Cell cycle checkpoints in bacteria

Bio~'hh~de ~19t)7) 7t). 549-.554

@ Soci~l~fi'an~'aisede biochim;e c,l bi~d~giemld~3culait~.,: El,.cvicr.Paris.

Review

Cell cycle checkpoints in bacteria S Aulret, A Levine, |B Holland, SJ Sfror* Instilut de G61idtiquc et Microhiologie. URA 2225. Univcrxitd Paris XI. BtPtmwnt 409. 91405 Or.~av ~ede.v. I"ram c

(Received 4 June 1997; accepted 13 November 1997) SUillnltil'y - - When DNA replication is iu,lerrup,lcd iu bac,leria, a specific illhil%,lor I SfiA), a coulpilnelit of ,lhc SOS systciu, is ~,,rnlhesised which tran,nieuily blllcks cell division, This i~ ,lhe pro,lolyp¢, disl~ensable, cell cycle checkpoin,l, e~,selilial for nl:iXilllal survival tinder a particular stress, In COlltr;.l~,l, uo pl'Ol:12Ss spccil'ically signalliug the ,lernlilla,li4,~nof chroulosolnal replicalion Io ac,livat¢ Ihe S,libkel}tw!ll di~ ision eveut, which luighl hi2 lernled ;.ill essci,l,lial checkpoint, has ye,l bCt~lldenlonMl'a,lcd, hi 1;' coli. a specific nleeh.tinislll is apparollly requhcd Io reactivate replicalilm I'ork~, blocked by damage, but )Is nloleeulta l,a,,is is un¢leiir, luducliou of Ihc S,ll'illg¢lll I'en1-~Olise, nlediatod by ReL,\ r/. Ihe level of ppGpp, presun/ably ,io op,lilni~,e nlacrolllOlt2¢illar syuthesis :iccording Io the availability of nulrienls, aclivales a conlrol syMelll which inhibits I)NA replica,lion in both E coli and II ",lihlilis, hi/'.~ coil this blocks new rOtulds el initialion al ori(', altholl~2h Ihe ilit.'c'haili~ll/ is not ele;u'. Conversely, iuilialion is not blocked in B ,llll/l.li/is, hut rt2plicalitul is blocked al)pareuily al a nuuthcr )if distinct ~itcs 10il 2(}1) kb dowll~treaill aud either side of eriC. This nutrieu,l-dependeu,l replicating checkpoint specifically requires RTP. ,lhe chrolnOSOl'blal ,lerrilinalor proieiu, alld new evidelac¢ indicates lhai specific RTP binding sites may be invtllw2d in Ihis posl-iifi,lialion control nlechanisnl. A ',iulilar post-initiation conU'ol inet-h~.ulislll ,'lppears itl bll.lck replicalion reversibly afio" prenli.llUrl2 hill)alien in B sithfili~, indieali!l.~ !hal this s)slem nlu), have u dual funetiou, Iimi,lhlg replication ill starvation eondilioiis und as ~1nlet'hanisul to conltlellSall2 for prenla,lure illlllallons.

checkpoints I SOS system I stringent irespollSe7'RTP Introduction Cell cycle "cbeckpoil,lis" were origiilally defined [ l l a s ~llrvcilh,lnce nlecllanisnls thai result ii,l specific cell cycle blocks in response to perturbation of a cell cycle process, slleh )is chrOlllOSlnllld replicatioil, ill)tests or cell division (cyiokil,lesis). In tile original strict SeliSe, sucll nlel:hlil,li,',M', might be dispeu,nable uuder lllOst COllditions and die prolo o lype ex;lnlple is lbe SOS-DNA replication checkpoint hn Escherlchia coil (for a review sec. 121). In this case DNA

damage, for example resulting from UV.irradiation, blocks replication fork movemeul, triggering a cascade leading to the activation of tbe RecA protein, plx)teolytic degrad.',ition of the LexA, SOS represser, and consequent ,l'apid synthesis of a division inhibito,l; SfiA (SulA), which specifically blocks the ability of FtsZ to lbrm a cytokinetic ring. This elegantly controlled syste,ln completely prevents division, but not mass increase 13I, whilst DNA repair is carried out by other enzymes uuder SOS control, thereby preventing the transmission of damaged chrmnosomes to daughter cells. Importantly. Ibis replication-division checkpoint control is completely dispensable, in that sfiA cells under normal growth conditions replicate and in particular divide as the wild type [41. The LexA-SfiA control mechanism to block

FtsZ action therefore plays absolutely no role in the normal temporal program of cell cycle events, hi contra~,l, cell cycle clleckl)oims, pariicularly vs applied to euk;`,ryolcs, since tllcir original concepttmlisalio,1 by Harllwell and colleagues. have been tl~,ed It) define es~,enfial cell cycle mechani,,ms which obligatorily control tile lransilioll fronl one cell cycle evel,ll [o ant)|her, i¢, process B i:allllO| pl'ocecd tlntil lll't~l-:es", A has been complcicd 151. In hacleria, thu,~ far. no such

process has been conchJsively demonslrated although the pt~ssibilily of an obligatory link between tern|(nation of re= plication and a subsequenl division (cytokinetic) event, remains a controversial possibility. In E colt, closer inspection of the state of DNA replicalion in UV-irradiated cells reve:,lls additional complexities not involving the SfiA checkpoint 1Ob Thus, al'ter UV, I)NA replication progressively is greatly reduced, prob~,bly as a res,dt of lesions or ttnusual structures in the geilor~le, rather than as a result of a specific SOS-induced mechanisn,l. In contrast, resumption of D N A replicalion is a controlled process. requiring ReeA protein and also an unidentified pro° |ein controlled in some way by RecA protein 171. However. the nature of this inechanfisnl, which may constitute a speo cil'ic replica)ion checkpoint, allowing I'eStlnlllliou of replL

cation only after DNA damage has been renloved, has remained illusive. In this review, we shall now c~msider a novel form of DNA replication checkpoint, which, p:a-ticularly in the case

of Bacillus s.btilis, may l~e sufficiently flexible to fnnction both in respon~ to environmental signals, and to internal sigruds in order to control the transition from one cell cycle stage to another, in this case the completion of full DNA r e p l k ' a t ~ rtmnd, in this e,xample, the cells are apparently ~l~'mding to nutritional States rather than DNA damage.

Prmmture i n ~ eheck~ control

of DNA replication and

DNA replication in bacteria is primarily regulated at the level of iniiiatkm. The initiator protein DnaA, well con~rved in Gram-negative and Gram-pusitive bacteria, is a key pre4ein in this process, Thus. binding of Dana to 9 mer repeats in the eriC region of E colt promotes initial DNA unwinding, allowing entry of the DauB helica,~ and sub+ w,,'quent asmmbly of the replisome 181. Nevertheless, how the action of DnaA at eric is restricted to one initiation event per cycle, at a specific initiation mass, remains unclear. It has ~ n propnsed that DnaA itself constitutes the ~ e cortroller (o~iltator] responsible for regulation of initiation through a mechanism, wbereby fluctuations in the concentration of flee Dana refl~n:t tile availability, during the cell+cycle, of DanA boxes (binding sites) on the chro+ mosom¢, in particular those with low affinity !t~ated close to eriC 191, However, thi~ view has not been confirmed and remains controversial, Indeed, recent studies I IOI demonstrafe that eric plasmids continue to replicate in a cell cycle.s~cific nmtmer, in cells where the cllrnmusomc itself, engineer~,d |o ~ independent of DanA, replicates ran+ domly in the cell cycle, Under these condititms, a DnaA:DnaA bo,,~ titration mechanisn~ el+ctnm'o! would he expected It+drastically wrturh the liuling of h+fitiationof the replica+donof th¢ m,iC pl+mid+ The ~sults of Eli+son at+d NoN~lr6m [10] therefore suggest that the activation of Duma ~ u i r e d Ibr eric plasmid replication remains subservieint to ~llceil CyCleclock linked to the attainment oF a critical ~ t l mass, in addRion to the control of initiation exerted at the origin its!f, evi~nc¢ lbr the existence of a regulatory mechanism (post+initiation control) for DNA replication downstream of the origin, has ~'~n descdhed in the Gram+l~sitive bacte+ rium, B s~+htilis. One line of evidence comes from the analysis of the temperature-sensitive mutant, throB37, defective in initiation. When this mutant is first grown at 3IYC. then transferred to the restrictive temperature. 45'~C for 30 min for completion of ongoing replication rounds and then re+ turned to 30"C. replication resumes synehronously I't~r at least two r~mnds, The fi~t cycle is ini- tinted a short time after return to the permissive temperature, even in the presence of ehlormllphenicol, since a sufficient amount of initialx~r pn~tein(s) has accumulated at the restrictive tem~mture for this round, The ~cond round of replication takes plate 45 rain latelz although the requirements for pro-

rein and RNA synthesis for initiation of this round were completed well betbre [ I I 1, Using different strategies to measure the increase in the tanGent of different DNA sequences during synchronous replication of dm~B37, Henckes et al[ 12] showed that the markers close to the origin and up to 100 kb and 175 kb, on the left and the right arms, respectively, were duplicated early, as expected but then, surprisingly, continued to accumulate during the first round (fig In), In contrast, DNA sequences located 220 kb downstream of the origin on the left arm and 330 kb on the right arm were replicated at the predicted time with a two-fold incremem during the first round amt only replicated again during the second replication rom~d (fig Ibt. Thus, an extra (premature) initiation eves ~'curred during the l*irst~:ycle, hut the extent of tbe overreplicated region was limited to a total of approximately 400 kb around the origin, These sequences present in excess during the first round were not h,,wever degraded, nor were they replicated again during the second cycle. In fac~, although the timing of premature initiation was not synchronous, replication of the second round still occurred synchronously at the expected time, as revealed by the measurement of the rate of DNA synthesis. Thus, an important conclusion from this experiment was that the second round of replication, despite being under control of the normal cell clock, apparently commenced from the preanatnrely initiated but arrested replication forks. it is less clear whether a post-initiation control mechanism exists in E colt, In the course of several studies involving over-expression of ¢hmA to increase initiation, it was demonstrated that this does not lead to a significant increase in DNA eoncentratiun tlbr example, see 1131), However, n|tder these conditions, a drastic reduction in leplisome veh~¢ity t+r even possible slalih+t~ tfl' replication forks, par + licularly close to eriC, was ohserved I 13, 14l. Nevertheless, it has not I+~Jcnestablished whether this represents a specific i~st+initiathm control for example, active after premature initiation or whether this simply reflects limitation of some factor essential for replication, when over-initiation occurs.

Stringent response and replication control The stringent system is one of the regulons used in bacteria to modulate the synthesis of many molecules as a lunction of the nutritional stage of the medium. During nutritional deprivation, the stringent response is triggered and the level of an alarmone, ppGpp, is increased in the cell. This leads to pleiotropic effects on a variety of processes, in both a i~+sitive and negative way, and in pmlieular, the inhibition of the synthesis of macromolecules inchiding stable RNA synthesis 115l, Two enzymes, RelA and SPOT regulate the intracellular concentratkm of ppGpp in E coll. The activity of these enzymes is modified in response to different stress conditions. High levels of ppGpp are promoted principally by the activation of RelA (ppGpp synthetase l) during

55t amino acid starvation and by the inhibition o f fl~e ppGpp degradative activhy o f SpoT during other stress conditions, including energy starvation. In contrast, a tow level of ppGpp daring bal;mced growth is primarily maintained by SpoT whict~ :tppears to have both ppGpp hydrolytic and synthetic activities 1161. lnteresth~gly, in an E co6 mutant which is apparently devoid of ppGpp, through deletion of both the relA and sp¢,T genes, the calculated initiation mass for DNA replication was reduced at fast growth rates II 71. This finding raises the interesting possibility that the level

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replic;flion, at least under certain conditions. Previous studies showed that ~!heexpression of the dFutA gent is repressed by a high level of ppGpp m E c , l i [18 I. In addition, IHF and Fis, two proteins ~hich bind to the e r i c l'cgion, and transcription of both mioC and gi&& t~ u genes flanking e r i C were shown to be subject tu stringent control [19-21 I. Moreover, studies in this laboratory ha~e shown that DNA replication is inhibited vdlen the concentration of ppGpp is increased in E colt or in B sub;ills by induction of the stringem response 1221. These studies invoh,ed the use of thermosensitive mutants, dmtA46 for E colt and &roB37 for B subtilis, allowing the synchronisation o1' DNA replication rounds by a simple transfer of the cells from restrictive to permissive temperature. In these condilions, where the pl'oteins required for initiation accuntulated before the induction of the stringent respo.~se, the resulting high level of ppGpp blocks DNA replication at the origin in E coll. In contrast, as shown ill figure 2, m B sulmli,~, initiation occnrred but the replication lk)rks were then blocked 100-20() kb dowustreaul of |he origin. This effect w;is reversible since, when the stringe,tt response was !ilied, replication restnned close to or at the blocked sites even in the presence of chloramphenicol or rifampicin 1221. This result suggested that post-initiation control provided a simple mechanism to halt replication lorks according to the nutritional state of the cell, and that this may constitute a checkpoint control. Precise attalysis of the sequences duplicated during the stringent response showed that the lbrks were stalled in specific regitms on each side o f eriC. designated LSTer and RSTer (left attd right stringent fermi,ill respectively. 1~'o arleSl regions were defined on the left al'lll. LSqkrrl ;tud LSter2, located apprt)xinialely 11111;ntd 130 kh li'out the origin. Similarly, two regions were identified on the right arm 1231. This Ictuls to a contpletc block uf ;,ill tlw lorks 13(1 kb downs;re;n; t)l Ihc oritain tm tllc h:ft ;.'nt n,ld 250 kh ~it

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Fig !. Over-replication of |he origin regitm in the dmdt37 inutallt of 13tlcillus sttblilis following ;iccalnul;ltitm of initiali~)n prot¢i|ls at ilOl|ollel'o missive temperature. The copy nulnber of different chromosumal markers was quantified tluring synchronnns replicqtion ~)1'the &roB37 illul;tnt. Precise qn:ttnificl|liOll wIis obtained by mmlsuring the i'eassuci;ltiml kinetics of denatured, 3.sS-Inhelled probes in the presence or ;ibsence ot chronlt)sonl,'tl DNA extracted 1'1'o111samples ~1 cuhnre harves|ed ;~t differe.lt times during the replication cycle, a, b, Cmnl~arisoll of the eXo perimental values with predicted values durin,d synchrolmus replication I'tm respeclively, a proximal marker (E6" located at 6 kb to the right of the origin) .'rod u distal marker As;cA hie;ted lit 220 kb to the left of the origin}. ¢. A schematic representation of chrnmosomal replication during the first ~lnd tile second rounds reflecting both Ihe synchrtmy !~t~DNA synthesis and the excess rcplic.'aion, based on the measurement of copy number of severtd markers, during the first round,

t ~ right. Interestingly, this inhibition of DNA replication Idler the induction of the stringent tx~sponse ~:curred in the regions of the ehromo~mle similar It) those intplicated ill replication arrest after premature initiation. This ~rengthcns the idea of the exist,race of a second tevel o|" { ~ , ~ t ~ a t k m } ~mtrol in B sz4btiliS, acting as a checklxfint t 0 -"-~ " i m pthe e d emovemem o f the replisome, when initiation at ~ origin is incorrectly tri~et~.xl. ~ w cells ma~ ~nse the inappropriate initiation event. ~bserved in dmtB37returned to 3()°C, is not clear. A single hypotMsis to e~plain both the observed checkpoint control in t~lation to the se~,xmd(and p~mature) initiatkm event in ~hm~Tat~c~ ~ttn:n to permissive temperature, or when superimposed on the first initiation event in dnaB37 cells at~tcr induction of tile stringent response, could involve the alarmtme ppGpp. Thus. a post-initiation checkpoint control process timid nornlally he sensitive to Iluctuations it) intracellular levels of ppGpp, inducing an RTP-dependent F--a J in replication, in reslxmse to an increased level of ppGpp. Levels of ppGpp could be expected to rise under conditions of ntttriont limitation and, in a given growth nlediunt, the level of pIK~pp might b¢ one of ,mveral factors whose concemr',~titm is tightly coupled to the initiation mass for initiation of DNA replication. only way to satisfactorily uncouple DNA ~plication from protein synthesis {required fin" initiation per set is to u~ the dnaB37 synchrnnised system. This is essential in o ~ r In analyse directly the effects of increased level,,, of

ppGpp (via inhibition of protein synthesisL An experimental system to test directly, under otherwise steady state conditions, the elfcct of all abrupt increase of ppGpp levels on replication forks passing tl~rough chromosomal STer sites, is not available. Thus, it in not possible, for the moment to analyse separately the requirements for post-initiation control and replication arrest induced by stringent response. Mechanism of the DNA replication checkpoint in B

s~btilis Role oI'RTP Alter premature initiation, as described ab~vc, the replicalion forks were stalled transiently and then c~mtinued Iheh" progression according to tile cell clock. In both this case and after intposi|ion of the strhlgent ix~xponse, replication appears to be blocked in specific regions. When the stringent response is rentoved, replication lh>m blocked fiwks resumes without ,de ~ l ' ~ protein or RNA synthesis. These results all indicated a specific, sequence-dependem, arrest mechanism, We have shown that the arrest of replication forks in these specific regions, lbllowing the induction of the stringem response requires RTP, a eontrahelicase previously shown to be involved in the an'esl of replication forks at the chromosomal terminus, following binding to a specific DNA terminator 124 I. In out" studies, in the absence

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FIll 2. Analysis oF DNA r~plication during the strhIBent l'~sl~,,us¢ in/~ ~olxfili,~. Au exponential culture of dll(d137 groWu at 30"(..', Was tr~nsl~rp~,dto 4,~( ' for 30 rnin and th~n returned tt~ ,~t~'~("in ~brdertu s~nehl'~miseDNA t\,plicatiou cycles. Arginiue hydroxalnate ~ArB-I-IXL u~d t~ Mdac~0the stringent resl~mne, ~as added 3 nfiu bel'twothe return to lx~rmixsivelenrl~rature to o n e part of the culture. DNA resulting fp,m~ ~t tlew r~md oF initiation of DNA ~plication was labelled with tmethyl 3H) thymidine fi~r 30 rain alter i~larn to the permissive ~mperalur¢, Frt~mcultures plus t~r minus Arg-tIX, chromosomal DNA wax then extracted, purified and sonicated. An identical portion el' each ,,~mplewas ~llow~ to ~ass~iatc in tile presence of an exces,~of dil'fizrentcold DNA probes fixed on nitrocellulose filters. Alter extensive wttshlng, filters.(in triplic~t.~) were countuxL Results were corral:ted for backg~mnd (amount of DNA binding to calF-thymus DNA) and are F~c~ntcd as percenta~,~of hybridisation ~lative to the cons'el e~l~rimen! without drug, versu,~prol~ positions in the chromosomal origin

553 of RTE the replication fi~rks progressed well beyoqd the STer regions [231, One ~f the STer regi~ms, LSTer 2, was cloned in a unidirectional theta replicating plasmid and a plasmid replication arrest, requiring both RTP and the augmentation of the intraeeHular level of ppGpp was demonstrated, The arrest of the reptisome apparently occurred al a discrete site nmpped to a 3.65 kb fragment from the chromosome. Furthermore, this arrest was detected only if the LSTer 2 region was cloned in the same orientation relative to the direction of the replication fork, as in the chromosome (Autret et al, submitted). This 365 kb fragment contains a sequence homologous (13/17 identical nucleotides) to the B fragment core sequence of the normal chromosomal terminator. Importantly, this sequence was shown to bind ffFP with high affinity in vim,. The presence of ppGpp did not iuere~,lsebinding affinity. From these results, we propose that this W-sequence is directly inw~lved in plasmid and chromosomal replication arrest and therefo,'e constitutes an essential part of the LSTer2 site.

site was fmmd in the adjacent sequences and the B'-sequence binds only one dimer of RTP in vitro. Con~,equentIy. we cannol rule out the possibilily that RTP. in this system, blocks replication by a novel mechanism, independent of the DnaB helicasc. As indicated above, the effect of a high level of ppGpp in replication fork arrest is still unknown, but we may speculate that this could involve inhibition of replication fork movement pet" se and/or the stabilisation of the RTP dimer at the STer site in the presence of an advancing replication fork. Other factors may also be required to effect an efficient arrest, in addition to the B'-sequences, including other as yet unidentified DNA sequences in the vicinity of the STer sites, the level of local supercoiling, or even the relative activity of transcription in the LSTer region~ An interesting possibility is that a second B' sequence, located some distance fi'om B'-I, might be recruiled via looping of the DNA, allowing binding of two adjacent RTP directs.

Role o.]'pl~Gpl~

Following the induction of the stringent response in dm~B37 containing a wild type level of RTP, replication forks initiated at the origin were stalled in the STer regions. However, even in the absence of RTP, whilst replication now proceeded through the STer region, the whole claromosmne was not fully duplicated (approximately 60%) following the induction of the stringent response 1231 One simple interpretation of these results is that high levels of ppGpp, which are generated during the sn'ingent response, retard replisome movement, with RTP ensuring that the final arrest takes place in specific regions. Alternatively, even in the absence of RTP, an addition:d lactor may participate with high levels of ppGpp to effect progressive slowing down of Ihe rcplisonte. Comparison between replication arrest at the STer sires and at the classical DNA terminator

RTP in the normal process o1' termination of DNA replication exerts its effect by specific binding to a terminator DNA element, containing two overlapping RTP binding sites A and B. The interaction of RTP with the two sites is not equivalent and the replisome is impeded only if it encounters first the B site on which RTP, as a dimer, is strongly bound 1251. The binding of the second dimer to the A site is absolutely required lbr the function of the nucleo-protein complex, and this may reflect the need to stabilise the interaction between the first RTP dimer on the B site and/or the need for the complex to adopt a unique conformation, able to block helicase activity at the terminus 126, 271. The sequence (B'-1) which binds RTP in the LSTer2 region and is probably implicated in the arrest of replication alter the induction of the stringent response, showed 75% homology with the B site (Autret et al, submitted). No discernible A

Other possible cell cycle checkpoints Although intuitively a specific cell cycle checkpoint controlling terntination of DNA replication and the subsequent initiation of partitioning (segregation) of the nucleoids and ultimately of cell division, remains a reasonable expectation, clear-cut evidence for such a process is still lacking in bacteria. In B .~ubtilis a developmental checkpoint has been proposed, coupling normal DNA replication but also initia° lion of replication to tile onsel of sporuhiliolL Thus, boliJ DNA damage, and induction of the SOS response 1281, attd inhibition of initiation of replication [291, block the ex~ pression ol' genes early in Ihe spomlalion pathway. These processes appear 1o involve different inechanisnls, RccA dependent and RecA-independent respectively, resulting in inhibition of the phosphorylation of the sporulationospecifie SpoOA transcription factol, However; the details of these apparent checkpoint systems are not yet clear. Recent studies in B subtilis 13[)-321 concerning SpoOL a DNA binding protein implicated in the paltitionhtg of nucleoids. indicate that this protein may constitute part of a checkpoint mechanism, ensuring that final entry into the sporulation pathway only takes place when segregation of nucleoids is proceeding normally. Grossman and colleagues and Errington and colleagues have shown that a fluorescent spot of SpoOL is seen to duplicate and to migrate, apparently localised to a fixed position in the nucleoid, to a region close to the cell poles before cells finally divide 131.32 I. SpoOJ has, howevel; another function, antagonising the action of Sol, a negative regulator of the production of SpoOA~P and theretore spoOJ null mutants are also sporulation-defective. Further details of this apparent checkpoint mechanism arc awaited ,'rod it appears likely that additional checkpoint controls affecting the cell cycle in prokaryotes can be anticipated.

5~ Acknow~nts We at~ p l e a ~ to acknowledge support from CNRS. Unive~ity Pads XL the Human Frontier Science Program (no RG-386/95M) ~nd Association pour la Recherche sur le Cancer.

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