiated would be in-creased, leading to even higher levels of unscheduled

fork collisions and therefore of 39 flaps. In the absence ofRecG, these flaps would also have a longer half-life,increasing the opportunity for their targeting by PriA orfor the loading of RecA. Furthermore, without RecG,any D-loops established by subsequent recombinationwould be stabilized, further increasing the likelihood ofperpetuating cycles of fork collisions and re-replication.

According to this scenario, 39–59 ssDNA exonucleasesmight be rather vital in the absence of RecG. In thisarticle we show that the presence of exonuclease I(ExoI), exonuclease VII (ExoVII), or the SbcCD nucle-ase, all of which can digest ssDNA from 39 ends, isneeded to keep DrecG cells alive. This requirement canbe overcome by eliminating the helicase activity of PriA,consistent with the idea that a major function of RecG isto curb pathological replication of the chromosome.The presence of at least one of these three enzymes isalso needed to help keep rep and uvrD cells alive, but fordifferent reasons consistent with ssDNA exonucleaseshaving multiple roles in DNA replication and repair.

MATERIALS AND METHODS

Strains: Bacterial strains are listed in supporting informa-tion, Table S1. All constructs used for synthetic lethality assaysare based on E. coli K-12 MG1655 DlacIZYA strains carryingderivatives of pRC7 (Bernhardt and De Boer 2004). Thequiescent rusA gene was activated to express the RusA Hollidayjunction resolvase using constructs carrying rus-2, an IS10insertion upstream of the coding sequence (Mahdi et al.1996). Chromosomal genes were inactivated using Tn10 or kaninsertions, conferring resistance to tetracycline (Tcr) andkanamycin (Kmr), respectively, or with deletions tagged withsequences encoding resistance to chloramphenicol (Cmr; cat),kanamycin (kan), trimethoprim (Tmr; dhfr), apramycin (Aprar;apra), or spectinomycin (Spcr; spc) (Mahdi et al. 2006; Zhang

et al. 2010). New deletion alleles of xseA (DxseATdhfr;DxseATcat), recA (DrecATspc; DrecATcat; DrecATkan), recQ(DrecQTapra), uvrD (DuvrDTcat), and sbcCD (DsbcCDTspc)were made using the one-step gene inactivation method ofDatsenko and Wanner (2000). The DuvrD, DrecA, and DxseAalleles remove all but 39, 45, and 48 bp, respectively, from the59 and 39 ends of the coding sequence. For the sbcCD deletion,50 bp at the beginning of sbcD and 100 bp at the end of sbcCwere retained.

Plasmids: pRC7 is a low-copy-number, mini-F derivative ofthe lac1 construct pFZY1 (Bernhardt and De Boer 2004).pJJ100 (recG1), pAM375 (recB1), pAM390 (ruvABC1), andpAM409 (recG1 ruvABC1) are derivatives of pRC7 encodingthe wild-type genes indicated. Their construction has beendescribed elsewhere (Mahdi et al. 2006; Zhang et al. 2010).pAM401 (sbcCD1) and pAM490 (rnhA1) are also derivatives ofpRC7. In each case, the indicated wild-type coding sequencewas PCR amplified from MG1655 using 59 and 39 primersincorporating ApaI sites, and the product was cloned into theApaI site within the lacIq gene of pRC7. The inserts aretranscribed in the same orientation as the disrupted lacIq.

Media and general methods: LB broth and 56/2 minimalsalts media and methods for monitoring cell growth and forstrain construction by P1vir-mediated transduction have beencited (Al-Deib et al. 1996; McGlynn and Lloyd 2000;Trautinger et al. 2005). The incidence of spontaneousresistance to rifampicin (rpoB mutants) was determined by

474 C. J. Rudolph et al.

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spreading 100-ml samples of broth cultures grown to�2 3 109

cells/ml on LB agar plates supplemented with rifampicin at afinal concentration of 50 mg/ml, which were then incubatedovernight. Wild-type strain MG1655 typically yields ,10 re-sistant colonies under these conditions whereas derivativeslacking a functional MutHLS system usually yield severalhundred (mutator phenotype).

Measuring sensitivity to DNA damage: Sensitivity to UVlight and ionizing radiation was measured using exponential-phase cells grown to an A650 of 0.4 (1–2 3 108 cells/ml forstrain MG1655). Samples of appropriate dilutions wereirradiated on the surface of LB agar plates and survivors werescored after 18–24 hr incubation. Survival data are means fromat least two, and usually three to six, independent experi-ments. Errors (SE) range between 5% and 15% of the mean.Sensitivity to mitomycin C was determined by growing culturesto an A650 of 0.4 and spotting 10 ml of serial 10-fold dilutionsfrom 10�1 to 10�5 on LB agar with or without mitomycin C at afinal concentration of 0.5 mg/ml and incubating at 37�. Plateswere photographed after 24 hr incubation, unless statedotherwise. Sensitivity to 2-aminopurine (2-AP; Sigma) wasdetermined by the same method, using LB agar containing2-AP at a final concentration of 300 mg/ml.

SOS induction: SOS induction was analyzed using sulA(sfiA)TlacZ fusion strains. Cultures were grown in broth to anA650 of 0.3 and split in two before adding mitomycin C to onehalf to a final concentration of 1 mg/ml. Incubation was thencontinued for 1 hr, and samples were assayed for b-galactosidaseactivity as described (Miller 1972).

Synthetic lethality assays: The rationale for synthetic lethal-ity assays has been described (Bernhardt and De Boer 2004;Mahdi et al. 2006). Essentially, a wild-type gene of interest iscloned in pRC7, a lac1 mini-F plasmid that is rapidly lost, andused to cover a null mutation in the chromosome in a Dlacbackground. A mutation in another gene of interest is thenintroduced into the chromosome. If the double mutant isviable, the plasmid-free cells segregated during culture willform Lac� colonies on agar plates. If synthetically lethal, theywill fail to grow and only Lac1 colonies formed by cellsretaining the plasmid will be observed. When viability isreduced but not eliminated, the colonies formed by cellsretaining the plasmid are notably larger than those formed byplasmid-free cells. To record the phenotype, cultures of strainscarrying the relevant pRC7 derivatives were grown overnight inLB broth containing ampicillin to maintain plasmid selection,diluted 80-fold in LB broth, and grown without ampicillin

Figure 1.—Models depicting possible outcomes of replication fork collision [adapted from Rudolph et al (2009b)]. (A) Forkmerging and nascent strand ligation. (B) Pathological replication resulting from unscheduled replication fork collisions or duringnormal termination: (B, i) Schematic of the E. coli chromosome showing normal replication from oriC and the presence of severaladditional replication forks initiated as a result of SDR induction. The opposed arrowheads indicate the positions of unscheduledfork 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 provokerecombination. (D) Termination achieved via a 59 ssDNA exonuclease after RecG converts a 39 flap to a 59 flap. (E) Terminationachieved via a 39 ssDNA exonuclease. (F) Pathological, PriA helicase-independent replication in the presence of RecG.

Exonuclease-Deficient DrecG Cells 475

selection to an A650 of 0.4 before spreading dilutions on LBagar or 56/2 glucose minimal salts agar supplemented with X-gal and IPTG. Plates were photographed and scored after 48 hr(LB agar) or 72 hr (56/2 agar) at 37�, unless stated otherwise.Plasmid-free cells forming small white colonies were re-streaked to see if they could be subcultured, and the streakedplates were photographed after incubation at 37� for 24–48 hr(LB agar) or 48–72 hr (56/2 glucose salts agar), as indicated. Incertain specified cases where plasmid-free segregants (Lac�

clones) form healthy colonies on 56/2 agar, but fail to appearon LB agar, sample colonies from the 56/2 agar plates weregrown in 56/2 glucose minimal salts medium to an A650 of 0.4and further tested to quantify the effect. Each culture wasdiluted in 10-fold steps from 10�1 to 10�5, and 10-ml aliquotswere spotted on both LB and 56/2 glucose minimal agar.Colony-forming ability was recorded by photographing theplates after incubation for 24 hr (LB agar) or 48 hr (56/2 agar),unless specified otherwise.

Identification of 2-AP-resistant suppressors: Samples fromseven independent cultures of strain N7037, which is deletedfor xonA, xseA, and sbcCD and therefore sensitive to 2-AP andmitomycin C, were spread on LB agar plates containing 2-aminopurine at a final concentration of 300 mg/ml. A fewcolonies of 2-AP-resistant derivatives were visible on each plateafter 24 hr at 37�. Seven resistant clones, one from each of theoriginal cultures, were purified for further analysis. Fourexhibited a strong mutator phenotype but remained assensitive to mitomycin C as the parent. The other three werenot mutators but exhibited increased resistance to mitomycinC. In one of these three, strain N7050, the mutation re-sponsible was identified as an allele of rpoC in the followingway: The DsbcCDTkan allele in N7050 was first replaced with adeletion tagged with resistance to spectinomycin (DsbcCDTspc),and the resulting construct (N7683) was transduced with P1phage grown on pools of cells carrying random kan insertionsin the chromosome generated in strain MG1655 using the EZ-Tn5 ,kan-2. Tnp Transposome system (Epicentre Technol-ogies). The Kmr transductants were screened for those thatwere also sensitive to mitomycin C and 2-AP on the basis thatsuch a clone would carry a kan insertion linked to the wild-typeallele of the suppressor locus. One candidate was identified(N7704) and shown by PCR sequencing to carry an insertion inyijC at minute 89.64 of the genetic map. P1 phage from thisclone was used to transduce N7683 to Kmr. Fifty-three percentof the transductants tested proved sensitive to mitomycin Cand to 2-AP; i.e., they had lost the suppressor. P1 grown on atransductant retaining the suppressor phenotype (N7711) wasused to transduce strain N7427, which is deleted for xonA, xseA,and sbcCD. In this case, 37% of the Kmr transductants selec-ted acquired resistance to mitomycin C and to 2-AP; i.e., theyhad inherited the suppressor. These proved as resistant asthe original isolate, N7050, from which we concluded that themutation linked to yijC was the sole factor responsible for thesuppression in that isolate. Further genetic analyses sug-gested a mutation in the vicinity of the rpoBC operon. PCRsequencing revealed a G-to-A transition at bp 2755 in rpoC,encoding an R919H substitution in RpoC, the b9-subunit ofRNA polymerase.

RESULTS

The scenario outlined in Figure 1 predicts that ssDNAexonucleases might be vital in the absence of RecG. Toinvestigate whether this is the case, we exploited asynthetic lethality assay based on a recG1 derivative of

pRC7, a lac1 mini-F plasmid that is rapidly lost. Theplasmid was used to cover DrecG in a strain also deletedfor the lac operon and carrying additional mutationsinactivating one or more of the enzymes with knownssDNA exonuclease activity. We tested ExoI (encoded byxonA), which attacks 39 ends (Lehman and Nussbaum

1964); RecJ, which attacks 59 ends (Lovett and Kolodner

1989); and ExoVII (encoded by xseA), which can targeteither end (Chase and Richardson 1974). We alsotested the SbcCD enzyme, which has multiple nucleaseactivities, including the ability to remove 39 overhangsfrom partial duplexes and to cut hairpin structures(Chalker et al. 1988; Connelly et al. 1998, 1999;Eykelenboom et al. 2008). Synthetic lethality betweenthe covered and the uncovered mutations is revealed ifthe construct fails to show growth of plasmid-free Lac�

clones (white colonies and white sectors within bluecolonies) on agar plates supplemented with X-gal andIPTG (Mahdi et al. 2006). A reduction in viability isindicated when the colonies formed by plasmid-freecells are smaller than the blue/sectored coloniesformed by those cells that retained the plasmid at thetime of plating. In such cases, viability can be evaluatedfurther by streaking samples of the colonies on therelevant agar media to see if they can be subcultured. Afailure to subculture indicates that the colonies formedwere the result of abortive growth following plasmid lossand dilution of the relevant plasmid-encoded geneproduct. The emergence of large colony variants in-dicates a viability defect that can be overcome by theacquisition of suppressors.

39 ssDNA exonuclease activity is vital for cells lackingRecG: The assays conducted revealed that DrecG cellslacking ExoI, ExoVII, and SbcCD are inviable on LBagar, showing no ability to form visible colonies without acovering recG1 plasmid (Figure 2A, i and ii; Figure S1,xiv). Some colonies of plasmid-free cells are detected onminimal salts agar, but these are tiny and tend toaccumulate suppressors, as evident from the appearanceof large colony variants (Figure 2A, iii and iv). Otherwise,DrecG cells lacking any one or two of ExoI, ExoVII, orSbcCD form colonies on both LB and minimal salts agarand can be subcultured on both types of media withoutacquisition of suppressors (Tables 1 and 2; Figure S1;data not shown). These observations reveal that DrecGcells need a 39 exonuclease to stay alive and that thisrequirement can be satisfied by any one of ExoI, ExoVII,or SbcCD. However, SbcCD alone is able to do soefficiently only if RecJ is present. Without RecJ, the cellsform small colonies on LB agar that take nearly 24 hr tobecome visible to the naked eye (Figure 2A, v; Figure 2C,strain N7317). ExoI and ExoVII show no such limitation(Figure S1, xv and xvi).

The only nuclease activity reported for ExoI is theability to digest unpaired ssDNA from a 39 end (Lehman

and Nussbaum 1964). Therefore, the fact that thisenzyme suffices to keep DrecG cells alive, and robustly





so (Table 1; Figure S1, x), is highly informative. It showsthat the viability of these cells can be maintainedprovided unpaired 39 ssDNA can be degraded, indicat-ing that the accumulation of such strands might havetoxic consequences. ExoVII and SbcCD appear to pro-vide the only other nucleases capable of eliminating thisthreat. None of the several other E. coli enzymesreported to attack ssDNA from 39 ends, such as exo-nuclease III, exonuclease IX, and exonuclease X(ExoX) (Viswanathan and Lovett 1999; Lombardo

et al. 2003; Centore et al. 2008), appears up to the task.

However, the ability to digest 39 ssDNA is vital only in theabsence of RecG. With RecG present, cells lacking ExoI,ExoVII, and SbcCD remain viable (Figure 2A, i), whichimplies that ssDNA species with exposed 39 terminieither do not accumulate or can be dealt with by othermeans. This observation also argues against the idea thatExoI is needed to keep some essential protein complexintact. For example, ExoI is known to bind the ssDNAbinding protein SSB, which itself interacts with anumber of other proteins associated with genome repli-cation and maintenance (Shereda et al. 2008).

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, andsimilar assays reported in subsequent figures, are described in detail in materials and methods. The relevant genotype of theconstruct used is shown above each photograph, with the strain number in parentheses. The fraction of white colonies is shownbelow with the number of white colonies/total colonies analyzed in parentheses. The spot assays in C are of cultures of the strainsindicated as serially diluted in 10-fold steps from 10�1 to 10�5 before spotting 10-ml samples on the media indicated, as described inmaterials and methods.



Assays with constructs based on an sbcCD1 derivative ofpRC7 revealed that ExoI� ExoVII� SbcCD� cells growvery slowly on LB agar if RecJ is eliminated. However,provided RecG is available, they form colonies onminimal salts agar that can be subcultured withoutdifficulty (Figure 2B, i–iv; Figure 2C, strain N7074). Fromthese data, and those in Table 1 and Figure S1, it seemsthat, whereas DrecG cells have a specific requirement for a39 ssDNA exonuclease, recG1 cells can be maintained byeither a 39 or a 59 activity. However, the 39 activity has to beExoI, ExoVII, or SbcCD. No other nuclease present inE. coli seems to be able to maintain viability.

Rep and UvrD promote viability in rich media: Weextended our studies to investigate whether DNAhelicases other than RecG are required to supportgrowth of ExoI� ExoVII� SbcCD� cells. RecQ, HelD,and DinG proved dispensable (Figure 2D, i–iii; Table 1).However, Rep, a 39–59 DNA helicase considered to beinvolved with DNA replication (Yarranton and Gefter

1979), proved essential for colony formation on LB agar,although dispensable on minimal salts agar (Figure 2D,iv and v), which contrasts with the requirement for RecGunder both conditions (cf. Figure 2D, iv and v, withFigure 2A, ii and iii). Likewise, UvrD, a 39–59 DNAhelicase associated initially with DNA repair (Matson

1986), is required specifically for good growth of

colonies, but only on LB agar (Figure 2D, vi and vii).As with cells lacking RecG, the presence of any one ofExoI, ExoVII, or SbcCD suffices to maintain robustviability on LB agar (Table 1 and data not shown). Theseobservations indicate that ExoI� ExoVII� SbcCD� cellsare likely to have multiples defects in DNA macromo-lecular metabolism.

ExoI� ExoVII� SbcCD� cells are sensitive to mito-mycin C and 2-aminopurine: The above data (Figure3C; Table 1) demonstrate that any combination of ExoI,ExoVII, and SbcCD can be eliminated from wild-typecells without obvious loss of viability, as reported(Dermic 2006). The mutants form healthy colonies onLB agar and are almost as resistant to UV light andmitomycin C as the wild type (Figure 3, A and C; Table 2),with the exception of a construct lacking all threenucleases. Cells lacking ExoI, ExoVII, and SbcCD arefairly resistant to UV light but proved sensitive toionizing radiation and mitomycin C, although not assensitive as a recB strain defective in DNA double-strandbreak repair (Figure 3, B and C; data not shown). Theyare also sensitive to the base analog 2-AP (Figure 3C). Astrain lacking ExoX in addition to ExoI, ExoVII, andSbcCD was made without difficulty (Table 2, strainN7007), but did not appear different from an ExoI�

ExoVII� SbcCD� strain in terms of sensitivity to UV,

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

Other chromosomal mutation(s)b

pAM401sbcCD1/c

pJJ100recG1/DrecG

pAM390ruv1/Druv

pAM490rnhA1/DrnhA

None, or any 1 or 2 of xonA, xseA, sbcCD, recJ 1 1 1 1

xonA xseA recJ 1 1 * �xonA sbcCD recJ 1 1 1 1

xseA sbcCD recJ 1 1 1 1

xonA xseA sbcCD 1 � � �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 1 1 1 1

xonA xseA sbcCD priA300 1 1 1d �e

a As determined by using a synthetic lethality assay (materials and methods). 1, plasmid-free segregants form well-developedcolonies on LB agar equal to or approaching in size those formed by cells retaining the plasmid (unless indicated otherwise inthe text). They also account for 25–75% of the total colonies observed and can be subcultured without difficulty. �, colonies ofplasmid-free segregants not detected on either LB or 56/2 minimal salts agar, except as indicated in the text. *, plasmid-freesegregants account for .20% of the total colonies observed on LB agar but establish colonies that are much smaller than thoseformed by cells retaining the plasmid; they establish much stronger colonies on 56/2 minimal salts agar and can be subculturedwithout 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, whichis 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.




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 ExoI� ExoVII�

SbcCD� strain to 2-AP and mitomycin C. (E) Synthetic lethality assays showing the effect of eliminating MutS on the viability ofExoI� ExoVII� SbcCD� strains lacking RecG, RecJ, Rep, or UvrD. (F and G) mutS derivatives of ExoI� ExoVII� SbcCD� cells lackingRecJ or Rep accumulate suppressors during growth on LB agar.


mitomycin C, or 2-AP (Table 2 and data not shown).Reducing both 39 and 59 activities increases sensitivity toUV, as reported (Viswanathan and Lovett 1998;Dermic 2006), but especially if RecG is also absent(Table 2; Figure 3A).

Samples of ExoI� ExoVII� SbcCD� cells spread on LBagar supplemented with 2-AP at 300 mg/ml give rise tocolonies of resistant derivatives within 24–48 hr. Thesesuppressors appear at a frequency of�1/106 initial cellsplated. We isolated seven independent isolates forfurther analysis. Four of these proved strong mutators(see materials and methods), consistent with a defectin mismatch repair. A mutS construct confirmed thatresistance to 2-AP is restored by inactivating theMutHLS-dependent and methyl-directed mismatch re-pair system (Figure 3D). These data indicate thatmismatch repair is compromised in ExoI� ExoVII�

SbcCD� cells and leads to inviability when mismatchesare increased by exposure to 2-AP. They fit with previ-ous studies demonstrating the involvement of ExoIand ExoVII in mismatch repair (Harris et al. 1998;Viswanathan and Lovett 1998; Burdett et al. 2001;Viswanathan et al. 2001) and suggest that SbcCD mayalso be engaged in this process. Eliminating MutS also

improves the ability of ExoI� ExoVII� SbcCD� cellslacking UvrD to form colonies on LB agar (Figure 3E, i;Table 3). Thus, the poor growth seen with MutS presentmay be attributed in part to some consequence ofabortive mismatch repair.

Eliminating MutS from ExoI� ExoVII� SbcCD� cellsdoes not restore resistance to mitomycin C (Figure 3D),nor does it eliminate the dependence on RecG, RecJ,and Rep for growth on LB agar (Figure 3E, ii–iv; Table3). There is some improvement in the recovery ofplasmid-free colonies of recJ and rep derivatives (Figure3E, iii and iv), but this is largely attributable to theoutgrowth of suppressors, which is not surprising, giventhe mutator phenotype of the mutS construct. Theoutgrowth of suppressors is very evident when cellssubcultured in minimal salts medium are plated on LBagar (recJ derivative; Figure 3F) or when white coloniesinitially detected on minimal salts indicator platesare streaked directly on LB agar (rep derivative; Figure3G).

One of the three suppressor strains that did notdisplay a mutator phenotype proved strongly resistantto both 2-AP and mitomycin C (Figure 4A, strainN7050). This isolate carries an rpoC mutation encoding

TABLE 2

Effect of RecG on UV sensitivity of exonuclease-depleted strains

xonA, xseA, sbcCD,exoX, recJ genotype

Exonucleasedeficiency (polarity)

rec1 constructs DrecG constructs

Strain Survivala Strain Survivala

Wild type None TB28 0.68 N5742 0.14xonA ExoI� (39–59) N6946 0.67 N7293 0.21xseA ExoVII� (39–59 and 59–39) N6951 0.68 N7297 0.11sbcCD SbcCD� (39–59) N5281 0.59 N7295 0.11recJ RecJ� (59–39) N4934 0.62 N7294 0.044xonA xseA ExoI� ExoVII� N6954 0.58 N7301 0.093xonA sbcCD ExoI� SbcCD� N6945 0.44 N7299 0.059xonA recJ ExoI� RecJ� N7065 0.22 N7298 0.028xseA sbcCD ExoVII� SbcCD� N6952 0.65 N7303 0.13xseA recJ ExoVII� RecJ� N7066 0.32 N7302 0.0028sbcCD exoX SbcCD� ExoX� N7004 0.80 NCb

sbcCD recJ SbcCD� RecJ� N7056 0.63 N7300 0.0067xonA xseA sbcCD ExoI� ExoVII� SbcCD� N6953 0.33 Inviablec

N7037 0.30xonA xseA recJ ExoI� ExoVII� RecJ� N7036 0.0072 N7317d 0.001d

xonA sbcCD exoX ExoI� SbcCD� ExoX� N7005 0.49 NCb

xonA sbcCD recJ ExoI� SbcCD� RecJ� N7063 0.17 N7312 0.011xseA sbcCD exoX ExoVII� SbcCD� ExoX� N7006 0.79 NCb

xseA sbcCD recJ ExoVII� SbcCD� RecJ� N7064 0.21 N7311 0.00025xonA xseA sbcCD exoX ExoI� ExoVII� SbcCD� ExoX� N7007 0.23 NCb

xonA xseA sbcCD recJ ExoI� ExoVII� SbcCD� RecJ� N7074d 0.001d inviablec

a Survival was determined at a dose of 30 J/m2. Except where indicated otherwise, survival was determined byusing cultures grown to exponential phase in LB broth and irradiated on the surface of LB agar plates. Valuesare 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 otherwiseidentical conditions.


an R919H substitution in the b9-subunit of RNA poly-merase. Genetic reconstructions established that thismutation alone is responsible for the suppression(materials and methods). In other work, a particularclass of stringent RNA polymerase (RNAP) mutations(rpo*) was shown to act as partial suppressors of the DNArepair-deficient phenotype of ruv mutants (McGlynn

and Lloyd 2000; Trautinger and Lloyd 2002). Theserpo* mutations also promote growth of uvrD rep doublemutants in rich media (Guy et al. 2009). They arethought to act by destabilizing RNAP transcriptioncomplexes, thereby reducing the likelihood of patho-logical consequences following collisions with replica-tion forks (Trautinger et al. 2005; Rudolph et al. 2007).

TABLE 3

Suppression of the reduced viability of exonuclease-deficient cells lacking recombination/repair activities

Other chromosomalmutation(s)b

Effect of mutS, rpoC[R919H], and priA300 mutations on colony formation byplasmid-free segregants of pAM401 sbcCD1/DsbcCD DxonA DxseA constructsa

mut1 rpo1 pri1 mutS rpoC[R919H] priA300

None 1 1 1 1

DrecG � �b 1

recJ284 * *,c 1 �d

Drep �d *,c,d 1

DuvrD *,c 1 1 1

DruvABC * *,c *,c 1

DruvABC rus-2 1

DrecA or recA269 �d �b,d *,e

a 1, �, 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.

Figure 4.—Mutations in subunits of RNAP modulate the phenotype of ExoI� ExoVII� SbcCD� strains. (A) Effect of RpoB andRpoC mutations on sensitivity to mitomycin C and 2-AP. (B) Synthetic lethality assays showing the effect of RpoCR919H on theviability of derivatives lacking RecG, RecJ, Rep, or UvrD. (B, ii, b) Abortive growth of a plasmid-free colony from B, ii, a.


We examined three rpoB alleles in a strain lacking ExoI,ExoVII, and SbcCD and found that each conferredresistance to mitomycin C and, to a lesser extent, to 2-AP(Figure 4A). Thus, the sensitivity of the ExoI� ExoVII�

SbcCD� strain may reflect in part a reduced ability toresolve conflicts between DNA replication and tran-scription, which are elevated when the template DNA iscorrupted.

However, none of the RNAP mutations tested elimi-nates the requirement for RecG to maintain viability.The synthetic lethality assays exploited revealed thatsome white colonies do appear on LB agar, but are tinyand cannot be subcultured without acquisition ofsuppressors (Figure 4B, i and ii; Table 3; data notshown). Therefore, although conflicts between replica-tion and transcription most probably contribute to thesensitivity of ExoI�ExoVII� SbcCD� cells to mitomycin Cand 2-AP, we suspect that such conflicts are not theprimary reason for the inviability observed when thecells are also depleted of RecG. However, they might bethe reason why the cells need Rep and UvrD to grow wellon LB agar as the rpoC suppressor abolishes the re-quirement (Figure 4B, iii and iv; Table 3). They may alsohelp to explain why the cells need RecJ, although suchconflicts cannot be the only reason as the recJ rpoCconstruct still shows a growth defect relative to the recJ1

control (Figure 4B, cf. i and v).39 ssDNA exonucleases limit recombination: Early

genetic studies indicated that ExoI and SbcCD eliminatesubstrates that RecA protein might otherwise exploit toinitiate homologous DNA pairing and strand exchange(Kushner et al. 1971; Lloyd and Buckman 1985;Kowalczykowski 2000). To determine whether suchsubstrates accumulate in ExoI� ExoVII� SbcCD� cells,we examined a DruvABC derivative on the premise thatthe initiation of recombination might be toxic withoutan efficient system for resolving Holliday junctions.Synthetic lethality assays revealed that a DruvABC de-rivative of an ExoI� ExoVII� SbcCD� strain grows verypoorly on LB agar, forming very small colonies without acovering plasmid, colonies that become overgrown withsuppressors (Figure 5A, i and ii; Table 1; data notshown). Activation of the RusA Holliday junctionresolvase (rus-2 insertion) largely abolishes this defect(Figure 5A, iii; Table 3), demonstrating that the poorgrowth is indeed most likely due to an accumulation ofHolliday junctions. However, the ruv cells have nodifficulty growing on minimal salts agar (Figure 5A,iv), indicating that the accumulation of Holliday junc-tions is a problem that arises during conditions permit-ting rapid growth. The rpoC[R919H] suppressorimproves colony growth on LB agar, but the effect ismarginal (Figure 5A, v; Table 3). Eliminating MutS alsoincreases colony growth (Figure 5A, vi; Table 3), butmuch of this increase can be attributed to the accumu-lation of suppressors, triggered no doubt by the mutatorphenotype (data not shown).

Any one of ExoI, ExoVII, or SbcCD is sufficient tomaintain robust viability in cells lacking RuvABC (Figure5A, i; Table 1). But, as we found with DrecG cells, RecJ isredundant, except when both ExoI and ExoVII aremissing, in which case the cells grow very slowly, evenmore slowly than their DrecG counterparts (Figure 5B, iand ii). Thus, it seems that, under conditions promotingrapid growth, recombination is provoked specifically incells lacking the 39 ssDNA exonuclease activities of ExoI,ExoVII, and SbcCD and for reasons that have little to dowith any defect in mismatch repair or with any conflictbetween replication and transcription.

Recombination is necessary in ExoI� ExoVII�

SbcCD� cells: We investigated recA derivatives of ExoI�

ExoVII� SbcCD� cells to see if the recombinationdetected using ruv constructs is needed to maintainviability. Synthetic lethality assays revealed that thesecells do need RecA to form colonies on LB agar,although not on minimal salts agar (Figure 6, A and B;Table 3), which is consistent with the absence of anyneed for RuvABC under these conditions. A constructmade using recATTn10 yields plasmid-free colonies onLB agar, but these appear at a reduced frequency, growvery slowly, and reveal large colony variants on sub-culture (Figure 6A, iii and iv). These variants proved tobe recA1 derivatives resulting from excision of Tn10(data not shown). Their accumulation under theseconditions emphasizes the need for RecA to maintainviability. The rpoC[R919H] suppressor does little toeliminate this requirement. Plasmid-free colonies aremore frequent, but still very small, and the emergence oflarge colony variants remains a major feature (Figure6C, i and ii; Table 3).

We made a lexA3 construct to investigate whether RecAmight be needed to induce the SOS repair response. SOSis induced when RecA is assembled on ssDNA exposedduring replication of damaged DNA or following DNAbreakage and triggers autocleavage of LexA protein, theSOS repressor (Sassanfar and Roberts 1990). ThelexA3 allele encodes a mutant repressor resistant toautocleavage. Consequently, lexA3 cells, like recA cells,cannot induce SOS (Sassanfar and Roberts 1990). Theconstruct made revealed that lexA3 does not preventExoI� ExoVII� SbcCD� cells from growing on LB agar.Indeed, they form fairly robust colonies (Figure 6C, iii).Furthermore, they still need RecG to do so (Figure 6C, iv)as well as RecA, Rep, and UvrD (data not shown). Somesmall white colonies are seen with the construct lackingRecG, but these are full of suppressors (Figure 6C, v).

From these data we conclude that excessive SOSexpression is not the main reason why the viability ofExoI�ExoVII� SbcCD� cells is so reduced in the absenceof RecG, Rep, or UvrD. The data also enable us toconclude that these cells rely on the recombinaseactivity of RecA to survive growth in rich media ratherthan on its ability to promote SOS induction. However,the recombinase activity requires assembly of a RecA


filament on ssDNA, in which case it might be expectedto cause some increase in the expression of SOS genes.We investigated this possibility and found that the basallevel of expression is increased quite substantially. Thisis clear from the approximately fivefold higher levels ofb-galactosidase seen in ExoI� ExoVII� SbcCD� cellscarrying the lacZ1 gene fused to the LexA-regulated sulA(sfiA) promoter (Table 4). It is also clear that thepresence of any one of ExoI, ExoVII, or SbcCD curbsthis increase, whereas the rpoC[R919] suppressor doesnot. The SOS response is induced strongly in every casefollowing exposure to mitomycin C.

These data demonstrate that ssDNA must be exposedand in a form that not only is vulnerable to attack byExoI, ExoVII, or SbcCD but also is amenable to RecAloading. Two pathways have been identified by which astable RecA filament can be assembled on ssDNA: onemediated by RecBCD enzyme and the other by theRecFOR proteins. RecBCD unwinds and resects duplexDNA ends and loads RecA at a 39 ssDNA overhangcreated after the enzyme activity has been modulatedat a x-sequence. The 39 end of the exposed strand maybe held within the RecBCD complex, protecting itfrom other nucleases, while RecB’s helicase activitymay strip away any SSB protein (Kowalczykowski

2000; Amundsen and Smith 2003; Singleton et al.2004). The RecFOR proteins normally load RecA atssDNA gaps. They enable RecA to displace SSB protein,which has a higher affinity for ssDNA (Umezu et al. 1993;Morimatsu and Kowalczykowski 2003; Cox 2007).However, RecFOR can also assemble RecA at ssDNAends generated independently of RecBCD, although

normally such DNA would be vulnerable to exonucleasedigestion.

It is therefore significant that even after 48 hrincubation, ExoI� ExoVII� SbcCD� cells lacking RecFform small and sickly colonies on LB agar, although theygrow well enough on minimal salts agar (Figure 6D, iand ii; Table 1), whereas those lacking RecB are quiteviable, forming colonies on both types of media asefficiently as the recB1 parent. Unlike the recF derivative,the colonies formed on LB agar are well developedafter only 24 hr incubation (Figure 6E; data not shown).The relevant recB construct was made both with andwithout the aid of a covering plasmid, but any coveringplasmid used is eliminated with such a high frequencyunder nonselective conditions that it prohibits use ofour standard assay for synthetic lethality. However, thisitself emphasizes the viability of the plasmid-free cells(Table S1, strains N7783 and N7789). We concludethat at least some of the ssDNA on which RecA isloaded to initiate recombination is generated by meansother than RecBCD-mediated digestion of duplex DNAends.

RecG limits recombination during normal growth: Acentral tenet of the model in Figure 1 is that recombi-nogenic 39 flaps are generated more frequently in cellslacking RecG because of the increased incidence ofunscheduled fork collisions. Accordingly, and on thebasis that a ruv mutant would have no means to resolveHolliday junctions efficiently by junction cleavage(Zhang et al. 2010), a Druv DrecG cell should be moresensitive to a reduction in 39 exonuclease activity than aruv single mutant. We used a ruv1 recG1 derivative of

Figure 5.—ExoI� ExoVII� SbcCD� cells need a Holliday junction resolvase to maintain rapid growth. (A) Synthetic lethalityassays revealing the extremely poor growth on LB agar compared with minimal agar of ExoI� ExoVII� SbcCD� cells lackingRuvABC. 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 ofDruvABC cells depends on RecJ.



pRC7 to test this possibility and examined the effect ofeliminating any two of ExoI, ExoVII, and SbcCD.Removing all three would be uninformative as we haveshown this to compromise the viability of both Druv andDrecG strains. With all three nucleases present, DruvABCDrecG cells show no major reduction in viability in thatthey form good colonies without the covering plasmid(Figure 7A, i and ii). If recombinogenic 39 flaps do arisemore frequently in the absence of RecG, then it seemsthat these can be removed efficiently by exonucleasedigestion.

ExoI proved sufficient to maintain robust viability(Figure 7A, iii). ExoVII also keeps the cells alive, but theslightly smaller size of the plasmid-free colonies observedwith the relevant construct suggests that it might be a littleless effective (Figure 7A, iv). SbcCD proved rather in-effective, with the cells forming small and rather sicklycolonies without the covering plasmid (Figure 7B, i).

Robust growth is maintained in this case if RecG is presentor RecA eliminated (Figure 7B, ii and iii).

Taken together, these data indicate that potentiallyrecombinogenic substrates do arise more frequently inthe absence of RecG but can be eliminated efficiently viathe action of 39 ssDNA exonucleases, particularly byExoI. However, the inability of ExoI� ExoVII� SbcCD�

cells lacking either RuvABC or RecA to establish goodcolonies on LB agar indicates that recombinogenicsubstrates are generated under conditions promotingrapid growth even with RecG present and that the cellsmust engage in and complete recombination if they areto stay alive. If these recombinogenic substrates are 39

flaps, or dsDNA branches arising from subsequent PriA-mediated replication, RecG is clearly unable to convertall 39 flaps to 59 flaps, or if it can, then RecJ cannoteliminate all the 59 flaps formed. The fact that elimi-nating RecA enables SbcCD to maintain robust viability

Figure 6.—The recombinase activity of RecA is needed to maintain rapid growth of ExoI� ExoVII� SbcCD� cells. (A and B)Synthetic lethality and spot dilution assays showing how ExoI� ExoVII� SbcCD� cells lacking RecA grow well on minimal agar butare inviable on LB agar. (C–E) Synthetic lethality and spot dilution assays showing the effects of RpoCR919H, lexA3, RecF, andRecB on the viability of ExoI� ExoVII� SbcCD� cells.


in the absence of RecG is significant (Figure 7B, iii). It isconsistent with the formation of dsDNA branches thatprovoke RecBCD-mediated recombination (Figure 1C,iii and iv). Without RecA to load on the 39 ssDNAexposed after an encounter with x, RecBCD recombi-nase activity is aborted and its dsDNA exonucleaseactivity (ExoV) becomes rampant (Dabert et al. 1992;Kuzminov and Stahl 1997). SbcCD removes thessDNA, enabling the DNA to be targeted and furtherdigested by another RecBCD molecule (Zahradka et al.2009). This combination of nuclease activities facilitatescomplete removal of the DNA branch, thus possiblyexplaining the restoration of robust viability.

PriA activates recombination in cells lacking RecG:Previous studies revealed that most, if not all, aspects ofthe recG mutant phenotype are suppressed by mutations

reducing or eliminating the helicase activity of PriA (Al-Deib et al. 1996; Jaktaji and Lloyd 2003; Rudolph et al.2009a; Zhang et al. 2010). We exploited priA300, whichencodes helicase-defective PriAK230R, to investigatewhether the same holds true for the inviability causedin ExoI� ExoVII� SbcCD� cells. Synthetic lethalityconstructs revealed that priA300 restores robust viability(Figure 8A, i–iv; Table 3). The priA300 allele alsoimproves the ability of ExoI� ExoVII� SbcCD� cellslacking RuvABC or RecA to form colonies on LB agar,although viability is still compromised in each case, asevident from the more robust growth of cells retainingthe covering plasmid (Figure 8A, v and vi; Table 3).However, priA300 does not confer viability in theabsence of both RecG and RecA (Figure 8, vii). It alsodoes not suppress the sensitivity of ExoI� ExoVII�

TABLE 4

Effect of ssDNA exonuclease deficiency on SOS expression

b-Galactosidase activitya

Exonuclease deficiency (additional genotype) Strain no. No. of experiments �MMC 1MMC

None N7843 10 77 6 8 1474 6 70ExoI� N7844 7 94 6 4 1591 6 96ExoVII� N7859 4 83 6 12 1343 6 60SbcCD� N7845 4 65 6 13 1384 6 61ExoI� ExoVII� N7860 4 136 6 6 1542 6 95ExoI� SbcCD� N7846 4 102 6 5 1354 6 82ExoVII� SbcCD� N7847 4 109 6 7 1309 6 47ExoI� ExoVII� SbcCD� N7837 7 398 6 28 1962 6 52None (rpoC[R919H]) N7888 4 135 6 7 2520 6 42ExoI� ExoVII� SbcCD� (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 andexpressed as Miller Units. Values are means 6 SE. MMC = mitomycin C.

Figure 7.—RecG limits the requirement for RuvABC to maintain rapid growth of ExoI� ExoVII� SbcCD� 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.


SbcCD� cells to mitomycin C and 2-AP (Figure 8B).These observations, together with the fact that priA300is not otherwise a suppressor of ruv or recA (Jaktaji andLloyd 2003), demonstrate that it is PriA, and inparticular some activity that depends on its ability tounwind DNA, that is responsible for the recombinationprovoked in cells lacking ExoI, ExoVII, and SbcCD andfor making these cells dependent on RecG for theirviability. However, it is also clear from the weak growth ofcells lacking RuvABC or RecA (Figure 8A, v and vi) andthe inviability of those lacking both RecG and RecA thateliminating PriA helicase activity does not preventrecombination altogether.

We also examined priA300 cells lacking ExoI, ExoVII,SbcCD, and UvrD and found that they, too, form robustcolonies on LB agar (Figure 8A, viii; Table 3). Theirvigorous growth contrasts sharply with the feeblecolonies formed by the equivalent priA1 cells (Figure2D, vi), which we have demonstrated to be due in largemeasure to some consequence of abortive mismatchrepair as it can be alleviated by eliminating MutS (Figure3E, i). Therefore, we suspect that the abortive mismatchrepair in these cells creates substrates that PriA canexploit via its helicase activity to load DnaB and thusinitiate replication in a manner that reduces viabili-ty. We were unable to investigate the effect of priA300on ExoI� ExoVII� SbcCD� cells lacking Rep, as wewere unable to make the relevant construct. Previousstudies established that priA300 rep double-mutant cells

have reduced viability (Sandler 2000; Mahdi et al.2006).

RNase HI-deficient cells require ssDNA exonu-cleases to stay alive: The data presented above fit withthe idea that the inviability of ExoI� ExoVII� SbcCD�

cells lacking RecG is a consequence of SDR initiated viaPriA helicase activity. However, we were conscious thatRecG is needed to facilitate recovery of recombinants incrosses with ruv strains, a fact previously interpretedas evidence that RecG and RuvABC provide alterna-tive ways for processing recombination intermediates(Lloyd 1991). To investigate whether it is SDR that isresponsible rather than some recombination defect,we analyzed exonuclease-deficient constructs lackingRNase HI (DrnhA). This enzyme degrades RNA fromRNA:DNA duplexes, including from R-loops, and itsabsence is known to trigger a high level of cSDR, highenough in fact to sustain viability in the absence oforigin firing (Kogoma 1997). There is no evidence thatit has any direct role in recombination.

We observed that removing RNase HI mimics theeffect of removing RecG in that DrnhA cells are inviableif ExoI, ExoVII, and SbcCD are eliminated. Thesynthetic lethality constructs that were exploited yieldedno plasmid-free colonies on either LB agar or minimalsalts agar (Figure 9, i–iii; Table 1; data not shown). Thisis consistent with SDR being the trigger for the in-viability of exonuclease-depleted DrecG cells. However,removing RNase HI differs in that priA300 does little to

Figure 8.—Effect of priA300 on ExoI� ExoVII� SbcCD� cells depleted of RecA, RecG, RuvABC, or UvrD. (A) Synthetic lethalityassays. (B) Spot dilution assays showing sensitivity to mitomycin C and 2-AP.


restore viability. The plasmid-free cells form tiny colo-nies on minimal agar but fail to grow on LB agar (Figure9A, iv and v; Table 3). The cells are also inviable on LBagar if ExoI, ExoVII, and RecJ are missing but grow wellon minimal agar (Figure 9B, i and ii). As with cellslacking RecG, the presence of either ExoI or ExoVII issufficient to maintain viability, even if both SbcCD andRecJ are missing (Figure 9B, iii and iv; Table 1; data notshown). The two differences may be explained by theincomplete suppression of SDR by priA300 and thepresence of RecG, which could convert some 39 flaps to59 flaps. Without RecJ and ExoVII, PriA might theninitiate pathological re-replication by targeting theseflaps (Figure 1F). The PriAK230R protein would retainthe ability to do so as the template for DnaB loading isalready single stranded, eliminating the need for heli-case activity.

Removing RNase HI also mimics the effect of re-moving RecG in that it increases the sensitivity of ruvmutant cells to killing by UV light. However, the effect isnot as severe (Figure 9C). On the other hand, even witha full complement of ssDNA exonucleases available,removing RNase HI makes DrecG cells inviable, asreported (Hong et al. 1995), whereas it only reduces

the viability of ruv cells (Figure 9B, v and vi). Thedifferences observed may be explained by the possibilitythat RNase HI may also help to process Okazakifragments during replication (Ogawa and Okazaki

1984; Kornberg and Baker 1992). Without it, gaps mayaccumulate in the lagging strand, compounding anydifficulties arising from the increase in SDR but alsocompromising viability even when SDR is reduced.

DISCUSSION

Recent studies of how the integrity of the genome andcell viability are maintained during the course of DNAreplication in bacteria have focused on describing whathappens to replication forks as they encounter blockinglesions in or on the DNA template or on dissecting themolecular mechanisms that enable cells to overcomesuch blocks and complete replication (Heller andMarians 2006b; Mahdi et al. 2006; Michel et al. 2007;Rudolph et al. 2007; Guy et al. 2009; Boubakri et al.

2010; Gabbai and Marians 2010). By comparison,much less attention has been paid to what happenswhen one fork runs into another, an event that usually

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.


happens only once at the termination of replicationduring the bacterial cell cycle, but which occurs manytimes along each chromosome during S-phase in eu-karyotes. In this article, we have presented evidenceconsistent with the idea that the termination of DNAsynthesis does not always run smoothly, at least in E. coli,and that, consequently, replication fork encountersneed to be limited.

The data presented demonstrate that DrecG cells needa 39 ssDNA exonuclease to stay alive and that thisrequirement can be satisfied by any one of ExoI, ExoVII,or SbcCD or eliminated by inactivating the helicaseactivity of PriA. The data are consistent with a model inwhich unscheduled initiation of replication via PriA-mediated replisome assembly at sites remote from oriC(SDR) compromises the highly evolved replichorearrangement that orchestrates duplication and trans-mission of the E. coli chromosome and that normallylimits fork collisions to a single event during each cycleof cell growth and division. The data fit with the idea thata major role of RecG is to limit such replication (Figure1). Taken together, the data suggest that the ability ofPriA to secure chromosome duplication in times ofreplicative stress comes at a price (Rudolph et al.2009a,b).

Previous studies demonstrated that mutations reduc-ing or eliminating PriA helicase activity suppress thesensitivity of recG mutants to mitomycin C (Al-Deib et al.1996; Gregg et al. 2002; Jaktaji and Lloyd 2003). Theyalso eliminate most of the delay in division observedfollowing irradiation of DrecG cells with UV light(Rudolph et al. 2009a). However, it is significant thatDrecG cells are quite healthy, with a doubling time closeto that of wild type. This implies that PriA becomes toxic

to DrecG cells only when these cells have suffered damageto their DNA. Thus, given that DrecG cells lacking ExoI,ExoVII, and SbcCD are inviable, it would seem that DNAmust somehow become ‘‘damaged’’ in these cells evenwithout application of external genotoxic agents, i.e., asa result of events that occur during normal growth.

We considered the possibility that this ‘‘damage’’might reflect abortive repair of DNA base-pair mis-matches generated during chromosome replication(Iyer et al. 2006). Both ExoI and ExoVII are implicatedin nascent strand removal following initiation of mis-match repair by the MutHLS proteins (Burdett et al.2001; Viswanathan et al. 2001). Inactivating these twoenzymes, and any possible substitute, might thereforeleave recombinogenic nicks or gaps in the nascentstrands, which might then account for the fact thathomologous recombination and means to resolve Holli-day junctions are essential for the survival of cellslacking ExoI, ExoVII, and SbcCD (Figures 5A and 6A).Indeed, we discovered that cells lacking ExoI, ExoVII,and SbcCD are sensitive to 2-AP, one of the hallmarks ofa DNA mismatch repair defect downstream of theinitiation step (Glickman and Radman 1980). Thesecells also need UvrD to grow well on LB agar, a DNAhelicase required for displacement of the nascent standcontaining the mismatched base (Iyer et al. 2006).Eliminating MutS restores resistance and reduces theneed for UvrD, establishing that the cells do indeedsuffer from abortive mismatch repair. However, elimi-nating MutS does not remove the requirement for RecGand RuvABC, indicating that the cells must have at leastone additional defect.

A reduced ability to resolve conflicts between DNAreplication and transcription is indicated by the fact

Figure 10.—Models illustrating how DNA molecules containing single-strand flaps might be processed by RecG translocase orby ssDNA exonucleases or targeted by RecA or PriA to provoke recombination (see discussion for further details).


that, in addition to UvrD protein, ExoI� ExoVII�

SbcCD� cells need Rep to grow on LB agar, but not onminimal salts agar. Previous studies revealed that repuvrD double mutants have the same growth constraintand that this can be alleviated by mutations shown todestabilize RNAP transcription complexes (Guy et al.2009). We suggest that, in the absence of ExoI, ExoVII,and SbcCD to remodel a fork stalled at a ternarytranscription complex, UvrD might have greater diffi-culty compensating for the absence of Rep and viceversa. Consistent with this, we discovered that therequirement for UvrD and Rep is alleviated in thepresence of an rpoC mutation encoding an R919Hsubstitution in the b9-subunit of RNAP. This mutationand other previously described rpoB alleles also allevi-ates the sensitivity of ExoI�ExoVII� SbcCD� cells to 2-APand mitomycin C. However, they do not eliminate theneed for RecG or for RecA, indicating that there is yetanother defect in these cells.

Studies with strains carrying sulATlacZ fusions re-vealed that the SOS response is constitutively elevated inExoI� ExoVII� SbcCD� cells and that ExoI, ExoVII, andSbcCD all contribute to keeping its expression at a lowlevel during normal growth in LB broth. Furthermore,SOS expression remains high in the presence of therpoC[R919H] suppressor mutation, indicating that theSOS-inducing signal is generated at the same high leveleven when conflicts between DNA replication andtranscription might be reduced.

The high level of SOS expression, coupled with thefact that cells lacking ExoI, ExoVII, and SbcCD becomeinviable if RecA, RecG, or RuvABC is eliminated issignificant. It indicates (1) that ssDNA species with free39 ends accessible to these nucleases are generated withor without RecG present; (2) that these strands enableRecA to initiate recombination with high efficiency; and(3) that this recombination is essential, providing theonly means apart from exonuclease digestion of dealingwith the exposed strands. So, how do these recombino-genic 39 strands arise? And why are they particularlyproblematic in the absence of RecG? There is clearly adeficiency in mismatch repair and most probably areduced ability to resolve conflicts between DNA repli-cation and transcription. However, neither defect issufficient to explain why ExoI� ExoVII� SbcCD� cellsneed RecA, RecG, and RuvABC to stay alive.

Chromosome breakage would most certainly expose aneed for RecA. Breakages occur when forks encounternicks or gaps in the template strands or when they stalland reverse to establish a Holliday junction structurethat could be targeted and cleaved by RuvABC (Kuzminov

1995; Seigneur et al. 1998). RecBCD enzyme wouldnormally be expected to unwind the duplex DNA endexposed in both cases and to degrade both strands viaits ExoV activity until it encounters a x-sequence in thestrand ending 39, whereupon its activity is modified insome manner that is not fully understood but that fo-

cuses subsequent degradation on the strand ending 59,leaving a 39 tail on which RecBCD then loads RecA toinitiate recombination (Smith 1990; Kowalczykowski

2000; Singleton et al. 2004; Amundsen et al. 2007;Dillingham and Kowalczykowski 2008). However,although RecBCD is crucial for the repair of DNAbreaks, recBC single mutants are viable, as are recAmutants, which would suggest that chromosome break-age is rather rare under normal growth conditions.Breaks are detectable in recBC cells (Seigneur et al.1998), and viability is reduced (Capaldo-Kimball andBarbour 1971), but some of the breaks may bepathological in origin, being a consequence of RecBCDinactivation. ExoV is thought to be particularly instru-mental in eliminating the Holliday junction structureformed at reversed forks, thus limiting fork breakage viaRuvABC (Seigneur et al. 1998). It is therefore signifi-cant that cells lacking ExoI, ExoVII, and SbcCD do notrequire RecBCD to stay alive. Furthermore, the viabilityof recB recG and recB ruv cells is no different from that ofrecBC single mutants (Lloyd et al. 1987; Lloyd andBuckman 1991). Taken together, these observationsindicate that increased chromosome breakage is not theprimary reason for the failure of cells lacking ExoI,ExoVII, and SbcCD to form colonies when RecG, RuvABC,or RecA is inactivated and that something other thanthe processing of duplex DNA ends is responsible forthe initial generation of potentially toxic 39 ssDNA.

Hiasa and Marians (1994) presented evidence in-dicating that a 39 flap might be generated when thereplisome of one replication fork displaces the 39 end ofthe nascent leading strand of the fork coming in theother direction. This led us to propose that 39 flapsmight be generated in this way quite frequently in aDrecG strain as a result of the increase in SDR, whichleads to additional and unscheduled replication forkencounters outside of the normal termination area(Rudolph et al. 2009a,b, 2010). Flaps of this naturewould be expected to be particularly problematic forcells lacking RecG and depleted of 39 ssDNA exonu-cleases. As outlined in Figure 10, they could providetemplates for RecA loading and strand exchange. Theloading of RecA might also provoke recombination.Otherwise, PriA could target the branch point to initiatefurther replication, which would convert the flap to adsDNA branch, thus providing an alternative route torecombination via RecBCD enzyme. Such exchangeswould generate a network of partially replicated chro-mosomes with numerous DNA branches (Figure 1C, ivand v), as observed (Rudolph et al. 2009b).

The suggestion that cells lacking RecG and depletedof 39 ssDNA exonucleases suffer damage to their DNA asa result of unscheduled replication fork collisions isconsistent with the fact that the problem largelydisappears when the helicase activity of PriA is elimi-nated (Figure 8), especially as PriA helicase mutantsreduce SDR (Tanaka et al. 2003). Given that DrecG and


DrnhA cells have in common a high level of cSDRinitiated at R-loops (Kogoma 1997), it is also consistentwith the fact that eliminating RNase HI mimics theeffect of eliminating RecG (Figure 9). However, priA300does little to eliminate the problem in this case.Furthermore, RecJ is also needed to maintain viability.These two observations are exactly what we wouldpredict if the RecG present in these cells were able toconvert a 39 flap to a 59 flap (Figure 10). PriA might thenbe able to initiate replisome assembly without need of itshelicase activity as there would be no lagging strand toget in the way of DnaB loading, but alternatively, PriCmight do so (Heller and Marians 2005, 2006a,b).However, cells lacking RNase HI not only are lesseffective in eliminating R-loops but also may be de-fective in Okazaki fragment processing (Ogawa andOkazaki 1984), which, if true, would create at least twodifferent problems for chromosome replication andthus make it difficult to draw definitive conclusionsfrom rnhA derivatives of ExoI� ExoVII� SbcCD� cells.

By reducing fork collisions to a single event per cellcycle and restricting termination to a defined area, thereplichore arrangement of the chromosome enablescomplete replication of the chromosome to be achievedwith minimum conflict with transcription and in a waythat allows the 8-bp KOPS (FtsK orienting polar sequen-ces) DNA elements to be arranged symmetrically in eachchromosome arm to direct efficient FtsK-mediatedchromosome segregation during cell division (Bigot

et al. 2005; Reyes-Lamothe et al. 2008). With RecGpresent to unwind R-loops, D-loops, and 39 ssDNA flaps,and a full complement of exonucleases with the ability todigest any ssDNA flaps and dsDNA branches that mayarise, wild-type cells are able to maintain the advantagesconferred.

However, we cannot exclude other interpretations ofthe data presented, especially because RecG, PriA,SbcCD, and ExoVII act on a variety of substratesin vitro. Thus, cells lacking multiple exonucleases mayaccumulate so much ssDNA that they simply die whenDNA macromolecular metabolism is further compro-mised by the elimination of RecG. We did not test theviability of DrecG cells lacking every possible combina-tion of ssDNA exonucleases to eliminate this possibility.Furthermore, the RecBCD enzyme has a potent ssDNAexonuclease activity that might be able to eliminate a 39

flap. Thus, the lesion responsible for the inviability ofexonuclease-depleted DrecG cells, rather than being a 39

ssDNA flap generated during unscheduled fork colli-sions as we suggest, may be some downstream conse-quence of replication fork blockage resulting from aninability to process DNA strands by nuclease digestion.RecG itself has been shown to drive replication forkreversal in vitro, a key feature of models for promotingreplication restart in both bacteria and eukaryotes thatincorporate the need to process the nascent DNAstrands (Seigneur et al. 1998; McGlynn and Lloyd

2000; Gari et al. 2008; Sun et al. 2008). However,although RecG might be targeted to replication forksvia its interaction with SSB (Lecointe et al. 2007), directevidence that it promotes replication fork reversal in vivois distinctly lacking. Furthermore, it exhibits a strongpreference in vitro for unwinding forks that mimica 39 flap structure of the type depicted in Figure 1(McGlynn and Lloyd 2001).

We thank Carol Buckman and Lynda Harris for excellent technicalhelp and colleagues identified in Table S1 for plasmids and strains.This work was supported by a program grant to R.G.L. from the UKMedical Research Council and by an early career fellowship to C.J.R.from the Leverhulme Trust.

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Communicating editor: G. R. Smith


GENETICSSupporting Information


RecG Protein and Single-Strand DNA Exonucleases Avoid Cell LethalityAssociated With PriA Helicase Activity in Escherichia coli

Christian J. Rudolph, Akeel A. Mahdi, Amy L. Upton and Robert G. Lloyd

Copyright � 2010 by the Genetics Society of AmericaDOI: 10.1534/genetics.110.120691

C. J. Rudolph et al. 2 SI

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

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


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

xseA::dhfr

N6949 P1.AM1874 to Tmr

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 sbcC201 exoX1::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

xseA::dhfr

N7008 x pAM401 to Apr

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)

xonA::apra

N6329 x P1.JIG486 to Aprar

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

sbcCD::kan

N7037 pAM401 to Apr

N7043 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat rus-2 (orf-56::IS10) N7031 x P1.AM1874 to Tmr


6 SI

xonA::apra xseA::dhfr

N7045 pAM390 (lac+ ruvABC+) / lacIZYA ruvABC::cat rus-2 (orf-56::IS10)

xonA::apra xseA::dhfr sbcCD::kan

N7043 x P1.N6978 to Kmr

N7050 lacIZYA xonA::apra xseA::dhfr sbcCD::kan rpoC[R919H] Selection on N7037 for resistance to 2-

AP


sbcCD::kan recJ284::Tn10

N7042 x P1. N4934 to Tcr

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


sbcCD::kan ruvABC::cat

N7042 x P1.N4884 Cmr

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

sbcCD::kan

Plasmid free derivative of N7045

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

recJ284::Tn10

N7266 P1.N4934 to Tcr

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

recJ284::Tn10

N7268 P1.N4934 to Tcr

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

recJ284::Tn10

N7274 P1.N4934 to Tcr

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

recJ284::Tn10

N7284 P1.N4934 to Tcr


sbcCD::kan

N7284 P1.N6978 to Kmr

N7291 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xseA::dhfr sbcCD::kan

recJ284::Tn10

N7286 P1.N4934 to Tcr

N7292 pJJ100 (lac+ recG+) / lacIZYA recG265::cat xonA::apra sbcCD::kan

recJ284::Tn10

N7281 P1.N4934 to Tcr


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

xseA::dhfr recJ284::Tn10

N6975 x P1.N4934 to Tcr

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

sbcCD::kan recJ284::Tn10


N7316 pAM401 (lac+ sbcCD+) / lacIZYA recG265::cat xonA::apra

sbcCD::kan recJ284::Tn10 xseA::dhfr

N7314 x P1.AM1874 to Tmr


recJ284::Tn10

Plasmid free derivative of N7289 (56/2)


sbcCD::kan recA269::Tn10

N7042 x P1.N3072 to Tcr

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

xseA::dhfr

N7325 pAM409 to Apr


xseA::dhfr recG263::kan

N7337 N3793 to Kmr (Apr)


sbcCD::kan mutS::spc/str

N7290 P1.E15 to Spcr


sbcCD::kan ruvABC::cat mutS::spc

N7060 x P1.E15 to Spcr

N7382 lacIZYA sbcCD::spc TB28 P1.AM1994 to Spcr


xseA::dhfr recG263::kan

N7338 x P1.AM1986 to Spcr

N7417 lacIZYA sbcCD::spc xonA::apra N7382 P1.JIG486 to Aprar

N7418 lacIZYA sbcCD::spc xseA::dhfr N7382 P1.AM1874 to Tmr


8 SI

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

recJ284::Tn10

N7424 pAM490 to Apr

N7452 pAM490 (lac+ rnhA+) / lacIZYA xonA::apra xseA::dhfr rnhA::cat

sbcCD::kan

N7441 x P1.N6978 to Kmr

N7457 pAM490 (lac+ rnhA+) / lacIZYA sbcCD::spc xonA::apra

recJ284::Tn10

N7438 pAM490 to Apr

N7458 pAM490 (lac+ rnhA+) / lacIZYA sbcCD::spc xseA::dhfr

recJ284::Tn10

N7439 pAM490 to Apr

N7481 pAM490 (lac+ rnhA+) / lacIZYA xonA::apra xseA::dhfr

recJ284::Tn10 rnhA::cat

N7447 P1.N4704 to Cmr (Apr)

N7482 pAM490 (lac+ rnhA+) / lacIZYA sbcCD::spc xonA::apra


N7457 P1.N4704 to Cmr (Apr)

N7483 pAM490 (lac+ rnhA+) / lacIZYA sbcCD::spc xseA::dhfr


N7458 P1.N4704 to Tcr (Apr)

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

recG263::kan

N6628 P1.N3793 to Kmr (Apras)


recG263::kan

N7504 P1.N3793 to Kmr

N7518 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xseA::dhfr

recG263::kan

N7505 P1.N3793 to Kmr

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

recG263::kan

N7561 x P1.N3793 to Kmr

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

rnhA::cat

N7576 P1.N4704 to Cmr

N7591 pAM490 (lac+ rnhA+) / lacIZYA priA300 xonA::apra xseA::dhfr

rnhA::cat sbcCD::kan

N7584 P1.N6978 to Kmr


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

sbcCD::kan

N7611 x P1.N6978 to Kmr


sbcCD::kan recA269::Tn10

N7615 x P1.N3072 to Tcr


sbcCD::kan recG265::cat

N7615 x P1.N4560 to Cmr


sbcCD::kan ruvABC::cat

N7615 x P1.N4884 to Cmr

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


sbcCD::kan recG265::cat recA269::Tn10

N7625 x P1.N3072 to Tcr


sbcCD::kan tna::Tn10 recF143

N7042 x P1.JC12334 to Tcr

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

xonA::apra


N7687 lacIZYA priA300 xonA::apra xseA::dhfr sbcCD::spc N7628 x P1.N7382 to Spcr


xonA::apra recQ::kan

N7684 x P1.N5602 to Kmr


xonA::apra recJ284::Tn10

N7684 x P1.N4934 to Tcr


recG263::kan sbcCD::spc

N7517 x P1.N7382 to Spcr

N7695 pAM409 (lac+ recG+ ruvABC+) / lacIZYA ruvABC::cat xseA::dhfr

recG263::kan sbcCD::spc

N7518 x P1.N7382 to Spcr


xonA::apra mutS::kan



xonA::apra recJ284::Tn10 mutS::kan

N7692 x P1.AM2014 to Kmr

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

mutS::kan

Plasmid free derivative of N7699

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

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+


xonA::apra yijC::kan rpoC[R919H] recA::Tn10

N7715 x P1.N3072 to Tcr


xonA::apra yijC::kan rpoC[R919H] recJ284::Tn10

N7715 x P1.N4934 to Tcr


xonA::apra yijC::kan rpoC[R919H] recG265::cat

N7715 x P1.N4560 to Cmr


xonA::apra yijC::kan rpoC[R919H] ruvABC::cat

N7715 x P1.N4884 to Cmr

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)

xonA::apra xseA::dhfr sbcCD::kan


N7783c lacIZYA sbcCD::spc xseA::dhfr xonA::apra recB268::Tn10 N7684 x P1.TRM308 to Tcr


xonA::apra helD::cat

N7684 x P1.SWM101 to Cmr

N7789 lacIZYA sbcCD::spc xseA::dhfr xonA::apra recB268::Tn10 N7427 x P1.TRM308 to Tcr


xonA::apra malE::Tn10 lexA3

N7684 x P1.N5123 to Tcr


xonA::apra malE::Tn10 lexA3 recG263::kan

N7794 x P1.N3793 to Kmr

N7806 pAM401 (lac+ sbcCD+) / lacIZYA sbcCD::spc xseA758::kan

xonA::apra

N7684 x P1.BW25113 to Kmr (Tms)


xonA::apra dinG::kan

N7684 x P1.HB169 to Kmr

N7816 lacIZYA xseA::dhfr TB28 x P1.AM1874 to Tmr


xonA::apra rep::cat

N7691 x P1.JJC735 to Cmr (Kms, recQ+)


xonA::apra recG265::cat

N7806 x P1.N4560 to Cmr


xonA::apra recA::cat

N7684 x P1.AM2037 to Cmr

N7837 lacIZYA sbcCD::spc xseA::dhfr xonA::apra

sulA::MudAplacMuB::Tn9

N7427 x P1.JJC1382 to Cmr (Apr)

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


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)


xonA::apra uvrD::cat



xonA::apra mutS::kan uvrD::cat



xonA::apra mutS::kan rep::cat

N7696 x P1.JJC735 to Cmr

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)


xonA::apra yijC::kan rpoC[R919H] rep::cat

N7715 x P1.JJC735 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]

sulA::MudAplacMuB::Tn9

N7861 x P1.JJC1382 to Cmr (Apr)

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

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.

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RecG Protein and Single-Strand DNA Exonucleases Avoid Cell ...pAM401 (sbcCD1) and pAM490 (rnhA1) are...

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