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Feb 23, 2004 - E-mail graumann@mailer.uni-marburg.de; Tel. (+49) 6421 2825747; Fax. (+49) 6421 .... It is assumed that RecBCD loads RecA onto free DNA ends, while RecFOR ..... behaved as a protein with an apparent mass of ª480 kDa,.
Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004? 200452616271639Original ArticleDNA double-strand break repair in live bacteriaD. Kidane, H. Sanchez, J. C. Alonso and P. L. Graumann

Molecular Microbiology (2004) 52(6), 1627–1639

doi:10.1111/j.1365-2958.2004.04102.x

Visualization of DNA double-strand break repair in live bacteria reveals dynamic recruitment of Bacillus subtilis RecF, RecO and RecN proteins to distinct sites on the nucleoids Dawit Kidane,1 Humberto Sanchez,2 Juan C. Alonso2 and Peter L. Graumann1* 1 Biochemie, Fachbereich Chemie, Hans-MeerweinStraße, Philipps-Universität Marburg, 35032 Marburg, Germany. 2 Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología-CSIC, Campus Universidad Autonoma de Madrid, Cantoblanco, E-28049 Madrid, Spain. Summary We have found that SMC-like RecN protein, RecF and RecO proteins that are involved in DNA recombination play an important role in DNA double-strand break (DSB) repair in Bacillus subtilis. Upon induction of DNA DSBs, RecN, RecO and RecF localized as a discrete focus on the nucleoids in a majority of cells, whereas two or three foci were rarely observed. RecN, RecO and RecF co-localized to the induced foci, with RecN localizing first, while RecO localized later, followed by RecF. Thus, three repair proteins were differentially recruited to distinct sites on the nucleoids, potentially constituting active DSB repair centres (RCs). RecF did not form regular foci in the absence of RecN and failed to form any foci in recO cells, demonstrating a central role for RecN and RecO in initializing the formation of RCs. RecN/O/F foci were detected in recA, recG or recU mutant cells, indicating that the proteins act upstream of proteins involved in synapsis or post-synapsis. In the absence of exogenous DNA damage, RCs were rare, but they accumulated in recA and recU cells, suggesting that DSBs occur frequently in the absence of RecA or RecU. The results suggest a model in which RecN that forms multimers in solution and high-molecular-weight complexes in cells containing DSBs initiates the formation of RCs that mediate DSB repair with the homologous sister chromosome, which presents a novel concept for DSB repair in prokaryotes. Accepted 23 February, 2004. *For correspondence. E-mail [email protected]; Tel. (+49) 6421 2825747; Fax (+49) 6421 2822191.

© 2004 Blackwell Publishing Ltd

Introduction All cells need to ensure the stability of their genetic material for cell survival. A major threat for the genomic integrity of DNA are double-strand breaks (DSBs). Such breaks are targets for exonucleases, and can thus lead to deletions or, if the wrong DNA ends are connected, chromosome rearrangements that are commonly found in tumours. DNA DSBs occur as a result of a variety of insults such as gamma or UV irradiation and chemicals that modify the DNA. For example, mitomycin C (MMC) cross-links DNA strands within the double helix, and excision repair of these adducts leads to breaks in both strands. Additionally, if a replication fork meets chemically altered bases or basepairs that cannot be replicated, the fork can collapse leading to a DSB (Cox et al., 2000). To cope with DSBs, cells have evolved two general repair pathways. Broken DNA ends can be directly reconnected by non-homologous end joining (NHEJ), or the break can be repaired using the intact DNA region on the sister chromosome, i.e. by homologous recombination (Cromie et al., 2001). NHEJ relies on a DNA ligase that is targeted to DNA ends by a conserved protein named Ku. Ku binds to DNA ends as a heterodimer in eukaryotes, and apparently as a homodimer in bacteria, protects the ends from exonucleolytic degradation and recruits DNA ligase (Paques and Haber, 1999; Weller et al., 2002). In contrast to this process in which wrong DNA ends can easily be connected, repair by homologous recombination requires a large number of proteins that act at various stages of the process (Cromie et al., 2001). The first stage, presynapsis, is the generation of 3¢ singlestranded (ss) DNA ends that can then be used for annealing with the homologous sequence on the sister chromosome. In Escherichia coli, the RecBCD protein complex binds to DNA ends and generates overhangs through its helicase/exonuclease activity. In eukaryotes, the Rad50–Mre11–Nbs1 complex has been implicated in presynapsis, with Mre11 conferring ssDNA endonuclease and 3¢ Æ 5¢ exonuclease activity in association with Rad50 (Paques and Haber, 1999; Trujillo and Sung, 2001). The second and most crucial step in DNA recombination is the introduction of the 3¢ DNA overhang into the homologous duplex of the sister chromosome, termed

1628 D. Kidane, H. Sanchez, J. C. Alonso and P. L. Graumann synapsis. This is performed by RecA in bacteria and by its homologue Rad51 in eukaryotes (West, 2003). RecA binds to ssDNA in an ATP-dependent manner and generates a right-handed helix that can invade homologous duplex DNA and mediate strand annealing, accompanied by extrusion of the other strand that can pair with the remaining 5¢ overhang of the DSB (called D-loop formation). Because SSB (single-strand binding protein) has a much higher affinity for ssDNA than RecA, RecA needs to be loaded on to the generated ssDNA that is coated with SSB. In E. coli, this task appears to be performed by the RecBCD system (that is not conserved in all bacteria) or, alternatively, by a second epistatic group of recombination proteins, RecF, RecO and RecR (Bork et al., 2001). It is assumed that RecBCD loads RecA onto free DNA ends, while RecFOR act on gapped DNA or on free 5¢ ends (Arnold and Kowalczykowski, 2000; Bork et al., 2001; Morimatsu and Kowalczykowski, 2003). During the third step in recombination, post-synapsis, RecA-promoted strand transfer produces a four-stranded exchange or Holliday junctions (HJs; true cross-overs), generated by the RecG and RuvAB helicases. Finally, RuvC resolves HJs in an orientation determined by RuvB (Cromie and Leach, 2000), and the remaining nicks are sealed by DNA ligase. In Bacillus subtilis, homologous recombination during chromosomal and plasmid transformation involves five different epistatic groups: a [comprising recFLOR (counterpart of the E. coli recFOR pathway) and recN], b (addAB, counterpart of the E. coli recBCD pathway), g (recP and recH), e (ruvA, recD and recU) and z (recS), all of which depend on RecA (Alonso et al., 1993; Fernandez et al., 1999; 2000; Chedin et al., 2000). Additionally, several other recombination gene products (recJ, recQ, recG) are also required for recombination, as well as DNA polymerases, SSB, histone-like protein HBsu, PcrA, PriA, Mfd and topoisomerases (Alonso et al., 1993; Fernandez et al., 1997). RecN has also been shown to be important for DNA recombination and DNA repair in other bacteria (Funayama et al., 1999; Skaar et al., 2002); however, the function of RecN is unclear. In eukaryotes, the Rad50–Mre11–Nbs1 complex plays a pivotal role in DSB repair by homologous recombination (Grenon et al., 2001). Additionally, Rad50 also appears to play a role in NHEJ, where it has been shown to interact with Dnl4 DNA ligase (Chen et al., 2001). Rad50 belongs to the SMC protein family, the members of which are also crucial components of protein complexes involved in sister chromosome cohesion (cohesin) and chromosome condensation (condensin) in eukaryotes (Strunnikov, 1998; Hirano, 1999), and chromosome segregation in bacteria (Graumann, 2001). SMC proteins are composed of dimers each consisting of an ATPase cassette head domain (Hopfner et al., 2000; Lowe et al., 2001) con-

nected to a central hinge region by an extremely large coiled-coil region. Dimerization is mediated by the hinge domains (Haering et al., 2002) or a Zn-hook in Rad50 (Hopfner et al., 2002), giving rise to flexible symmetric molecules. Cohesin and bacterial SMC complex bind to DNA as a ring-like structure (Volkov et al., 2002; Gruber et al., 2003), in which DNA is embraced by the long coiledcoil arms, and ring closure occurs at the head domains through dimerization. In B. subtilis, SMC forms a complex with ScpA and ScpB that is required for proper chromosome compaction and segregation (Britton et al., 1998; Graumann, 2000; Mascarenhas et al., 2002; Soppa et al., 2002; Volkov et al., 2002). The SMC complex localizes to discrete sites on the nucleoids (that contain the chromosomes), each in one cell half, from where it is thought to condense locally and organize DNA. The replication machinery is a stationary complex in the middle of the cell (Lemon and Grossman, 1998), such that the chromosome moves through the centrally located polymerase (DNA PolIII) as it is replicated (Lemon and Grossman, 2000). Soon after initiation of replication, origin regions on the chromosomes are rapidly separated towards opposite cell poles (Sharpe and Errington, 1998; Webb et al., 1998), and then all other regions are separated soon after their duplication. This gives rise to an ordered chromosome arrangement (Teleman et al., 1998) that depends on the action of the SMC complex (Graumann, 2000). Interestingly, RecN is also a member of the SMC protein family, showing that several SMC-like proteins play important functions in DNA recombination and repair. In spite of this large body of genetic and biochemical data, the mode of DSB repair in vivo is still rather unclear. We have sought to gain further insight by investigating the role of DSB repair proteins within the three-dimensional context of the cell. We have been able to visualize discrete RecN foci that are formed in response to various conditions inducing DSBs, and that RecF and RecO colocalized with, and to track the function of the proteins in time and space. Our results show that DSBs lead to the induction of foci that contain several DNA repair enzymes, suggesting that defined DNA DSB repair centres exist in prokaryotic cells.

Results RecN, RecO and RecF play an important role in DNA DSB repair We wished to investigate the function in repair of DNA DSBs of proteins reported to be important for repair of DNA lesions and for homologous recombination. Therefore, we grew wild-type (wt) and various mutant strains in the presence of different concentrations of mitomycin C (MMC), which leads to the formation of DSBs. As shown © 2004 Blackwell Publishing Ltd, Molecular Microbiology, 52, 1627–1639

DNA double-strand break repair in live bacteria 1629 Fig. 1. Growth of B. subtilis strains on different concentrations of mitomycin C (MMC). A. Strains PY79 (wt), DrecN and DrecO growing on LB with no MMC (top), 50 ng ml-1 (bottom left) or 100 ng ml-1 (bottom right). B. Strains PY79 (wt), DK10 (recN–cfp, recF– yfp) and DK9 (recF–cfp, recO–yfp) cells growing on 50 ng ml-1 (top) or 100 ng ml-1 of MMC (bottom).

in Fig. 1A, wild-type B. subtilis cells were able to form normal colonies on medium containing 50 ng ml-1 MMC, while a concentration of 100 ng ml-1 resulted in a slightly smaller colony size. MMC (200 ng ml-1) did not allow for growth of B. subtilis cells (Table 1). In contrast, a recN deletion strain already showed strongly compromised growth on 50 ng ml-1 and formed few colonies on 100 ng ml-1 MMC (Fig. 1A), while a recO deletion strain hardly grew on 50 ng ml-1 and not at all on 100 ng ml-1. In a recF15 mutant strain (the mutation results in a null allele), growth was also compromised at 50 ng ml-1, comparable to recN cells (Table 1). DSB repair was most severely affected by a recA deletion: the mutant strain did not form any colonies on 50 ng ml-1 MMC (Table 1). We also tested an addAB mutant strain, which is the counterpart of a recBCD mutant strain of E. coli. This strain showed a growth defect at 100 ng ml-1, while growth was similar to that of wild-type cells at 50 ng ml-1 (Table 1). Table 1. Growth of different strains on plates containing mitomycin C (MMC) and survival after MMC treatment. MMC (ng ml-1)

Survival rate

Strain (mutation)

None

50

100

200

%50a

%100a

wt (PY79) DrecN recN–yfp DrecO recO–yfp recF15 addA5 addB72 DrecA recF–cfp, recN–yfp recF–yfp, recO–cfp

+++ +++ +++ ++ +++ +++ +++ ++ +++ +++

+++ + +++ (+) +++ + ++ – ++ +

+++ – ++ – ++ – + – + –

– – – – – – – – – –

50 22 49 6 50 24 29 450 cells analysed). Strikingly, 60 min after addition of 100 ng ml-1 MMC, ª70% of the cells showed fluorescent foci (Fig. 2B). In general, cells contained one visible focus and, rarely, two or three foci. MMC treatment led to a strong decrease in the growth rate, and cells elongated for some time without dividing (which is apparent because treated cells are much longer than untreated cells, compare Fig. 2A and B). In contrast to uninduced cells, in which ª30% contain two separated nucleoids (Fig. 2A, right), MMC-treated cells contained almost exclusively single elongated nucleoids (Fig. 2B, middle), suggesting that nucleoids fuse upon induction of DSBs. RecN foci were generally present on the nucleoids (Fig. 2B, right), although foci were occasionally found in spaces where DNA stain was undetectable, possibly because of DNA ends that have diffused away from the nucleoids. To verify that RecN forms foci in response to DSBs rather than to DNA modifications, we exposed exponentially growing cells to different doses of hard X-rays from a linear electron accelerator. In contrast to exponentially growing cells (Fig. 2D, top), this treatment induced fluorescent RecN–YFP foci indistinguishable from that caused by MMC, with a similar number of cell containing one to, rarely, three foci (Fig. 2D). Additionally, we treated cells with inhibitors of DNA gyrase. Nalidixic acid inhibits gyrase through a mechanism that generates DSBs, while inhibition through novobiocin does not cause DSBs. Addition of nalidixic acid to growing cells induced RecN foci in 75% of the cells, while addition of novobiocin led to RecN foci in only 3% of the cells (most likely through inhibition of DNA replication, which can lead to DSBs) (Fig. 2E and F). These results show that RecN foci are induced by true DSBs, and not solely by an effect specific to MMC. Similar to RecN–YFP, RecO–YFP and RecF–YFP accumulated in discrete fluorescent foci after treatment with MMC (Fig. 3A–C). To test whether RecN, RecO and RecF are all recruited to the same sites after MMC treatment, we created CFP fusions of RecN, RecO and RecF for simultaneous visualization of the proteins in the cells. RecN–CFP and RecO–YFP co-localize to the MMCinduced foci (Fig. 2C, grey arrows), with RecO–YFP being present in all RecN–CFP foci (in 55 cells containing clear foci). It was difficult to co-localize RecN and RecF, because fluorescent foci were very faint, but in all cells with clear foci (25), RecN–CFP and RecF–YFP were coincident (data not shown). Although RecF–CFP or RecO– YFP formed foci separately after MMC treatment, cells expressing both RecF–CFP and RecO–YFP did not form any foci for both fusions (Fig. 3J; instead, RecF–CFP localized all over the nucleoids, left), suggesting that CFP tagging of both proteins interferes with their direct inter-

action. Interestingly, a RecN–CFP/RecF–YFP strain was more sensitive to 50 ng ml-1 MMC than wild-type cells, whereas a RecF–CFP/RecO–YFP strain was as sensitive to MMC as the recO null mutant (Fig. 1B, Table 1), showing that an impairment in the formation of foci is deleterious for cells to cope with DSBs. These data suggest that RecN, RecO and RecF form active repair centres (RCs) that play a vital role during DSB repair. RecN, RecO and RecF are differentially recruited to putative DSB repair centres We performed time course experiments to assess the time-dependent accumulation of RecN, RecO and RecF in the RCs. Clear RecN foci were visible as soon as 20– 30 min after treatment with MMC, with 35% of the cells containing visible foci (Fig. 3A, left) after 30 min, which increased to 60% after 60 min (Fig. 3A, middle) and to 70–75% after 120 min (Fig. 3A, right). RecN foci became fainter 3 h after induction of DSBs (with growth resuming 2–3 h after MMC treatment), but could still be observed for as long as 5 h. In contrast to RecN, foci of RecO–YFP were first visible 60 min after induction (Fig. 3B, middle), with peak induction after 120 min, whereas RecF–YFP foci were observed after about 90–120 min (Fig. 3C, right). Intensity of RecF–YFP foci also peaked at 120 min. Interestingly, RecF first appeared to localize to whole nucleoids before clear foci were apparent (Fig. 3C, middle). In dual-labelling experiments, cells were frequently observed that contained RecN–CFP foci but no RecO– YFP foci (in Fig. 2C, two RecN–CFP foci are indicated by white arrowheads in which RecO–YFP is not detectable), supporting the finding that RecO localizes to the foci after RecN. Likewise, RecN–YFP foci were visible that did not contain RecF–CFP fluorescence (data not shown), verifying the temporal order of recruitment to RCs. These results show that B. subtilis has a dynamic response to DSBs in which proteins are differentially recruited to defined RCs. Interdependence of repair proteins for proper formation of RCs To investigate a possible dependence of localization of RecNOF and other known repair proteins, we moved the YFP fusions into various mutant backgrounds. RecN, RecO and RecF formed inducible foci indistinguishable from those in wild-type cells in the absence of RecG, RecH and RecU (Fig. 3D, Table 2), which are members of three other epistatic groups of recombination proteins. In agreement with these findings, RecG and RecU (counterparts of RecG and RuvC from E. coli) are proposed to act during post-synapsis and thus downstream of RecNOF. RecN and RecO also formed normal foci in the absence of RecF © 2004 Blackwell Publishing Ltd, Molecular Microbiology, 52, 1627–1639

DNA double-strand break repair in live bacteria 1631

Fig. 2. Fluorescence microscopy of B. subtilis strains. A. Strain AV1 (recN–yfp) growing exponentially and (B) 60 min after the addition of 100 ng ml-1 MMC. Note that a fluorescent focus in exponentially growing cells (in 0.05% of the cells, indicated by an arrow in A) is an extremely rare event. Left: YFP/membrane overlays, DAPI–DNA stain. (B) right: YFP/DAPI overlay, arrows point out fluorescent foci. C. Strain DK8 (recN–cfp, recO–yfp) 90 min after 100 ng ml-1 MMC. Grey arrows point out coincident CFP and YFP foci; white arrowheads indicate RecN–CFP foci lacking RecO–YFP fluorescence. D. Strain AV1 (recN–yfp) 60 min after treatment with different doses of hard X-rays. E. Strain AV1 (recN–yfp) 60 min after treatment with novobiocin. F. Strain AV1 (recN–yfp) 60 min after treatment with nalidixic acid. White bars indicate 2 mm; white lines ends of cells. © 2004 Blackwell Publishing Ltd, Molecular Microbiology, 52, 1627–1639

1632 D. Kidane, H. Sanchez, J. C. Alonso and P. L. Graumann

Fig. 3. Fluorescence microscopy of B. subtilis strains. A. Strain AV1 (recN–yfp). B. strain DK2 (recO–yfp). C. strain DK4 (recF–yfp) at time points indicated above after the addition of MMC. Arrows point out fluorescent foci. D–G, I and J. Images taken 2 h after addition of MMC. D. Strain DK19 (recN–yfp, DrecU). E. Strain DK30 (recN–yfp, addA5 addB72). F. DK25 (recF–yfp, DrecN). G. DK33 (recF–yfp, DrecO). H. DK14 (recN–yfp, DrecA) in the absence or (I) in the presence of MMC. J. Strain DK9 (recF–cfp, recO-yfp). © 2004 Blackwell Publishing Ltd, Molecular Microbiology, 52, 1627–1639

DNA double-strand break repair in live bacteria 1633 Table 2. Localization of RecN/F/O in different mutant backgrounds using 100 ng ml-1 MMC. Mutant

RecN–YFP

RecF–YFP

RecO–YFP

DrecN DrecO recF15 addA5 addB72 recH342 DrecU a DrecG DrecAa

NA ++ + + ++ + ++ +

(++) – NA + ++ ++ ++ NA

+ NA + + ++ ++ ++ NA

++, normal foci (number and intensity); +, faint foci; (++), aberrant foci; –, no foci. a. Foci also in the absence of MMC. At least 300 cells have been scored for each fusion in each mutant background.

(Table 2), whereas RecF failed to form foci in the absence of RecO (Fig. 3G, note the higher background fluorescence compared with wild-type cells in Fig. 3C; Table 2), suggesting that RecO recruits RecF to the repair foci. This is consistent with a direct interaction of RecO and RecF found in several biochemical studies (Hegde et al., 1996; Bork et al., 2001) and in this work (see above). RecO formed foci in recN mutant cells upon addition of MMC; however, frequently, foci appeared to be rather patchy (data not shown; Table 2), while RecF–YFP was predominantly found in large patches on the DNA rather than in defined foci in recN cells (Fig. 3F). This shows that RecN is important for the proper formation of RCs. Severe damage to DNA induces the SOS response, resulting in an increase in transcription of genes involved in DNA repair (including RecN) and error-prone replication (Sutton et al., 2000). The SOS response is abolished in the absence of RecA and, indeed, the number of RecN molecules (a few hundred in the absence of damage) increases several fold after the addition of MMC, dependent on RecA (S. Ayora, personal communication). To test whether an increase in the level of RecN is required for the formation of foci, RecN–YFP was monitored in a recA deletion strain. After the addition of MMC, >90% of the recA cells contained RecN–YFP foci (often multiple); however, the foci were usually fainter than those observed in cells carrying a functional recA gene (except for addAB cells, see below). These data show that RCs can form in the absence of RecA, and thus under a limited number of

RecN molecules. Interestingly, even in the absence of MMC during exponential growth, RecN–YFP foci were present in about 35% of recA null cells. Likewise, 5% of exponentially growing cells carrying a recU deletion contained RecN–YFP foci, which increased to more than 70% of mutant cells after the addition of MMC. These findings suggest that DSBs occur frequently in the absence of RecU and RecA during normal growth, which explains at least in part why both deletions result in slow growth and in a chromosome segregation defect (Pedersen and Setlow, 2000; Sciochetti et al., 2001). Additionally, RecN– YFP foci were detected in addAB mutant cells, but they were much fainter compared with wild-type cells (Fig. 3E). This can be explained by our finding that the SOS response is strongly reduced in addAB cells, resulting in lower levels of RecN (data not shown). These data underscore the interpretation that RecN/O/F form defines RCs that probably act upstream of RecG, RuvAB and RecU, and show that RecO plays a central role in the formation of the structures, while RecN appears to be important for the organization of these subcellular structures. Induction of RCs does not depend on the dosage of MMC We performed induction of DSBs by increasing concentrations of MMC to assess the dosage dependence of foci formation. Sixty minutes after the addition of the sublethal dose of 50 ng ml-1 MMC, about 55% of the cells showed clear RecN–YFP foci, with 2% of the cells two or three foci (Fig. 4, left). Addition of the lethal dose of 250 ng ml-1 MMC resulted in the formation of RCs in 72% of the cells (Fig. 4, right), with 3% containing two or three foci. These results reveal that the number of cells containing foci increases slightly with an increase in MMC concentration, whereas surprisingly, the number of foci per cell does not increase markedly with a higher number of DSBs. Likewise, we did not observe a strong dosage dependence after X-ray irradiation. While irradiation with 2 Gy or 8 Gy did not cause severe reduction in doubling time, 20 Gy caused pronounced retardation of growth, suggesting that the latter is restrictive for growth of B. subtilis cells (12 Gy is lethal for fibroblasts). However, the number of foci per cell did not increase more than 1.5-fold between 2 Gy and

Fig. 4. Fluorescence microscopy of strain AV1 (recN–yfp) 60 min after the addition of 50 (left l) or 250 ng ml-1 (right) MMC. White bars 2 mm; white lines indicate ends of cells.

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 52, 1627–1639

1634 D. Kidane, H. Sanchez, J. C. Alonso and P. L. Graumann 20 Gy (Fig. 2D). Possibly, B. subtilis cells have only a limited capacity to form DSB RCs, such that the cells can only deal with a limited number of DNA DSBs. Alternatively, RCs could contain several DSBs that are repaired within the superstructure, which might be able to accommodate a limited number of breaks. RecN forms multimers in solution and DSB-induced high-molecular-weight complexes The fluorescence of RecN foci relative to the background levels of the surrounding regions indicates that the foci might represent large RecN aggregates. As a first step to shed light on the mode of action of RecN, a plasmid-borne recN gene was overproduced in and purified from B. subtilis cells. RecN has a pI of 4.5 and a molecular weight (Mr) of 64 000 under denaturing SDS-PAGE. However, the protein eluted exclusively in a single peak with Mr of ª500 000 in the presence of 50–500 mM NaCl during gel filtration chromatography (Fig. 5A). RecN protein eluted with Mr of ª500 000 at a concentration as low as 10 nM (ruling out an artifact through high protein concentration), and no RecN protein was found in the range of 64 kDa (Fig. 5). Assuming that RecN has a spherical structure, our analysis would suggest that RecN forms an octamer in solution. However, RecN is predicted to form an elongated head–coil–hinge structure similar to SMC proteins and, indeed, electron microscopy (EM) studies have shown that RecN is present as a short rod-shaped or U-shaped extended flexible rod-like oligomer (R. Lurz and J. C. Alonso, unpublished results). Because gel filtration does not reflect the true size of elongated molecules, we also confirmed the molecular mass of RecN by analytical centrifugation. After centrifugation to equilibrium, RecN behaved as a protein with an apparent mass of ª480 kDa, but fractions containing RecN protein with lower molecular masses were also observed (Fig. 5B, top). This supports the notion that RecN forms octamers in solution, that is, based on the EM studies, four RecN dimers, as well as intermediates (e.g. tetramer and dimers). Using SMC as a model, the (smaller) coiled-coil arms of a RecN dimer could form a ring with a diameter of 20 nm that can easily accommodate two DNA molecules plus additional proteins. To analyse whether RecN accumulates even in higher order structures in cells after induction of DSBs, cell extract from MMC-treated cells was loaded onto a glycerol gradient. Indeed, RecN was found to shift to higher molecular weight fractions (Fig. 5B, bottom, lanes 1 and 10–12), and even formed high-molecular-weight structures that were resistant to SDS, most probably nucleoprotein complexes (NCP, lane 1). These data show that RecN is able to form large structures, which are strongly enriched in the presence of damaged DNA and might form a platform for the organization of the observed DSB RCs.

Fig. 5. Native molecular mass of B. subtilis RecN protein. A. Elution profile of purified RecN on a Superose 12 HR 10/30 column in buffer containing 500 mM NaCl. Protein standards, in kDa, are indicated by arrows. The insert shows relevant fractions as separated by SDS-PAGE. B. Pure RecN protein (top) and an enriched fraction of an MMCdamaged cell (bottom) were applied on to 15–30% glycerol gradient in buffer B and separated by centrifugation. The samples were collected from the bottom of the tube, separated by SDS-PAGE, electroblotted and the RecN protein was highlighted with anti-RecN polyclonal antibodies. The fractions containing RNAP (400 kDa) and ALD (158 kDa) are indicated. The BSA protein (69 kDa) eluted at fraction 16 (not shown). The positions of the RecN protein and of the putative NPCs are denoted.

Discussion This report provides evidence for a dynamic recruitment of DNA repair proteins to distinct sites on the nucleoids in response to DSBs in a prokaryote. RecN, RecO and RecF, © 2004 Blackwell Publishing Ltd, Molecular Microbiology, 52, 1627–1639

DNA double-strand break repair in live bacteria 1635 which we found to be important for DNA DSB repair, localized to distinct foci on the DNA in B. subtilis cells after induction of DSBs. Several lines of evidence indicate that the proteins might form active DSB RCs in which DSBs are repaired through homologous recombination with the sister chromosome. RecN/O/F are involved in and are essential for efficient DNA recombination in vivo (Fernandez et al., 2000) and co-localized in the induced foci. Recruitment of proteins to the RCs was dynamic, with RecN localizing 20–30 min after induction of DSBs, whereas RecO localized later, followed by RecF. Because loading of RecA onto ssDNA (that is coated with SSB because SSB has much higher affinity to ssDNA than RecA) depends on RecO and RecR in vitro (Webb et al., 1997; Morimatsu and Kowalczykowski, 2003), RecA is likely to be recruited to the RecNOF foci. Our preliminary results suggest that, indeed, RecA forms foci upon induction of DSB. RecF was shown to interfere with the RecO– RecR interaction in vitro, possibly limiting the loading and spreading of RecA (Webb et al., 1997; Morimatsu and Kowalczykowski, 2003), which is supported in vivo by the recruitment of RecF to RCs. In the absence of RecN, RecF and RecO formed irregular patches on the DNA, while RecF localized throughout the cells in a recO deletion strain. This is consistent with the findings that RecF has similar affinity to ssDNA and dsDNA in vitro, without a clear preference for gapped DNA (Ayora and Alonso, 1997), and suggests that RecN organizes the RCs, and that RecO recruits RecF (as well as RecA). The idea that RecNOF initiate DSB repair through recombination is supported by our findings that RCs were formed in the absence of RecA, RecG and RecU, which act at the synapsis and post-synapsis stage of recombination (Cromie et al., 2001). Further, several observations show that the observed foci are required for DSB repair. First, in the absence of any of the proteins, B. subtilis cells become highly sensitive to DSB-inducing agents. Secondly, simultaneous GFP tagging of RecF and RecO abolished the formation of both RecO and RecF foci, resulting in MMC sensitivity similar to the recO null phenotype (while each fusion by itself is fully functional). A less pronounced but noticeable defect was also found in a RecN–YFP/RecF–CFP dual tag strain. Thus, RecO and RecF, and RecF and RecN interact in vivo, and interference with the formation of RCs is deleterious for DSB repair. Along the same lines, we found that the intensity of RecN foci (and thus the amount of RecN in the RCs) was reduced in addAB mutant cells. In these cells, the increase in the amount of RecN in response to DSBs is lessened, leading to enhanced sensitivity at high, but not at low, concentrations of MMC. Our data show that, unlike E. coli cells in which repair of ssDNA gaps is thought to be recF dependent and healing of DSBs recBCD dependent (Kowalczykowski et al., © 2004 Blackwell Publishing Ltd, Molecular Microbiology, 52, 1627–1639

1994), B. subtilis RecN, RecO and RecF, in an otherwise wild-type background, play important roles in the repair of DSB promoted by MMC. The induced foci started to dissipate around the time at which growth resumed after DSB induction, suggesting that the putative RCs are active throughout repair during growth arrest. In eukaryotic cells, Rad51 (homologous to bacterial RecA) and two homologues have been shown to form foci after induction of DSBs (Liu et al., 1999; Essers et al., 2002), similar to Rad50 that was shown to colocalize with its complex partner Mre11 (Maser et al., 1997; Nelms et al., 1998). Mre11 migrated to the DSB sites within 30 min, thus within a similar time frame to RecN in the bacterial cells. Recently, Rad52 has been shown to relocate to sites of DSBs, and several DSBs can be accommodated within the Rad52 foci (Lisby et al., 2003). If Rad52 is indeed the eukaryotic counterpart of RecO (Kantake et al., 2002), eukaryotic cells might have a RecN equivalent that is loaded before Rad52. In contrast to eukaryotic cells, in which sister chromosomes are paired from S phase through G2 to anaphase in mitosis, growing bacterial cells segregate their chromosomes during ongoing DNA replication (Graumann, 2001). Soon after initiation of replication, duplicated chromosome regions are actively and rapidly separated towards opposite cell poles and, therefore, duplicated regions are usually located within each cell half (Fig. 6).

Fig. 6. Model for DSB repair in prokaryotic cells. The replicative DNA polymerase (Pol III) machinery is located at the cell centre, and sister chromosomes are separated towards opposite cell poles soon after initiation of, and thus during, replication (indicated by horizontal arrows). The SMC-like RecN protein is the first observed to localize as discrete foci on the DNA, most probably at the site of the DSB. RecO is recruited to the putative repair centre (RC) somewhat later, being required for recruitment of RecF as shown in this work, and of RecA as shown in several biochemical studies (see text for references). Because RecA mediates homologous recombination, the intact region on the sister chromosome (indicated by !) must be paired with the site of the DSB, and thus either one or both sites must be translocated for synapsis (indicated by dashed arrow).

1636 D. Kidane, H. Sanchez, J. C. Alonso and P. L. Graumann The importance in DSB repair of proteins that are involved in DNA recombination suggests that at least some DSBs are repaired through homologous recombination with the intact sister chromosome, which is a novel concept for prokaryotic cells (see Fig. 6 for a model). Another striking observation of this study is the apparent dosage independence of the formation of RCs. That is, usually one and seldom two or three centres were observed in cells, regardless of whether the cells were treated with a sublethal or a lethal dose of MMC or hard X-rays. Thus, although the number of breaks must increase according to the concentration of MMC or irradiation, the number of RecNOF foci did not increase more than 1.5fold. Although it is possible that B. subtilis cells can only deal with one or up to three DSBs, we favour the view that several DSBs could be organized or even recruited into main RCs by RecN. We have found that the amount of RecN is one rate-limiting step in DSB repair, and that purified RecN forms multimers (octamers) in solution. We speculate that RecN multimers recognize or are recruited to individual DSBs that are combined and organized into RCs by further RecN protein–protein interactions, which would explain the limited number of RecN foci. Our data suggest that DNA DSBs occur in growing cells in the absence of an external threat, because RecN or RecF foci were present in 0.05% of normally growing cells. In contrast, RecN foci were present in 5% of cells containing a recU deletion and even more frequently in a recA deletion strain, suggesting that, in both cases, DSBs occur during normal growth, which leads to compromised growth seen in both mutant backgrounds. It is likely that arrest and reversion of replication forks followed by repair through homologous recombination gets stuck in recA and recU mutant cells, leading to DSBs. This indicates that the event of a stalled replication fork occurs in growing cells, but is efficiently repaired in normally growing cells. Combining the data from this work, it is possible to describe the following scenario after the occurrence of DNA DSBs. RecN is an initiator of putative RCs that accumulates or assembles at DSB sites (Fig. 6). We speculate that RecN induces or maintains a DNA topology at a DSB that facilitates loading of RecO/F and thus RecA, and organizes DSBs in large RCs based on its ability to form multimers and high-molecular-weight complexes after induction of DSBs. RecN is similar to SMC proteins (but with shorter coiled-coil arms than SMC) that condense DNA or hold sister chromosomes together by embracing DNA with their long arms, and influence DNA topology, e.g. condensin introduces positive writhe into DNA (Kimura et al., 1999). RecO is recruited to DSBs somewhat later and facilitates loading of RecA to ssDNA, aided by RecR. RecF is recruited by RecO (Fig. 6), possibly limiting the spreading of RecA. At this point, the homologous region on the sister chromosome that is

located in the other cell half (Fig. 6, indicated by a !) must be positioned in proximity to the DSB site. This mechanism is still to be elucidated but, in any event, our data argue in favour of the existence of it. Ensuing presynapsis by RecNOFR and synapsis by RecA, RecG and other repair proteins such as RecU apparently act downstream of RecNOFR (because RecNOF foci formed independently of several of these proteins) and mediate postsynapsis with final resolution of cross-overs. DNA Pol I and DNA ligase are needed for filling of gaps and might also be recruited to the putative RCs. Further work will shed more light on the repair of DSBs in live cells. Experimental procedures Bacterial strains and media Escherichia coli XL1-Blue (Stratagene) was grown in Luria– Bertani (LB) rich medium supplemented with 50 mg ml-1 ampicillin where appropriate. B. subtilis strains were grown in LB rich medium at 37∞C. All strains used in this study are listed in Table 3. For assay of DSB survival rate, cells growing Table 3. Strains used in this study. Strains

Relevant genotypes

References

BG129 BG119 BG189 BG439 BG427 BG190 BG277 BG691 DK34 DK35 DK36 AV1 DK1 DK2 DK3 DK4 DK5 DK8 DK9 DK10 DK12 DK13 DK14 DK15 DK19 DK20 DK21 DK22 DK23 DK24 DK25 DK26 DK27 DK28 DK29 DK30 DK31 DK32 DK33

recF15 recH342 addA5 addB72 recO::cat (DrecO) recU::cat (DrecU) recA::cat (DrecA) recN::cat (DrecN) recG::cat (DrecG) recF::tet (DrecF) recN::tet (DrecN) recO::tet (DrecO) recN–yfp recN–cfp recO–yfp recO–cfp recF–yfp recF–cfp recO–yfp, recN–cfp recO–yfp, recF–cfp recN–yfp, recF–cfp recN–yfp DrecG recN–yfp, DrecR recN–yfp, DrecA recN–yfp, DrecO recN–yfp, DrecU recO–yfp, DrecU recF–yfp, DrecU recN–yfp, recH342 recO–yfp, recH342 recF–yfp, recH342 recF–yfp, DrecN recO–yfp, DrecN recN–yfp, recF15 recN–yfp, recF7 recO–yfp, recF15 recN–yfp, addA5 addB72 recF–yfp, addA5 addB72 recO–yfp, addA5 addB72 recF–yfp, DrecO

Alonso et al. (1988) Alonso et al. (1988) Alonso et al. (1988) Alonso et al. (1991) Alonso et al. (1991) Alonso et al. (1991) Alonso et al. (1991) This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 52, 1627–1639

DNA double-strand break repair in live bacteria 1637 at 37∞C with an OD of 0.5 were either directly plated or treated with 50 or 100 ng ml-1 MMC for 20 min. After serial dilution, cells were plated and incubated at 37∞C overnight. For microscopy, cells were grown in S750 defined medium (Jaacks et al., 1989) at 25∞C. For induction of DNA DSBs, 100 ng ml-1 MMC was added.

PCR and construction of fluorescence tag vectors and strains To create C-terminal fusions of RecN with YFP or CFP for single cross-over integration into the chromosome, the 3¢ (600 bp) region of the recN gene was amplified by polymerase chain reaction (PCR) using primers 5¢-ATTGGTACC TGTACATGGAAAAATCAAC-3¢ and 5¢-TTAGAATTCTCCGC CCCCAGTTGTTTTGACTTGATC-3¢ and was cloned into KpnI and EcoRI sites of pSG1186 or pSG1187 (Feucht and Lewis, 2001), resulting pcDK5 and pyAV1. C-terminal fusions of recO with yfp and cfp were created by cloning of the 3¢ (700 bp) region of the recO gene (amplified by PCR using primers 5¢-ATATGGGCCCTAACATTACTGACAAGAGAAC AC-3¢ and 5¢-CCATCGATACTTTTGTTTTCACCCATAAGA TG-3¢) into ApaI and ClaI sites of pSG1186 and pSG1187 (Feucht and Lewis, 2001), resulting in pcDK6 and pyDK7. A C-terminal fusion of recF with yfp or cfp was created by cloning of the 3¢ (500 bp) region of the recF gene (amplified using primers 5¢-GCAGGGCCCGATCAGCTTGTAGTAGA AGTTG-3¢ and 5¢-TATGAATTCACCCC-CCTTCACTAACGC ACCATTTTG-3¢) into EcoRI and ApaI sites of modified pSG1164y and pSG1164c (Lewis and Marston, 1999), resulting pyDK8 and pcDK9. To move fluorescent protein fusions of RecN, RecO or RecF into different mutant backgrounds, strains DrecN, DrecO, recF15, recH342 and addA5 addB72 were transformed with chromosomal DNA from DK34, DK35, DK36 (recN–yfp, recO–yfp or recF–yfp respectively), and strains DK34, DK35 and DK36 (recN–yfp, recO–yfp or recF– yfp respectively) were transformed with DNA of DrecU, DrecG and DrecA mutant strains (Table 3).

Image acquisition Fluorescence microscopy was performed on an Olympus AX70 microscope. Cells were mounted on agarose pads containing S750 growth medium on object slides. Images were acquired with a digital MicroMax CCD camera; signal intensities and cell length were measured using the METAMORPH 4.6 program. DNA was stained with 4¢,6-diamidino-2phenylindole (DAPI; final concentration 0.2 ng ml-1), and membranes were stained with FM4-64 (final concentration 1 nM).

Purification of RecN protein Bacillus subtilis RecN protein was overexpressed from YB886 containing a pSOS18-borne recN gene, which is under the transcriptional control of the SOS-inducible recA promoter. YB886 bearing pSOS18 was grown at 37∞C in LB broth with neomycin (5 mg ml-1), and expression of RecN was induced by the addition of MMC to a final concentration of 250 ng ml-1 for 2 h. Cells were harvested, © 2004 Blackwell Publishing Ltd, Molecular Microbiology, 52, 1627–1639

resuspended in buffer A [50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 5% glycerol] containing 150 mM NaCl and disrupted by addition of lysozyme (500 ng ml-1) followed by sonication. The 64 kDa RecN protein was found in the soluble fraction. Polyethylenimine was added, and the mixture was spun at 30 000 g for 30 min. The supernatant was subjected to ammonium sulphate precipitation (60% saturation). The pellet was recovered, dialysed against buffer A to give a final concentration of 150 mM NaCl and loaded onto a Q-Sepharose column equilibrated with the same buffer. The column was washed with buffer A containing 175 mM NaCl and eluted by a step gradient from 175 to 500 mM NaCl. Fractions containing RecN, which eluted at 350 mM, were recovered and loaded onto a Superose 12 HR 10/30 column (Pharmacia). The peak fractions were pooled, aliquoted and stored at -20∞C. The N-terminus of the purified protein was sequence by automatic Edman degradation. The RecN protein was also purified towards homogeneity from E. coli overexpressing cells with the aim of producing high-quality polyclonal antibodies. Rabbit polyclonal antibodies against B. subtilis RecN protein purified from an E. coli overexpression system were obtained using conventional techniques.

RecN molecular mass determination The native molecular mass of RecN protein was analysed by gel filtration fast protein liquid chromatography (FPLC) using a Superose 12 HR 10/30 column (Pharmacia). Chromatography was carried out in buffer A containing 500 mM NaCl at a flow rate of 0.5 ml min-1, and the A280 was recorded. A standard curve of Kav versus log10 relative mobility was determined as recommended by the manufacturer. Protein standards were obtained from Pharmacia (chymotrypsinogen A, 25 kDa; ovalbumin, 43 kDa; albumin, 67 kDa; aldolase, 158 kDa; catalase, 232 kDa; ferritin, 440 kDa; and thyroglobulin, 669 kDa). Purified RecN was injected on to a Superose 12 column pre-equilibrated in buffer A containing 500 mM NaCl, and the collected fractions were analysed by SDS-PAGE. Pure RecN or an enriched extract containing RecN was applied onto a 5 ml linear gradient of 15–30% glycerol that was prepared in buffer B (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA and 7 mM b-mercaptoetanol). B. subtilis RNA polymerase (RNAP, 400 kDa), aldolase (ALD, 158 kDa) and BSA (69 kDa; not shown in Fig. 5B) were used as protein standards. After a 17 h centrifugation at 200 000 g at 4∞C, 200 ml fractions were collected, separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) Immobilon-P transfer membrane (Millipore) according to standard procedures. The membrane was probed with anti-RecN polyclonal antibodies, and the antibody detection was performed according to standard techniques.

Acknowledgements We thank Myriam Calonge for the gift of pSOS18 plasmid, Silvia Ayora and Rudi Lurz for the communication of unpublished results, Ashwani Verma for technical assistance, and

1638 D. Kidane, H. Sanchez, J. C. Alonso and P. L. Graumann Hans-Otto Neidel for assistance with the linear electron accelerator. This work was supported by the Deutsche Forschungsgemeinschaft (Emmy Noether Programme) and the Fonds der Chemischen Industrie to P.L.G., and BMC200300150 and BIO2001-4342-E from DGCICYT and QLRT2000-00365 from the EU to J.C.A.

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