Bacillus subtilis RecA and its accessory factorsRecF RecO RecR and RecX are required for sporeresistance to DNA double-strand breakIgnacija Vlasic12 Ramona Mertens1 Elena M Seco3 Begona Carrasco3 Silvia Ayora3

Gunther Reitz1 Fabian M Commichau4 Juan C Alonso3 and Ralf Moeller1

1Radiation Biology Department German Aerospace Center Institute of Aerospace Medicine Linder HoheD-51147 Cologne (Koln) Germany 2Division of Molecular Biology Laboratory of Evolutionary Genetics RuderBoskovic Institute Bijenicka cesta 54 10000 Zagreb Croatia 3Department of Microbial Biotechnology CentroNacional de Biotecnologıa CSIC Darwin 3 28049 Madrid Spain and 4Department of General MicrobiologyUniversity of Gottingen Grisebachstrasse 8 D-37077 Gottingen Germany

Received September 10 2013 Revised October 22 2013 Accepted November 1 2013

ABSTRACT

Bacillus subtilis RecA is important for spore resist-ance to DNA damage even though spores contain asingle non-replicating genome We report that inacti-vation of RecA or its accessory factors RecF RecORecR and RecX drastically reduce survival of maturedormant spores to ultrahigh vacuum desiccation andionizing radiation that induce single strand (ss) DNAnicks and double-strand breaks (DSBs) The presenceof non-cleavable LexA renders spores less sensitiveto DSBs and spores impaired in DSB recognition orend-processing show sensitivities to X-rays similar towild-type In vitro RecA cannot compete with SsbAfor nucleation onto ssDNA in the presence of ATPRecO is sufficient at least in vitro to overcomeSsbA inhibition and stimulate RecA polymerizationon SsbA-coated ssDNA In the presence of SsbARecA slightly affects DNA replication in vitro butaddition of RecO facilitates RecA-mediated inhibitionof DNA synthesis We propose that repairing of theDNA lesions generates a replication stress togerminating spores and the RecAmiddotssDNA filamentmight act by preventing potentially dangerous formsof DNA repair occurring during replication RecAmight stabilize a stalled fork or prevent or promotedissolution of reversed forks rather than its cleavagethat should require end-processing

INTRODUCTION

A DNA double-strand break (DSB) represents a poten-tially lethal form of DNA damage in all living organisms

(12) These lesions which mainly occur as by-products ofnormal cell metabolism can arise from external sourcessuch as exposure to ionizing radiation (IR) and chemicalmutagens (13) Cells rely on two major DSB repairpathways homologous recombination (HR) and non-homologous end joining (NHEJ) Many cells can alsouse a minor pathway known as single-strand DNA an-nealing to repair two-ended DSBs (24ndash8) DSB repairvia error-free HR requires many steps including but notlimited to DSB recognition long-range end-processingand the presence of an intact homologous template Ineukaryotes error-free HR is constrained to the S and G2phases of the cell cycle and in bacteria occurs during vege-tative growth when replication is active and the homolo-gous sister chromatid (chromosome) is present (46ndash12)In contrast by NHEJ two-ended DSBs can be joinedwith minimal end-processing NHEJ is the predominantform of DSB repair during G1 in eukaryotes or duringgrowth phases when bacteria have a single genomic copy(5913ndash15) When classical NHEJ is impeded single-strand DNA annealing which relies on redundant 30-or50-end-processing functions and on terminal tracts withdiscrete homology can anneal the DNA ends followedby ligation (6ndash8111415) Thus depending on theorganism and growth environment cells have severalstrategies to repair DSBs that have occurredIn certain bacteria including Bacillus subtilis nutritional

starvation triggers the formation of two types of cells alarger mother cell and smaller prespore or forespore eachof which contains a single genome equivalent (reviewed in16) During spore development asymmetric cell divisionis followed by different morphological events and eventu-ally liberation of a dormant spore that has only one non-replicating genome copy (1718) Bacillus subtilis dormantspores protect their chromosome from DNA damage due

To whom correspondence should be addressed Tel +34 91585 4546 Fax +34 91585 4506 Email jcalonsocnbcsicesCorrespondence may also be addressed to Ralf Moeller Tel +49 2203 601 3145 Fax +49 2203 61970 Email ralfmoellerdlrde

Published online 26 November 2013 Nucleic Acids Research 2014 Vol 42 No 4 2295ndash2307doi101093nargkt1194

The Author(s) 2013 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc30) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly cited For commercialre-use please contact journalspermissionsoupcom

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to its specific structure (1920) and process DNA damageunder unfavourable metabolic conditions (2122) Themechanisms used for dormant B subtilis spores toremove DSBs generated by IR (eg X-rays) or single-stranded nicks generated by ultrahigh vacuum (UHV) des-iccation upon spore germination are poorly understood(1923ndash27) In the presence of two-ended DSBs thespore-encoded NHEJ system reconnects the brokenends and it depends on two proteins YkoV (alsotermed Ku) which binds to dsDNA ends and YkoU(LigD) a protein that processes and ligates the dsDNAends (212728) Dormant spores lacking RecA are sensi-tive to DNA DSBs (272930) This result is interestingbecause spores should have one genomic DNA copyprecluding a role for RecA in HR Furthermore sporesdefective in NHEJ and lacking RecA are more sensitive toDNA DSBs than null recA (recA) spores (27283132)Therefore the role of RecA in spore resistance to DSBs isunclearIn bacteria RecA is the central player in HR (101233)

RecA with the help of accessory proteins (eg RecFRecO RecR and RecX) nucleates and polymerizes onthe single stranded (ss) DNA coated by a single-strandedbinding protein (SSBSsbA) forming a RecAmiddotssDNA nu-cleoprotein filament (NPF) (34ndash38) Up to now five func-tions were reported for Escherichia coli RecAmiddotssDNANPF First it catalyses DNA strand exchange reactionin the presence of an intact homologous template (10)Second it promotes induction of the SOS response bypromotion of the autocatalytic cleavage of the LexA re-pressor (3940) Third RecA (41ndash43) can displace the rep-licase (DNA polymerase III) when lesions or obstacles areencountered (44) Fourth RecA mediates activation ofUmuD0 by mediating autocatalytic cleavage of UmuD(45) Fifth the RecAmiddotssDNA NPF participates in SOSmutagenesis by activating Pol V (4546) In B subtilisonly two of these five RecA functions have been docu-mented (947) Here the RecAmiddotssDNA NPF formedwith the help of mediators (eg RecF RecO RecR)and modulators (eg RecF RecX etc) is involved inhomology search and DNA strand exchange in thepresence of an intact homologous DNA molecule duringrecombinational repair and it also plays an important rolein the SOS response (4849) Among the SOS-regulatedgenes no homologue of E coli UmuD (UmuDEco) hasbeen detected in the B subtilis genome therefore the ex-istence of a UmuD-like function in this bacterium is stillan open question (50) and the role of RecAmiddotATPmiddotMg2+

in the generation of an active mutasomal complex remainsto be documented (45)We aimed to investigate how RecA contributes to spore

survival after DSBs or nicks generated by X-rays or UHVtreatment respectively in the presence of one genomecopy We report that in the absence of DSB recognition(recN spores) or end-processing (addAB recQ recS recJor addA recJ mutant spores) germinated spores are as sen-sitive to X-ray treatment as the wild-type (wt) strain Theabsence of RecA or its accessory factors (RecF RecORecR RecX) render germinated spores extremely sensitiveto X-rays and UHV treatment suggesting that DNArepair in the spore requires a RecAmiddotssDNA NPF rather

than DSB repair via DNA strand exchange Sporesimpeded in SOS induction were sensitive to X-rays andUHV treatments suggesting that increased levels ofRecA or of an unknown modulator (eg PcrA) mightbe also involved in spore survival In the presence ofATP RecA cannot compete with SsbA for ssDNAbinding and slightly inhibits PolC-DnaE-promotedDNA synthesis SsbA blocks RecA polymerization onssDNA and in vitro RecO is sufficient to overcome theSsbA barrier RecA can nucleate and filament on theRecOmiddotssDNAmiddotSsbA complexes and it can inhibitleading and lagging strand DNA synthesis in thepresence of RecO Our results establish an intimateconnection between resistance of dormant sporesRecAmiddotssDNA NPF formation on SsbA-coated ssDNAtracts and the inhibition of DNA synthesis during sporegermination We propose that a RecAmiddotssDNA NPFshould work as a replicase auxiliary protein and thatthe RecAmiddotssDNA NPF might act by preventing poten-tially dangerous forms of DNA repair occurring duringa replication stress RecA by inhibition of DNA replica-tion might stabilize a stalled fork or prevent or promotedissolution of a reversed fork rather than its cleavage thatshould require end-processing

MATERIALS AND METHODS

Bacterial strains and spore preparation

All bacterial strains used in this study are derivatives ofBG214 or 168 strains and are listed in SupplementaryTable S1 Bacillus subtilis strains were grown overnightto stationary phase in the NB medium supplementedwith appropriate antibiotic For spore preparation200 ml of overnight cultures were plated evenly onSchaefferrsquos sporulation medium (SSM) plates (51) andplates were stored at 37C for 7 days The fully formedspores were harvested and purified by adjusted protocol(52) The purified spores were resuspended in 5ml ofdesterilized water and stored until final usage at 4C

Spore X-ray irradiation

The 200 ml of purified spores were transferred to PCRtubes and irradiated at room temperature (RT) withX-rays (200 kV15mA) generated by an X-ray tube(Gulmay Xstrahl RS225 A Gulmay Medical Inc GAUSA) The appropriate dilutions of treated and untreatedspore samples were plated on NB agar plates in order tomeasure spore survival by counting colony-forming units(CFUs) The survival curves were obtained by plotting thelogarithm of survival percentage versus applied X-ray ra-diation dose Each X-ray irradiation experiment wasrepeated at least three times and the data presented areexpressed as average values with standard deviationsAdditionally spore resistance after X-ray radiation wasexpressed as D10-value which represents the X-ray dosethat reduces spore survival to 10 The D10-values oftreated spores were compared statistically usingStudentrsquos t-test and differences with P-values of 005were considered statistically significant (3253)

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Spores UHV desiccation

Spore samples consisted of air-dried spore monolayersimmobilized on 7-mm quartz discs were exposed 7 daysto UHV produced by an ion-getter pumping system(400 ls Varian SpA Torino Italy) reaching a finalpressure of 3 106Pa (325455) The spores immobilizedon quartz discs were recovered by 10 aqueous polyvinylalcohol solution as described previously (3253) The ap-propriate dilutions of treated and untreated spore sampleswere plated on NB agar plates in order to count CFUs asa measure of spore survival The CFUs of untreated sporesamples were represented as 100 survival The UHVexperiment was performed in triplicate The CFUs ofUHV-treated spores were divided with the averageCFU-value of untreated spore samples in order toobtain the survival after UHV The data presented areexpressed as average values with standard deviationsThe percentage of survivals of treated spores wascompared statistically using Studentrsquos t-test and differ-ences with P-values of 005 were considered statisticallysignificant (32)

RecA nucleation and filament formation

A continuous ATP hydrolysis assay was used to analysethe extent of RecA nucleation and filament formationon ssDNA (56) SsbA RecO and RecA proteins andthe pGEM3-Zf(+) ssDNA substrate were purified asdescribed (5758) RecA protein concentration is expressedas moles of protein as monomers RecO as dimers andSsbA as tetramers (56) The rate of ssDNA-dependentRecA-mediated ATP hydrolysis and the nucleation lagtime were measured in buffer A (50mM TrisndashHCl [pH75] 1mM DTT 90mM NaCl 10mM MgOAc [magne-sium acetate] 50 mgml BSA 5 glycerol) containing5mM ATP for 25min at 37C as described (56) The nu-cleation time was deduced from the time-intercept of alinear regression of the steady state portion of data inATP hydrolysis assays as reported (5659)

SsbA and RecO (1 SsbA33 nt and 1 RecO50 nt)proteins did not exhibit ATP hydrolysis activity whencompared to the mock reaction in the absence of bothproteins or when BSA was added instead of bothproteins (data not shown) The orders of addition of3199-nt pGEM3-Zf(+) ssDNA (10 mM innt) the purifiedproteins (RecA SsbA and RecO) and their concentrationsare indicated in the text

DNA synthesis assay

A B subtilis replisome was reconstituted in vitro withpurified SsbA DnaG DnaC PriA DnaD DnaB DnaIPolC DnaE t d d0 and b subunits of the replicase and themini-circular DNA template as described (60) PolCDnaE DnaG PriA d and d0 are expressed as moles ofprotein monomers b as dimers t DnaB and DnaD astetramers and DnaC and DnaI as hexamers (60)Reaction conditions for DNA synthesis assays were15 nM DnaE 20 nM PolC 8 nM DnaG 25 nM t 25 nMd 25 nM d0 24 nM b 30 nM DnaC 15 nM PriA 50 nMDnaD 100 nM DnaB 40 nM DnaI 5 nM mini-circular

DNA template in molecules 350 mM ATP 100 mM CTPGTP and UTP 48 mM dNTPs (excepts 18 mM dCTP ordGTP for the leading- and lagging-strand DNA synthesisrespectively) and 15 mCireaction [a-32P]dCTP or[a-32P]dGTP all in BsRC buffer (60) The DNAtemplate was a 409-bp circle containing a 396-nt tail pre-viously described (60) that it has a strong (501) GC strandbias so labelled dCTP was incorporated mostly into thenascent leading strand and labelled dGTP into the laggingstrand (60)An enzyme mix containing all protein components

except SsbA RecA RecO was prepared in buffer BsRCat 4C The substrate mix contained the DNA substrateand 90 nM SsbA 1 mM RecA 01 mM RecO as indicatedand prepared on ice The enzyme mix was added to thesubstrate mix and reactions were then pre-incubated for5min at 37C in presence of 5 mM ATPgS and rNTPsexcept ATP The reactions were initiated by the additionof dNTPs and ATP and aliquots were withdrawn at theindicated times and incubated for 20min with an equalvolume of a stop mix consisting of 40mM TrisndashHCl(pH80) 02 wv SDS 100mM EDTA and 50 mgmlproteinase K Samples were applied onto Sephadex G-50columns to eliminate non-incorporated dNTPs Theextent of DNA synthesis in leading- and lagging-strandswas then quantified by scintillation counting as described(61) For the analysis of the size of replication productssamples were brought to 50mM NaOH 5 vv glyceroland 005 bromphenol blue and fractionated in alkaline045 agarose gels for 5 h at 60V as described (60)Alkaline agarose gel buffer consisted of 30mM NaOHand 05mM EDTA Gels were fixed in 7 (wv) trichloro-acetic acid dried autoradiographed on storage phosphorscreens and analysed with Quantity One (Bio-Rad)software

RESULTS AND DISCUSSION

Experimental system

RecA was found to be important for processing the DNAdamage of fully formed B subtilis spores upon germin-ation (2732) Spores contain a single non-replicatinggenome (1718) and recombinational repair requires anintact homologous template (6ndash947) suggesting afunction for RecA other than recombinational repairTo investigate the importance of HR functions in sporesurvival we have used fully formed spores with inactivatedrecombination gene products involved in DSB recognition(recN) long-range end-processing (addA5 addA5addB72 recJ recQ recS and addA5 recJ) RecAaccessory factors that act before (recO recR andrecF15) or during homology search (recX recF15 andrecX recF15) and the recombinase itself (recA) Inaddition we analysed the role of the SOS induction inspore survival (lexA (Ind-) (Supplementary Table S1)As a control of genuine two-ended DSB repair wehave inactivated NHEJ (ykoV also termed ku)(Supplementary Table S1) The spores were subjected toX-ray radiation which induces two-ended DSBs andUHV treatment which induces single-strand nicks that

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were then converted into one-ended DSBs during sporegermination

Spore resistance after X-ray radiation and UHVtreatment depends on RecA

X-ray radiation generates two-ended DSBs that shouldbe bound by YkoV (Ku) and should be processedand ligated by the YkoU (LigD) enzyme (2728) Wehave previously reported that the ykoV single or ykoVykoU double mutation render spores sensitive to DSB in-duction by X-ray radiation (32536263) As revealed inSupplementary Figure S1A the ykoV single mutationrender spores sensitive to DSB induction by IR TheykoV mutant spores however showed similar survivalas wt spores after UHV exposure for 7 days(Supplementary Figure S1B Table 1) It is likely thatthe majority of the DSBs formed upon UHV treatmentfor 7 days and detected upon germination were ssDNAnicks that cannot be repaired by NHEJRecA is a non-essential product but examination of

recA mutants revealed extensive chromosomal fragmenta-tion even in the absence of exogenous stress (64)Furthermore exponentially growing recA cells accumulateRecN-YFP foci (a sensor of DSBs) in 30 of total cellswhereas RecN-YFP foci accumulation is rare in wt cells(lt05) (65) and recA colonies have 5-fold less cellsthan wt colonies (6667) It is likely that RecA isrequired for genomic stability and in its absence the rep-lication fork might be more susceptible to breakageInactivation of RecA which does not block spore forma-tion drastically sensitized dormant spores to X-ray

radiation and UHV treatment (SupplementaryFigure S1 Table 1) Supplementary Figure S1A showsthat the recA mutant spores had considerable lowersurvival after X-ray radiation relative to wt spores Incomparison to the wt spores the recA mutant sporesshowed a significant decrease in the D10-value (2-fold)(Table 1) The experiments were performed in the BG190genetic background that also lacks prophages aconjugative transposon and it is impaired in the generalstress response Similar results were observed when theWN463 strain which contains prophages a conjugativetransposon and it is proficient in the general stressresponse was tested (Table 1) Supplementary FigureS1A shows that the recA mutant spores had consider-able lower survival after X-ray radiation relative to wtspores We have previously reported that YkoV andRecA contribute additively to spore resistance afterX-ray radiation or high vacuum treatment (6263)We corroborated the additive effect of these two muta-tions after X-ray radiation (Supplementary Figure S1ATable 1)

When spores were exposed to UHV we observed thatthe recA mutant spores showed significant decrease(3-fold) in survival when compared to wt spores(Table 1 Supplementary Figure S1B) From obtainedresults we concluded that recA gene product is importantfor spore resistance to X-ray radiation and UHVtreatment

This result is in agreement with previous studiesshowing that NHEJ is an important DNA repair mechan-ism for spore resistance after X-ray (induction of

Table 1 Survival characteristics of B subtilis spores after X-ray radiation and UHV

Genotype Dose (Gy) to D10a D10

REMD10wt Surv UHVb SREMSwt

BG214 genetic backgroundwt 10622plusmn2050 1 802plusmn34 1recA 4822plusmn480 05 289plusmn48 04ykoV 5717plusmn171 05 889plusmn176 11ykoV recA 2307plusmn1029 02 ND NDrecN 10069plusmn829 09 835plusmn188 10addA5 addB72 10775plusmn3029 10 548plusmn168 07addA5 13038plusmn854 12 564plusmn285 07recJ 11529plusmn1233 11 1004plusmn105 13addA5 recJ 8506plusmn1717 08 396plusmn87 05recQ 11889plusmn1048 11 781plusmn120 10recS 9680plusmn1322 09 832plusmn48 10recF15 4790plusmn656 05 0003plusmn0005 000004recO 3847plusmn503 04 372plusmn49 05recR 3216plusmn776 03 209plusmn46 03recX 10564plusmn2391 10 191plusmn125 02recX recF15 5234plusmn414 05 504plusmn224 06168 genetic backgroundwt 15255plusmn6710 1 960plusmn75 1recA 3914plusmn193 03 92plusmn34 01wtc 19215plusmn5411 13 1003plusmn212 10lexA (Ind-)c 5084plusmn181 03 502plusmn94 05

Data are averages of at least three independent experiments with standard deviations Asterisks indicate D10 or UHV survival values that weresignificantly different (P-values 005) from values for wt spores (BG214168)aThe dose that it reduced the survival of the given spore population to 10 is indicated (D10)bSurvival () after 7 days of UHV exposure with final pressure 3 10ndash6PacBoth strains carry upp mutation (Supplementary Table S1)REM repair mutant spore ND not determined

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two-ended DSBs) However NHEJ plays a minor rolein the repair of spore after exposure to UHV for 7 daystreatment (induction of nicks) (SupplementaryFigure S1B Table 1) (213253)

Spore resistance after X-ray radiation and UHVtreatment is independent of RecN

In response to replication fork collapse in exponentiallygrowing cells a complex molecular machinery involved inDNA repair is recruited and cell proliferation is arrested(948) One of the first responders to the formed DSBs isthe RecN protein RecN which assembles on one- or two-ended DSBs choreographs the DSB response (656869)Damage-inducible RecN in concert with polynucleotidephosphorylase degrades the 30-ends and specificallyremoves the 30 dirty ends and generates the substratefor long-range end-processing (7071)

To determine whether RecN is necessary for sporeresistance we exposed dormant recN mutant spores toX-ray radiation and UHV (Table 1 Figure 1A and B)The survival pattern after X-ray radiation of recNmutant and wt spores was similar (Figure 1A Table 1)Similar results were obtained after UHV treatment(Table 1 Figure 1B) These indicate that inactivation ofRecN does not influence spore survival to X-ray radiationor UHV treatment

Inactivation of end-resection does not influence sporeresistance after DSBs

During exponential growth after generation of one- ortwo-ended DSBs basal and long-range end-processingsteps are required for accurate DNA repair (948)Long-range end-resection involves redundant pathwaysconsisting of nuclease(s) DNA helicase(s) and a single-stranded binding protein (94748) The AddAB helicasendashnuclease complex (functional analogue of E coli RecBCDand of mycobacterial AdnAB) in concert with SsbA cata-lyses the formation of the 30-tailed ssDNA (7273) TheRecA loading into the generated ssDNA via AddABremains to be documented (72) The second long-rangeprocessing pathway includes the RecJ nuclease a RecQ-like helicase (RecQ or RecS) and SsbA (47ndash49)Furthermore the addA5 recJ double mutant strain isas sensitive to DNA damaging agents as exponentiallygrowing recA cells (69)

To determine whether spore resistance after X-ray radi-ation and UHV treatment depends on the proteinsinvolved in DSB end-processing we exposed addA5addA5 addB72 recQ recS recJ or addA5 recJmutant spores to X-ray radiation and UHV (Table 1Figure 1C and D) Figure 1C shows X-ray survival ofspores with inactivated helicase functions (addA5 addA5addB72 recQ and recS mutant spores) and nucleasefunctions (addA5 addA5 addB72 recJ and addA5 recJmutant spores) Survival capability of addA5 addA5addB72 recQ recS recJ or addA5 recJ mutantspores after X-ray radiation was comparable to wtspores having similar survival patterns and D10-values(Table 1 Figure 1C) Survival levels of recQ recSand recJ mutant spores obtained after UHV treatment

were similar to wt survival levels (Table 1 Figure 1D) Onthe other hand the introduction of addA5 addA5 addB72and addA5 recJ mutations showed a moderate effectimplying a direct or indirect involvement of AddAB inspore resistance to UHV treatment (Table 1 Figure 1D)The main conclusions from the presented results are

that inactivation of functions involved in one (AddABRecQ RecS or RecJ) or both (AddA and RecJ) end-re-section pathways did not influence the DSB repair capabil-ity of spores exposed to X-ray radiation when comparedto wt spores (Table 1 Figure 1C) Finally single-strandDNA annealing which relies on redundant end-processingfunctions and on terminal tracts with discrete homology(67111415) can be a very minor pathway if at allbecause absence of AddA and RecJ does not significantlyimpairs spore resistance to X-rays treatment (Figure 1C)The role of the AddAB complex in the survival of mutantspores upon UHV is poorly characterized It is stillunknown whether AddAB possesses a RecA loadingfunction as its E coli counterpart (RecBCD) (72)

Spore survival depends on RecF RecO RecR and RecX

In exponentially growing cells concomitantly with long-range end-processing RecN promotes tethering of DNAends to form a repair centre (RC) (69) RecO RecR andRecA are recruited to RecN-mediated RC and the SOSresponse is induced (949) Then RecA forms highlydynamic filamentous structures termed threads acrossthe nucleoid and RecF forms a discrete focus in thenucleoid which are coincident in time with RecX fociin response to DNA damage RecN co-localizes with theRecO RecR RecA and RecF focus (656874) DNAdamage-induced RecX foci co-localize with RecAthreads that persisted for a longer time in the recXcontext (6874) suggesting that RecO RecR and RecFaccessory proteins contribute to RecA nucleation ontoSsbA-coated ssDNA and that RecF and RecX contributeto RecAmiddotssDNA NPF formation (6574)To investigate whether inactivation of RecA accessory

proteins influence spore survival after DSBs the recF15recO recR and recX mutant spores were exposed toX-ray radiation and UHV treatment (Table 1 Figure 2AndashD) Inactivation of RecO RecR or RecF rendereddormant spores equally extremely sensitive to X-ray radi-ation with survival levels similar to recA mutant spores(Figure 2A) UHV treatment produced a drastic reductionof survival of recF15 mutant spores whereas the recO orrecR mutant spores showed survival level similar to therecA mutant spores (Table 1 Figure 2B) Since dormantspores have a single none replicating genome (17) andend-processing functions are not involved in spore resist-ance to DSBs (Figure 1) we have to assume that the majorrole of RecF RecO and RecR is to promote RecA nucle-ation onto SsbA-coated ssDNA and subsequent filamentgrowth during early steps of germination AlternativelyRecF RecO and RecR should protect the nascent DNAfrom degradation during spore germination but absenceof the degrading function (eg absence of RecJ andAddA) showed no phenotype (75) We favour the hypoth-esis that RecA loading on ssDNA (by the help of RecOR

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or RecFOR complexes) and filament growth (RecF) areessential for spore survival after X-ray radiation duringspore germination and outgrowthRecent in vivo data showed that RecX facilitates

whereas RecF delays RecA-mediated LexA self-cleavagewith subsequent induction of the SOS response and in theabsence of both proteins (ie in recF15 recX cells) SOSinduction is wt-like but the double mutant strain isblocked in DNA repair suggesting that RecF and RecXhave counteracting functions (74) Similarly RecXEco dis-assembles the RecAmiddotssDNA NPF and RecF stabilizes itsassembly or prevents its disassembly in vitro (76) TherecF15 or recX mutant spores were more sensitive toUHV treatment than recA spores (Figure 2B and DTable 1)To test whether the dynamic assembly and subsequent

disassembly of RecA filaments contributed to sporesurvival the sensitivity of the recX recF15 mutantspores was measured (Figure 2C and D) The absence ofRecX did not sensitized spores to X-ray radiation when

compared to wt spores The recXmutation partially sup-pressed the sensitivity to X-ray or UHV in the recXrecF15 context (Figure 2C and D)

Spore resistance after X-ray radiation and UHVtreatment depends on the SOS response

In exponentially growing cells mutation of recF recO orrecR shows a drastically reduction in the DNA repaircapacity (77ndash79) and results in a delay and reduction ofSOS induction (80) However these phenotypes are sup-pressed by specific mutations in the recA gene or by ex-pressing heterologous SSB proteins in the background(81) Under similar conditions the absence of RecXreduces DNA repair but facilitates SOS induction (74)The absence of both RecF and RecX renders exponen-tially growing cells extremely sensitive to DNAdamaging agents but the SOS induction is similar to wtcells (74) The results obtained in the previous section sug-gested that the role of RecA in spore survival is not onlythe induction of SOS response because the double recF15

Figure 1 Survival of B subtilis spores deficient in DSB recognition and end-processing after X-ray and UHV treatment (A) X-ray dose-dependentsurvival of wt (filled square) recA (filled triangle) and recN mutant (filled diamond) spores The wt and recA mutant spores were used ascontrols (B) Viability (white column) and survival (grey column) after 7 days UHV exposure of wt recA mutant and recN mutant spores (C) X-ray dose-dependent survival of wt (filled square) recA (filled triangle) addA5 (open diamond) addA5 addB72 (cross) recJ (filled diamond)recQ (filled circle) recS (asterisk) or addA5 recJ (open circle) spores (D) Untreated (white column) and treated (grey column) after 7 days UHVexposure of wt recA addA5 addA5 addB72 recJ recQ recS or addA5 recJ mutant spores

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recX mutant has normal levels of SOS induction but isnot spore repair proficient

Constitutive levels of RecA are sufficient to facilitate therepair of one- or two-ended DSBs via HR during vegeta-tive growth (82) However the SOS regulon was expressedin germinating spores exposed to space conditions (2983)which are known to cause DSBs in spores (26)

To test whether some SOS-regulated function is neededfor spore resistance after DSB introduction we used aspecific lexA mutant variant lexA (Ind-) resulting in anon-cleavable LexA repressor and therefore unable toinduce the SOS response (82) The lexA (Ind-) mutantspores and related wt spores carry upp mutation butthe upp mutation does not reduce spore survival afterX-ray radiation (Table 1) As revealed in Figure 3A theabsence of SOS significantly reduced X-ray survival oflexA (Ind-) mutant spores with the survival level similarto recA mutant spores when spores were treated with anX-ray dose of 500Gy With increasing dose lexA (Ind-)mutant spores turned to be less sensitive than the recA

mutant spores but still very sensitive compared to wtspores (Figure 3A) Comparable results were obtainedfor D10-values where lexA (Ind-) mutant spores showedslightly increased D10-value than the recA mutantspores but they were still significantly decreased (3-fold)compared to the D10-values of the wt spores (Table 1)Similar results were obtained after UHV treatmentwhere lexA (Ind-) mutant spores turned to be significantlysensitive than the wt spores (Figure 3B Table 1) Fromobtained results we conclude that SOS response is import-ant for spore survival after X-ray radiation and UHV It islikely that increased RecA levels might contribute to sporesurvivalAmong the SOS genes previously identified (84) only

four of them correspond to genes whose roles in error-freerecombinational repair error-prone translesion synthesisor error-prone recombinational repair (recA ruvAB pcrA[uvrDEco]) polY2 [umuC]) have been identified(5067828485) In addition we cannot exclude that in-duction of other B subtilis SOS genes with unknown

Figure 2 Survival of B subtilis spores deficient in RecA loading after X-ray and UHV treatment (A) X-ray dose-dependent survival of wt (filledsquare) recA (filled triangle) recF15 (filled diamond) recO (filled circle) or recR (asterisk) spores (B) Viability (white column) and survival(grey column) after 7 days UHV exposure of wt recA recF15 recO or recR spores (C) X-ray dose-dependent survival of wt (filled square)recA (filled triangle) recF15 (filled diamond) recX (filled circle) or recX recF15 (cross) spores (C) Untreated (white column) and treated (greycolumn) after 7 days UHV exposure of wt recA recF15 recX or recX recF15 spores

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function could be required for spore resistance to X-rayradiation or UHV treatment Since spore survival is notaffected in the addA5 recJ context and the involvementof RuvAB in spore resistance requires end-processing itsrequirement was tentatively ruled out Pol Y2 shareshomology with UmuCEco (50) and no homologue ofUmuDEco has been detected in the B subtilis genome sug-gesting that a mutasome similar to the one described inE coli does not exist in B subtilis (see Introductionsection) The contribution of PcrA cannot be analysedbecause absence of PcrA renders cells synthetically lethalunless a mutation in recF recO or recR accumulates in thebackground (86)

RecAmiddotATPmiddotMg2+ cannot nucleate and polymerize ontoSsbA-coated ssDNA in vitro

Previously it has been shown that the essential SsbA(counterpart of SSBEco) is involved in different stages ofDNA metabolism including DNA repair recombinationand replication The template for B subtilis RecAassembly is SsbA-coated ssDNA in vivo but RecA inthe ATP bound form cannot nucleate on theSsbAmiddotssDNA complexes (87) However RecA efficientlynucleates onto SsbA-coated ssDNA in the presence ofdATP (5658) The relationship between SsbA and RecAis complex SsbA (SSB) helps RecA to self-assemble ontoSsbA (SSB)-coated ssDNA by removing secondary struc-tures and competes with RecA for ssDNA binding(101233) Genetic data revealed that a recA73 mutationpartially overrides the recF recO or recR defect (81) sug-gesting that this mutation conferred RecA the ability toremove SsbA from the ssDNA To gain insight into themolecular basis of the inability of RecA to assemble ontoSsbA-coated ssDNA conditions where RecA efficientlyassembles onto SsbA-coated ssDNA in the presence ofATP were investigatedIn the absence of SsbA at a ratio of 1 RecA monomer

8 nt RecA nucleation onto ssDNA is not apparently ratelimiting and ATP hydrolysis increased with time

approaching the levels previously observed with a Kcat

of 117plusmn03min1 (Supplementary Figure S2A) (87BC T Yadav and JCA unpublished results) In thepresence of limiting SsbA (1 SsbA550 nt 18 nM)RecAmiddotATPmiddotMg2+ nucleated onto ssDNA andpolymerized on ssDNA with similar efficiency than inthe absence of SsbA (106plusmn03min1 versus117plusmn03min1) (Supplementary Figure S2A) Atincreased ratios (1 SsbA275ndash133 nt 37ndash75 nM) SsbAincreased the lag time of the reactions which correspondwith the RecA binding onto ssDNA (nucleation step) andreduced the maximal rate of ATP hydrolysis(83plusmn02min1 and 72plusmn03min1 respectively) sug-gesting that limiting SsbA partially competed withRecAmiddotATPmiddotMg2+ for binding to ssDNA(Supplementary Figure S2A) In the presence of stoichio-metric concentrations (1 SsbA 66 nt) SsbA-blockedRecA catalysed hydrolysis of ATP (14plusmn01min1)(Supplementary Figure S2A 150 nM) Similar resultswere observed when saturating SsbA (1 SsbA33 nt) con-centrations were used (BC T Yadav and JCA unpub-lished results) It is likely that both RecA and SsbA bindcompetitively to ssDNA and RecA cannot compete withSsbA bound to ssDNA as previously proposed (T YadavBC and JCA unpublished results)

RecO was necessary to overcome the inhibition of RecAassembly onto SsbA-coated ssDNA In the presence ofstoichiometric SsbA concentrations (1 SsbA66 nt) RecAcannot nucleate but addition of RecO as low as 1 RecO100 nt to the preformed SsbAmiddotssDNA complex signifi-cantly stimulated RecA nucleation and RecAmiddotssDNAfilament formation (Supplementary Figure S2B) Similarresults were observed when RecO and saturating SsbA(1 SsbA33 nt) concentrations were used (BC T Yadavand JCA unpublished results)

RecA inhibits DNA replication in the absence of SsbA

Previously it has been shown that (i) RecAEco binds andreleases a halted replicase at a replication fork stalled by

Figure 3 Survival of B subtilis spores unable to induce the SOS response (A) X-ray dose-dependent survival of wt (filled square) recA (filledtriangle) and lexA (Ind-) (filled diamond) mutant spores (B) Untreated (white column) and treated (grey column) after 7 days UHV exposure of wtrecA or lexA (Ind-) spores

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the inactivation of the replicative helicase (DnaB6)in vivo (43) Here replicase clearance is not affected bythe absence of RecFEco RecOEco or RecREco function(43) suggesting that RecA can assemble onto the SSBEco-coated lagging-strand template forming short RecA fila-ments in the absence of accessory factors in vivo(ii) RecAEco alone strongly inhibited the advance of a rep-lication fork when added prior initiation of DNA synthe-sis (42) To test whether B subtilis RecA inhibits thereplisome during DNA synthesis we have used areconstituted in vitro replication system (6061)

The Firmicutes replicase is composed of two distinctpolymerases (PolC and DnaE) the t d and d0 subunitsof the clamp loader and the sliding clamp processivityfactor b (6088) The B subtilis core replicase [PolC-t-d-d0-b] acts in concert with other nine proteins (DnaChelicase and its loading system composed by DnaBDnaD DnaI and PriA in addition to the DnaGprimase and SsbA) (60) To test whether the replisomecould directly load RecA as it proceeds to unwoundDNA or if RecA could bind to the ssDNA tracts in adirect competition with SsbA during replisome progres-sion we have measured DNA synthesis (60) in the pres-ence or absence of SsbA andor RecA proteins (Figure 4Supplementary Figure S2)

In the absence of RecA leading- and lagging-strandsynthesis was 2-fold reduced in the absence of SsbAwhen compared with the DNA synthesis observed in thepresence of SsbA (Figure 4A lanes 1ndash3 and 7ndash9 versusFigure 4C lanes 13ndash15 and 25ndash27 and SupplementaryFigure S3A versus Supplementary Figure S3B) (61) Thisis consistent with the observation that in the DNA sub-strate used the DNA helicase can self-assemble bythreading over the exposed 50-end of the flap of theforked substrates in the absence of SsbA (6189) In theabsence of SsbA addition of RecA reduced leading-strandsynthesis 15-fold but blocked lagging-strand synthesis(Figure 4B lanes 4ndash6 and 10ndash12 and SupplementaryFigure S3A) It is likely that RecA bound to ssDNA cancompete with the DNA helicase for substrate bindingThese results are similar to the ones described in vivoand in vitro E coli DNA replication in the presence ofthe SSB protein (4243)

In the presence of SsbA the addition of RecA did notsignificantly alter leading-strand synthesis but reducedlagging-strand synthesis 15-fold (Figure 4C and Dlanes 16ndash18 and 28ndash30 and Supplementary Figure S3B)It is likely that RecA bound to the dATP present inthe reaction mix (60) can at least partially assemble onSsbA-coated ssDNA and it can reduce lagging-strandsynthesis (5658)

RecO-mediated RecA filament growth onto ssDNAinhibits DNA replication in vitro

In the previous section it has been shown that RecAcannot be efficiently loaded and polymerize onto theSsbA-coated ssDNA substrate in the presence of ATPand in the absence of the RecO accessory factor

To determine whether RecO could participate withRecA in the modulation of the activity of the replisome

we tested the effect of adding RecO (1 RecO20 nt) andRecA (1 RecA3 nt) to a replication reaction containingSsbA (Figure 4C and D) The result shows that RecOdid not affect DNA synthesis in the presence of SsbA(Figure 4C and D Supplementary Figure S3B) Theweak insolubility of RecR and the strong one of RecFand the buffers used for maintaining them in a soluble (orpartially soluble) state (9091) restrained us to includethem in the complex in vitro replication reaction toanalyse their contributionIn the presence of sub-stoichiometric amounts of RecA

and RecO a significant inhibition of leading-strand DNAsynthesis was observed (Figure 4C lanes 23ndash26 andSupplementary Figure S3B) Addition of RecO- andRecA-blocked lagging-strand synthesis (Figure 4D lanes34ndash36 and Supplementary Figure S3B) It is likely thatRecO facilitates the efficient nucleation and RecAfilament growth onto SsbA-coated ssDNA ConverselyRecAEco filament formation was not a pre-requisitionfor replicase dislodging in vivo (43) Here upon inactiva-tion of the hexameric helicase RecAEco nucleatesprobably onto the lagging strand in the absence of theRecFOREco complex and facilitates the dislodging of thereplisome perhaps by facilitating fork reversal (43)Alternatively thermal DnaBEco inactivation leads touncoupling of the moving polymerases with the DNAhelicase and any asynchrony might lead to the generationof large tracts of protein-free ssDNA to which RecAEco

can be loaded with no mediator requirement

CONCLUSIONS

We have shown that spore resistance after X-ray radiationor UHV treatment depends on RecA its accessoryproteins namely RecF RecO RecR and RecX thatfacilitate RecA nucleation and filament growth ontossDNA and the induction of the SOS response(Table 1) However dormant spores have a single non-replicating genome (1718) and recombinational repairis constrained by the requirement of an intact homologoustemplate (1033) How can we rationalize the need ofRecA and its accessory factors for spore resistance toDSBs in the absence of a homologous template Fromthe results obtained previously and in this work wepropose that (i) an uncharacterized DNA damage check-point function(s) should inhibit DNA end-resection(which represents the primary regulatory step towardsHR) increase end-protection and contribute to thechoice of NHEJ during spore survival (ii) the two-endedDSBs were sealed by NHEJ and the ssDNA nicks shouldbe repaired by specialized repair pathways (Table 1)(13142047) (iii) during germination the replisomemight encounter lsquobarriersrsquo or base damage in the DNAtemplate (92) leading to replication fork stalling and(iv) a RecAmiddotssDNA NPF formed on ssDNA with thecontribution of RecO RecR RecF and RecX might becrucial for spore survival by stabilizing a stalled replica-tion fork This activity appears to be separate from RecA-mediated DNA pairing and strand exchange

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The replisome is inherently tolerant after collision withDNA lesions (44) and RecAmiddotssDNA NPF formation isrequired to inhibit replication fork progression uponstress and this novel RecA activity is necessary for

recovery during germination RecF RecO RecR andRecX which enable RecA assembly on SsbA-coatedssDNA in vivo are required for spore resistance Then aRecAmiddotssDNA NPF promotes the LexA self-cleavage and

Figure 4 RecAmiddotssDNA NPF inhibits DNA synthesis in the presence of RecO (A) Diagram of the DNA template and the B subtilis replisomeassembly scheme in the presenceabsence (plusmn) of SsbA RecA andor RecO The mini-circular DNA template has an asymmetric GC ratio (501)between the two strands permitting quantification of leading- and lagging-strand synthesis by measuring radioactive dCMP and dGMP incorpor-ation (B) Time course of the leading- and lagging-strand synthesis by the reconstituted replisome in the absence of SsbA and without (lanes 1ndash3 and7ndash9) or with RecA (1mM lanes 4ndash6 and 10ndash12) (C) Time course of the leading-strand synthesis by a replisome containing SsbA (009 mM) (lanes13ndash24) with RecA (1mM lanes 16ndash18) with RecO (01 mM lanes 19ndash21) and in the presence of both RecO and RecA (lanes 22ndash24) (D) Time courseof the lagging-strand synthesis by a replisome containing SsbA (lanes 25ndash36) in the presence of RecA (lanes 28ndash30) RecO (lanes 31ndash33) or both(34ndash36) M indicates the 30-labelled HindIII-digested DNA used as a size marker

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induction of the SOS response and works as a genuineauxiliary component of the replisome (Figure 4) by pre-venting recombinational repair to occur (Figure 1) Thepotential SOS-induced contributor(s) to spore resistanceare RecA itself PcrA and Pol Y2 (see above) although thepotential contribution of SOS genes of unknown functioncannot be ruled out Pol Y2 however does not contributeto spore survival upon induction of DSBs at least afterH2O2 treatment (93) The role of PcrA is to dismantle aRecAmiddotssDNA NPF (9495) but the absence of PcrArenders cells synthetically lethal and for viability muta-tions in recF recO or recR gene accumulated in the back-ground (86) The phenotype of the pcrA (recF) mutationprecluded any further studies to address the potentialmechanism of PcrA in spore resistance to DSBs

Why RecA accessory proteins are requiredRecAmiddotATPmiddotMg2+ which cannot compete with SsbAbound to ssDNA (Supplementary Figure S2A) hassome effect on DNA synthesis even in the presence ofSsbA but stoichiometric concentrations of RecO andRecA inhibit DNA synthesis (Figure 4C and D) In vitroRecO is sufficient to overcome the kinetic barriersimposed by SsbA on RecA assembly onto ssDNA andfacilitates RecA nucleation and filament growth(Supplementary Figure S2B) although genetic datasupport that in vivo the RecFOR complex is required forRecAmiddotssDNA NPF assembly (9)

The RecAmiddotssDNA NPF by inhibition of DNA replica-tion might prevent potentially dangerous forms of DNArepair from occurring during replication of germinatingspores Alternatively RecA contributes to convert thestalled fork to a lsquochicken-footrsquo structure which is subjectto cleavage causing DSBs (96) If fork reversal takes placewe have to assume that the RecAmiddotssDNA NPF mightstabilize a stalled fork preventing or promoting dissol-ution of a reversed forks rather than its cleavage whichshould require end-processing functions for recovery andend-processing mutants were only slightly impaired inspore survival On the basis of an Occamrsquos razor-like ra-tionale we assume that (i) the replication speed thresholdsdictate RecA assembly on the ssDNA (ii) a RecAmiddotssDNAfilament which is crucial for spore survival upon gener-ation of DSBs inhibits replication fork progression in amanner that resembles replication through barriers (egfragile sites etc) and (iii) RecA assembled on ssDNAtriggers SOS induction and inhibits DNA replication Itis likely that PcrA or any other helicase might dismantlethe RecAmiddotssDNA NPF followed by de novo re-startedand high-speed replication

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Onlineincluding [97ndash99]

ACKNOWLEDGEMENTS

We thank Prof C McHenry (Univ of Colorado Boulder)for providing us with materials for in vitro replicationProf L A Simmons (Univ of Michigan) for strains

and helpful discussions and we thank Andrea SchroderChiara Marchisone and Petra Schwendner for their excel-lent technical assistance Ralf Moeller gratefullyacknowledges DLRrsquos ForschungssemesterprogrammIgnacija Vlasic expresses gratitude to DLRrsquoslsquoGastwissenschaftlerprogrammrsquo

FUNDING

DLR-FuE-Projekt ISS Nutzung in der BiodiagnostikProgramm RF-FuW Teilprogramm 475 (to GR andRM) Ministerio de Economıa y CompetitividadDireccion General de Investigacion [BFU2012-39879-C02-01 to JCA BFU2012-39879-C02-02 to SA]Funding for open access charge Ministerio deEconomia y Competividad Direccion General deInvestigacion DLR-FuE-Projekt lsquoISS Nutzung in derBiodiagnostikrsquo Programm RF-FuW Teilprogramm 475

Conflict of interest statement None declared

REFERENCES

1 LindahlT (1993) Instability and decay of the primary structureof DNA Nature 362 709ndash715

2 CicciaA and ElledgeSJ (2010) The DNA damage responsemaking it safe to play with knives Mol Cell 40 179ndash204

3 FriedbergEC WalkerGC SiedeW WoodRD SchultzRAand EllenbergerT (2006) DNA Repair and Mutagenesis ASMPress Washington DC

4 BranzeiD and FoianiM (2010) Maintaining genome stability atthe replication fork Nat Rev Mol Cell Biol 11 208ndash219

5 LieberMR (2010) The mechanism of double-strand DNA breakrepair by the nonhomologous DNA end-joining pathway AnnuRev Biochem 79 181ndash211

6 San FilippoJ SungP and KleinH (2008) Mechanism ofeukaryotic homologous recombination Annu Rev Biochem 77229ndash257

7 WestSC (2003) Molecular views of recombination proteins andtheir control Nat Rev Mol Cell Biol 4 435ndash445

8 WymanC and KanaarR (2006) DNA double-strand breakrepair allrsquos well that ends well Annu Rev Genet 40 363ndash383

9 AyoraS CarrascoB CardenasPP CesarCE CanasCYadavT MarchisoneC and AlonsoJC (2011) Double-strandbreak repair in bacteria a view from Bacillus subtilis FEMSMicrobiol Rev 35 1055ndash1081

10 KowalczykowskiSC DixonDA EgglestonAK LauderSDand RehrauerWM (1994) Biochemistry of homologousrecombination in Escherichia coli Microbiol Rev 58 401ndash465

11 PaquesF and HaberJE (1999) Multiple pathways ofrecombination induced by double-strand breaks in Saccharomycescerevisiae Microbiol Mol Biol Rev 63 349ndash404

12 CoxMM (2007) Motoring along with the bacterial RecAprotein Nat Rev Mol Cell Biol 8 127ndash138

13 PitcherRS BrissettNC and DohertyAJ (2007)Nonhomologous end-joining in bacteria a microbial perspectiveAnnu Rev Microbiol 61 259ndash282

14 ShumanS and GlickmanMS (2007) Bacterial DNA repair bynon-homologous end joining Nat Rev Microbiol 5 852ndash861

15 SymingtonLS and GautierJ (2011) Double-strand break endresection and repair pathway choice Annu Rev Genet 45247ndash271

16 PiggotPJ and HilbertDW (2004) Sporulation of Bacillussubtilis Curr Opn Microbiol 7 579ndash586

17 LindsayJA and MurrellWG (1985) Changes in density ofDNA after interaction with dipicolinic acid and its possible rolein spore heat resistance Curr Microbiol 12 329ndash334

18 ErringtonJ (2001) Septation and chromosome segregation duringsporulation in Bacillus subtilis Curr Opn Microbiol 4 660ndash666

Nucleic Acids Research 2014 Vol 42 No 4 2305

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19 SetlowP (2006) Spores of Bacillus subtilis their resistance to andkilling by radiation heat and chemicals J Appl Microbiol 101514ndash525

20 SetlowP (2007) I will survive DNA protection in bacterialspores Trends Microbiol 15 172ndash180

21 WangST SetlowB ConlonEM LyonJL ImamuraDSatoT SetlowP LosickR and EichenbergerP (2006) Theforespore line of gene expression in Bacillus subtilis J Mol Biol358 16ndash37

22 VeeningJW MurrayH and ErringtonJ (2009) A mechanismfor cell cycle regulation of sporulation initiation in Bacillussubtilis Genes Dev 23 1959ndash1970

23 DoseK Bieger-DoseA KerzO and GillM (1991) DNA-strandbreaks limit survival in extreme dryness Origins Life Evol B 21177ndash187

24 DoseK Bieger-DoseA LabuschM and GillM (1992) Survivalin extreme dryness and DNA-single-strand breaks Adv SpaceRes 12 221ndash229

25 PottsM (1994) Desiccation tolerance of prokaryotes MicrobiolRev 58 755ndash805

26 NicholsonWL MunakataN HorneckG MeloshHJ andSetlowP (2000) Resistance of Bacillus endospores to extremeterrestrial and extraterrestrial environments Microbiol Mol BiolRev 64 548ndash572

27 WellerGR KyselaB RoyR TonkinLM ScanlanEDellaM DevineSK DayJP WilkinsonA drsquoAdda diFagagnaF et al (2002) Identification of a DNA nonhomologousend-joining complex in bacteria Science 297 1686ndash1689

28 de VegaM (2013) The minimal Bacillus subtilis nonhomologousend joining repair machinery PLoS One 8 e64232

29 SetlowB and SetlowP (1996) Role of DNA repair in Bacillussubtilis spore resistance J Bacteriol 178 3486ndash3495

30 CarrascoB CozarMC LurzR AlonsoJC and AyoraS(2004) Genetic recombination in Bacillus subtilis 168 contributionof Holliday junction processing functions in chromosomesegregation J Bacteriol 186 5557ndash5566

31 MascarenhasJ SanchezH TadesseS KidaneDKrishnamurthyM AlonsoJC and GraumannPL (2006)Bacillus subtilis SbcC protein plays an important role in DNAinter-strand cross-link repair BMC Mol Biol 7 20

32 MoellerR StackebrandtE ReitzG BergerT RettbergPDohertyAJ HorneckG and NicholsonWL (2007) Role ofDNA repair by nonhomologous-end joining in Bacillus subtilisspore resistance to extreme dryness mono- and polychromaticUV and ionizing radiation J Bacteriol 189 3306ndash3311

33 RaddingCM (1991) Helical interactions in homologous pairingand strand exchange driven by RecA protein J Biol Chem 2665355ndash5358

34 SandlerSJ and ClarkAJ (1994) RecOR suppression of recFmutant phenotypes in Escherichia coli K-12 J Bacteriol 1763661ndash3672

35 UmezuK ChiNW and KolodnerRD (1993) Biochemicalinteraction of the Escherichia coli RecF RecO and RecRproteins with RecA protein and single-stranded DNA bindingprotein Proc Natl Acad Sci USA 90 3875ndash3879

36 MorimatsuK and KowalczykowskiSC (2003) RecFOR proteinsload RecA protein onto gapped DNA to accelerate DNA strandexchange a universal step of recombinational repair Mol Cell11 1337ndash1347

37 SakaiA and CoxMM (2009) RecFOR and RecOR as distinctRecA loading pathways J Biol Chem 284 3264ndash3272

38 MorimatsuK WuY and KowalczykowskiSC (2012) RecFORproteins target RecA protein to a DNA gap with either DNA orRNA at the 5rsquo terminus implication for repair of stalledreplication forks J Biol Chem 287 35621ndash35630

39 LittleJW (1991) Mechanism of specific LexA cleavageautodigestion and the role of RecA coprotease Biochimie 73411ndash421

40 SassanfarM and RobertsJW (1990) Nature of the SOS-inducing signal in Escherichia coli The involvement of DNAreplication J Mol Biol 212 79ndash96

41 McInerneyP and OrsquoDonnellM (2007) Replisome fate uponencountering a leading strand block and clearance from DNA byrecombination proteins J Biol Chem 282 25903ndash25916

42 IndianiC PatelM GoodmanMF and OrsquoDonnellME (2013)RecA acts as a switch to regulate polymerase occupancy in amoving replication fork Proc Natl Acad Sci USA 1105410ndash5415

43 LiaG RigatoA LongE ChagneauC Le MassonMAllemandJF and MichelB (2013) RecA-promoted RecFOR-independent progressive disassembly of replisomes stalled byhelicase inactivation Mol Cell 49 547ndash557

44 YeelesJT and MariansKJ (2011) The Escherichia colireplisome is inherently DNA damage tolerant Science 334235ndash238

45 PatelM JiangQ WoodgateR CoxMM and GoodmanMF(2010) A new model for SOS-induced mutagenesis how RecAprotein activates DNA polymerase V Crit Rev Biochem MolBiol 45 171ndash184

46 CoxMM (2007) Regulation of bacterial RecA protein functionCrit Rev Biochem Mol Biol 42 41ndash63

47 LenhartJS SchroederJW WalshBW and SimmonsLA(2012) DNA repair and genome maintenance in Bacillus subtilisMicrobiol Mol Biol Rev 76 530ndash564

48 AlonsoJC CardenasPP SanchezH HejnaJ SuzukiY andTakeyasuK (2013) Early steps of double-strand break repair inBacillus subtilis DNA Repair 12 162ndash176

49 CarrascoB CardenasPP CanasC YadavT CesarCEAyoraS and AlonsoJC (2012) Dynamics of DNA Double-strandBreak Repair in Bacillus subtilis 2nd edn Caister Academic PressNorfolk

50 DuigouS EhrlichSD NoirotP and Noirot-GrosMF (2004)Distinctive genetic features exhibited by the Y-family DNApolymerases in Bacillus subtilis Mol Microbiol 54 439ndash451

51 SchaefferP MilletJ and AubertJP (1965) Catabolic repressionof bacterial sporulation Proc Natl Acad Sci USA 54 704ndash711

52 NicholsonWL and SetlowP (1990) Sporulation germinationand outgrowth In HarwoodCR and CuttingSM (eds)Molecular Biological Methods for Bacillus J Wiley amp SonsChichester UK pp 391ndash450

53 MoellerR SetlowP HorneckG BergerT ReitzGRettbergP DohertyAJ OkayasuR and NicholsonWL (2008)Roles of the major small acid-soluble spore proteins and spore-specific and universal DNA repair mechanisms in resistance ofBacillus subtilis spores to ionizing radiation from X rays andhigh-energy charged-particle bombardment J Bacteriol 1901134ndash1140

54 HorneckG (1993) Responses of Bacillus subtilis spores to spaceenvironment results from experiments in space Origins Life EvolB 23 37ndash52

55 MunakataN SaitouM TakahashiN HiedaK andMorohoshiF (1997) Induction of unique tandem-base changemutations in bacterial spores exposed to extreme dryness MutatRes 390 189ndash195

56 YadavT CarrascoB MyersAR GeorgeNP KeckJL andAlonsoJC (2012) Genetic recombination in Bacillus subtilis adivision of labor between two single-strand DNA-bindingproteins Nucleic Acids Res 40 5546ndash5559

57 CarrascoB AyoraS LurzR and AlonsoJC (2005) Bacillussubtilis RecU Holliday-junction resolvase modulates RecAactivities Nucleic Acids Res 33 3942ndash3952

58 ManfrediC CarrascoB AyoraS and AlonsoJC (2008)Bacillus subtilis RecO nucleates RecA onto SsbA-coated single-stranded DNA J Biol Chem 283 24837ndash24847

59 HobbsMD SakaiA and CoxMM (2007) SSB protein limitsRecOR binding onto single-stranded DNA J Biol Chem 28211058ndash11067

60 SandersGM DallmannHG and McHenryCS (2010)Reconstitution of the B subtilis replisome with 13 proteinsincluding two distinct replicases Mol Cell 37 273ndash281

61 SecoEM ZinderJC ManhartCM Lo PianoAMcHenryCS and AyoraS (2013) Bacteriophage SPP1 DNAreplication strategies promote viral and disable host replicationin vitro Nucleic Acids Res 41 1711ndash1721

62 MoellerR ReitzG LiZ KleinS and NicholsonWL (2012)Multifactorial resistance of Bacillus subtilis spores to high-energyproton radiation role of spore structural components and the

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homologous recombination and non-homologous end joiningDNA repair pathways Astrobiology 12 1069ndash1077

63 MoellerR SchuergerAC ReitzG and NicholsonWL (2011)Impact of two DNA repair pathways homologous recombinationand non-homologous end joining on bacterial spore inactivationunder simulated martian environmental conditions Icarus 215204ndash210

64 MichelB BoubakriH BaharogluZ LeMassonM andLestiniR (2007) Recombination proteins and rescue of arrestedreplication forks DNA Repair 6 967ndash980

65 KidaneD SanchezH AlonsoJC and GraumannPL (2004)Visualization of DNA double-strand break repair in live bacteriareveals dynamic recruitment of Bacillus subtilis RecF RecO andRecN proteins to distinct sites on the nucleoids Mol Microbiol52 1627ndash1639

66 SanchezH CarrascoB CozarMC and AlonsoJC (2007)Bacillus subtilis RecG branch migration translocase is required forDNA repair and chromosomal segregation Mol Microbiol 65920ndash935

67 SanchezH KidaneD ReedP CurtisFA CozarMCGraumannPL SharplesGJ and AlonsoJC (2005) TheRuvAB branch migration translocase and RecU Holliday junctionresolvase are required for double-stranded DNA break repair inBacillus subtilis Genetics 171 873ndash883

68 KidaneD and GraumannPL (2005) Dynamic formation ofRecA filaments at DNA double strand break repair centers inlive cells J Cell Biol 170 357ndash366

69 SanchezH KidaneD CozarMC GraumannPL andAlonsoJC (2006) Recruitment of Bacillus subtilis RecN to DNAdouble-strand breaks in the absence of DNA end processingJ Bacteriol 188 353ndash360

70 CardenasPP CarrascoB SanchezH DeikusGBechhoferDH and AlonsoJC (2009) Bacillus subtilispolynucleotide phosphorylase 3rsquo-to-5rsquo DNase activity is involvedin DNA repair Nucleic Acids Res 37 4157ndash4169

71 CardenasPP CarzanigaT ZangrossiS BrianiF Garcia-TiradoE DehoG and AlonsoJC (2011) Polynucleotidephosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN SsbA and RecAproteins Nucleic Acids Res 39 9250ndash9261

72 YeelesJT and DillinghamMS (2010) The processing of double-stranded DNA breaks for recombinational repair by helicase-nuclease complexes DNA Repair 9 276ndash285

73 NiuH RaynardS and SungP (2009) Multiplicity of DNA endresection machineries in chromosome break repair Genes Dev23 1481ndash1486

74 CardenasPP CarrascoB Defeu SoufoC CesarCE HerrKKaufensteinM GraumannPL and AlonsoJC (2012) RecXfacilitates homologous recombination by modulating RecAactivities PLoS Genet 8 e1003126

75 ChowKH and CourcelleJ (2004) RecO acts with RecF andRecR to protect and maintain replication forks blocked by UV-induced DNA damage in Escherichia coli J Biol Chem 2793492ndash3496

76 LusettiSL HobbsMD StohlEA Chitteni-PattuSInmanRB SeifertHS and CoxMM (2006) The RecF proteinantagonizes RecX function via direct interaction Mol Cell 2141ndash50

77 AlonsoJC TailorRH and LuderG (1988) Characterization ofrecombination-deficient mutants of Bacillus subtilis J Bacteriol170 3001ndash3007

78 AlonsoJC StiegeAC and LuderG (1993) Geneticrecombination in Bacillus subtilis 168 effect of recN recF recHand addAB mutations on DNA repair and recombination MolGen Genet 239 129ndash136

79 FernandezS KobayashiY OgasawaraN and AlonsoJC(1999) Analysis of the Bacillus subtilis recO gene RecO formspart of the RecFLOR function Mol Gen Genet 261 567ndash573

80 GasselM and AlonsoJC (1989) Expression of the recE geneduring induction of the SOS response in Bacillus subtilisrecombination-deficient strains Mol Microbiol 3 1269ndash1276

81 AlonsoJC and LuderG (1991) Characterization of recFsuppressors in Bacillus subtilis Biochimie 73 277ndash280

82 SimmonsLA GoranovAI KobayashiH DaviesBWYuanDS GrossmanAD and WalkerGC (2009) Comparisonof responses to double-strand breaks between Escherichia coli andBacillus subtilis reveals different requirements for SOS inductionJ Bacteriol 191 1152ndash1161

83 NicholsonWL MoellerR and Protect Team and HorneckG(2012) Transcriptomic responses of germinating Bacillus subtilisspores exposed to 15 years of space and simulated martianconditions on the EXPOSE-E experiment PROTECTAstrobiology 12 469ndash486

84 AuN Kuester-SchoeckE MandavaV BothwellLECannySP ChachuK ColavitoSA FullerSN GrobanESHensleyLA et al (2005) Genetic composition of the Bacillussubtilis SOS system J Bacteriol 187 7655ndash7666

85 Fernandez De HenestrosaAR OgiT AoyagiS ChafinDHayesJJ OhmoriH and WoodgateR (2000) Identification ofadditional genes belonging to the LexA regulon in Escherichiacoli Mol Microbiol 35 1560ndash1572

86 PetitMA and EhrlichD (2002) Essential bacterial helicases thatcounteract the toxicity of recombination proteins EMBO J 213137ndash3147

87 CarrascoB ManfrediC AyoraS and AlonsoJC (2008)Bacillus subtilis SsbA and dATP regulate RecA nucleation ontosingle-stranded DNA DNA Repair 7 990ndash996

88 BruckI and OrsquoDonnellM (2000) The DNA replication machineof a Gram-positive organism J Biol Chem 275 28971ndash28983

89 ManhartCM and McHenryCS (2013) The PriA replicationrestart protein blocks replicase access prior to helicase assemblyand directs template specificity through its ATPase activityJ Biol Chem 288 3989ndash3999

90 AlonsoJC StiegeAC DobrinskiB and LurzR (1993)Purification and properties of the RecR protein from Bacillussubtilis 168 J Biol Chem 268 1424ndash1429

91 AyoraS and AlonsoJC (1997) Purification and characterizationof the RecF protein from Bacillus subtilis 168 Nucleic Acids Res25 2766ndash2772

92 DurkinSG and GloverTW (2007) Chromosome fragile sitesAnnu Rev Genet 41 169ndash192

93 Rivas-CastilloAM YasbinRE RobletoE NicholsonWLand Pedraza-ReyesM (2010) Role of the Y-family DNApolymerases YqjH and YqjW in protecting sporulating Bacillussubtilis cells from DNA damage Curr Microbiol 60 263ndash267

94 FagerburgMV SchauerGD ThickmanKR BiancoPRKhanSA LeubaSH and AnandSP (2012) PcrA-mediateddisruption of RecA nucleoprotein filaments ndash essential role of theATPase activity of RecA Nucleic Acids Res 40 8416ndash8424

95 ParkJ MyongS Niedziela-MajkaA LeeKS YuJLohmanTM and HaT (2010) PcrA helicase dismantles RecAfilaments by reeling in DNA in uniform steps Cell 142 544ndash555

96 SeigneurM BidnenkoV EhrlichSD and MichelB (1998)RuvAB acts at arrested replication forks Cell 95 419ndash430

97 CeglowskiP LuderG and AlonsoJC (1990) Genetic analysisof recE activities in Bacillus subtilis Mol Gen Genet 222441ndash445

98 FernandezS SorokinA and AlonsoJC (1998) Geneticrecombination in Bacillus subtilis 168 effects of recU and recSmutations on DNA repair and homologous recombinationJ Bacteriol 180 3405ndash3409

99 AlonsoJC ShirahigeK and OgasawaraN (1990) Molecularcloning genetic characterization and DNA sequence analysis ofthe recM region of Bacillus subtilis Nucleic Acids Res 186771ndash6777

Nucleic Acids Research 2014 Vol 42 No 4 2307

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