RecA Protein Recruits Structural Maintenance of Chromosomes (SMC)-like RecN Protein to DNA Double-strand Breaks*

Background: RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA double-strand breaks (DSBs) repair. Results: We identified a RecA mutant that is deficient in interacting with RecN. Conclusion: A functional interaction between RecN and RecA is required for assembly of RecN at the sites of DSBs. Significance: RecN is critical for protecting the structural integrity of chromosomes during DSBs repair. Escherichia coli RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA double-strand break (DSB) repair. Previous studies show that GFP-RecN forms nucleoid-associated foci in response to DNA damage, but the mechanism by which RecN is recruited to the nucleoid is unknown. Here, we show that the assembly of GFP-RecN foci on the nucleoid in response to DNA damage involves a functional interaction between RecN and RecA. A novel RecA allele identified in this work, recAQ300R, is proficient in SOS induction and repair of UV-induced DNA damage, but is deficient in repair of mitomycin C (MMC)-induced DNA damage. Cells carrying recAQ300R fail to recruit RecN to DSBs and accumulate fragmented chromosomes after exposure to MMC. The ATPase-deficient RecNK35A binds and forms foci at MMC-induced DSBs, but is not released from the MMC-induced DNA lesions, resulting in a defect in homologous recombination-dependent DSB repair. These data suggest that RecN plays a crucial role in homologous recombination-dependent DSB repair and that it is required upstream of RecA-mediated strand exchange.

dependent repair of DSBs (31)(32)(33). Thus, RecN plays a specific role in the repair of DNA DSBs, and its role is not limited to a single branch or subpathway of HR.
In this study, we examine the mechanism by which RecN is recruited to the nucleoid in response to DNA damage. We show that a functional interaction between RecN and RecA is required for assembly of RecN foci at MMC-induced DSBs; conversely, conditions that abrogate or disrupt a stable RecN-RecA interaction lead to chromosome fragmentation and loss of cell viability in cells exposed to MMC. The RecN ATPase is not required for formation of RecN-DSB foci, but is required for release of RecN from DSBs and completion of RecA/HR-dependent DSB repair. These data demonstrate that the SMC-like protein RecN plays a crucial role in promoting RecA-dependent DSB repair.

EXPERIMENTAL PROCEDURES
Media and General Methods-Standard methods for E. coli genetics and recombinant DNA techniques were as described by Miller (34) and Sambrook et al. (35). Ampicillin (50 g/ml), tetracycline (10 g/ml), chloramphenicol (100 g/ml), and kanamycin (30 g/ml) were used where indicated. Mitomycin C (2.5 mg/ml) was dissolved in 10 mM Tris-HCl (pH 8.5) buffer. Sensitivity to UV damage was measured as described previously (36). To measure sensitivity to MMC, cultures were grown in LB broth to an A 650 of ϳ0.4, serially diluted, spotted onto LB medium containing the indicated concentration of MMC, and incubated at 37°C.
Bacterial Strains and Plasmids-Strains used in this study were isogenic with BW25141 (37) except for strains with P BAD -I-SceI. Wild-type strains and deletion mutants were provided by the National BioResource Project (NBRP) (38). The strains carrying the P BAD -I-SceI were a gift from S. M. Rosenberg (39). The strains carrying the inducible fluorescent repressor gene (araC PBAD-lacI-ecfp) and the lacO array were described previously (40). A fragment containing the SOS promoter and open reading frame of recN was cloned into the low copy plasmid pSCH19, generating pRecN (27). RecN was tagged with an enhanced GFP cassette at its NH 2 terminus to generate pSG101. Arabinose-inducible pBAD GFP-recN (pTF271) was constructed as described previously (27). recN K35A was generated from pUC19-recN by site-directed PCR mutagenesis, and it was substituted for wild-type recN in pSG101 to generate pSG105. The structures of recombinant plasmids were confirmed by DNA sequencing.
Isolation of recA Mutant-A library of recA mutants was generated by carrying out PCR-mediated random mutagenesis, as described previously (36). The resultant recA mutant clones were transformed into a recA strain. The transformants were resuspended in M9 salts, plated on LB plates containing ampicillin, and then irradiated with UV (20 J/m 2 ). After overnight incubation, UV-resistant colonies were replica-plated on LB plates containing MMC (1 g/ml). Clones that grew very poorly or did not grow at all on the MMC plates were selected.
SOS Induction-SOS induction was assessed by measuring the degradation of LexA as described previously (36). Log phase cultures were treated with MMC (0.5 g/ml), and aliquots were taken at multiple time points for immunoblot analysis with anti-LexA (BioAcademia), anti-RecA (BioAcademia), or anti-RecN (26). LexA resynthesis was inhibited by adding chloramphenicol (100 g/ml) to the cultures 10 min before adding MMC.
Fluorescence Microscopy and Localization Analysis of GFP-RecN-Exponentially growing cultures were treated with 0.5 g/ml MMC at 37°C. Cells were fixed with methanol, stained with 1 g/ml DAPI (4Ј, 6Ј-diamidino-2-phenylindole), and spread (1-2 l) on a cover glass. GFP fluorescence was not affected by prior fixation of the sample. Fluorescence microscopy was performed on a Zeiss Axioplan2 (41). Scale bars of 5 or 10 m are shown in the figures. Images of both nucleoids (blue as a color signal) and GFP-RecN foci (green as a color signal) were merged on identical cells. In the merged image, GFP-RecN foci localized on the nucleoid appeared as a light blue color, whereas GFP-RecN foci in the cytoplasm appeared as a green color. The localization of GFP-RecN foci was determined based on these visual criteria, and more than 150 individual cells were scored for each strain.
To observe the ori1 loci (15 kbp distance from oriC) on chromosomes, we constructed wild-type and ⌬recN strains carrying both a lacO array inserted into the ori1 locus and an inducible fluorescent repressor gene (araC PBAD-lacI-ecfp) (40,42,43). Cells were grown at 37°C for 90 min in LB medium containing 0.2% arabinose and 1 g/ml MMC and then analyzed by fluorescence microscopy.
Effect of I-SceI-induced DSBs on the Localization of RecN-To detect DSB-induced RecN foci, ⌬recA strains carrying the P BAD I-SceI cassette in the chromosome and a single I-SceI recognition site at the codA21 locus in the FЈ episome (39) were transformed with pSG101 (gfp-recN) and with pRecA or pRecA Q300R . When cultures had reached early log phase, I-SceI was induced by the addition of 0.2% arabinose (w/v). One hour after the addition of arabinose, aliquots were taken and examined under the microscope.

RESULTS
RecA Is Required for the Formation of Nucleoid-associated GFP-RecN Foci in MMC-treated Cells-When DNA is damaged or replication is inhibited, ssDNA-bound RecA becomes conformationally active and promotes cleavage of the LexA repressor, which results in the induction of SOS genes including recN (21,44). Previously, we showed that GFP-RecN formed foci on nucleoids after DNA damage (27). This implied that RecN could be recruited to the nucleoid at a step after RecA is loaded onto damaged DNA. To specifically examine this sequence of events, we measured GFP-RecN foci in a ⌬recA strain. Wild-type recN is a part of the SOS regulon, and its expression is completely dependent on activated RecA. Therefore, this experiment was performed in cells that expressed GFP-recN under control of the inducible PBAD promoter. Fig.  1A shows that arabinose-inducible GFP-recN fully complements the MMC sensitivity of ⌬recN, when cells are grown in the presence of arabinose, but not when cells are grown in the presence of glucose. Furthermore, GFP-RecN foci form in the cytoplasm of both MMC-treated and untreated wild-type cells and ⌬recA cells, whereas nucleoid-associated GFP-RecN foci form in wild-type MMC-treated cells but are absent in ⌬recA MMC-treated cells (Fig. 1, B and C). These results suggest that RecA is required to recruit GFP-RecN to DNA damage sites in MMC-treated cells.
Isolation of a recA Mutant That Mimics the Phenotype of ⌬recN-Although RecA plays the central role in recombinational repair and is the master inducer of the SOS pathway, it is unclear what functions of RecA are required to recruit RecN to the nucleoid in MMC-treated cells. The phenotype of ⌬recA or SOS-deficient lexA3 cells (45) differs from the phenotype of ⌬recN cells; the former are hypersensitive to MMC and UV, whereas the latter are hypersensitive to MMC but insensitive to UV ( Fig. 2A). Therefore, a library of recA mutants was generated and screened for mutants that provide resistance to UV but confer sensitivity to MMC. Three candidate mutants were isolated from ϳ2,000 clones. All three mutants carry an arginine substitution at the highly conserved C-terminal Gln-300 of RecA (Fig. 2B). Fig. 2C shows that this allele, recA Q300R , fully complements the UV sensitivity of ⌬recA cells, but does not complement their MMC sensitivity. In the wild-type strain, MMC-induced DNA damage leads to proteolytic cleavage of LexA, the repressor of the SOS regulon, and induces the SOS response (Fig. 2D). Similarly, recA Q300R is capable of inducing expression of the SOS regulon and specific proteolytic cleavage of LexA in MMC-treated cells (Fig. 2D). Thus, recA Q300R is proficient in the DNA damage-induced SOS response, and its phenotype is similar to the phenotype of ⌬recN.
Nucleoid Fragmentation in MMC-treated ⌬recN and recA Q300R Cells-To explore these results further, ⌬recA, recA Q300R , ⌬recN, and ⌬ruvB cells were stained with DAPI and examined by fluorescence and phase-contrast microscopy for genome integrity and cell morphology. Under conditions of exponential growth in the absence of MMC, all cells had a normal morphology, with two centrally located nucleoids per cell (Fig. 3A). Wild-type cells treated with MMC for 90 min became highly filamented with elongated, evenly spaced nucleoids (Fig.  3A). This morphology is typical of SOS-activated cells (46). By contrast, a large fraction of MMC-treated SOS-defective ⌬recA were anucleate, and filamentous cells were hardly detected (Fig.  3A). RuvABC resolvasome branch-migrates and resolves Holliday junctions, and inactivation of any of the three Ruv functions blocks resolution of recombinational repair intermediates (3). Therefore, RuvB plays a role in the later steps of HR. MMCtreated ruvB mutants formed both filamentous and anucleate cells. As reported previously (47), the nucleoids of filamentous ruvB cells were centrally located and little to no DNA migrated to cell poles, which is in contrast to the morphology of filamentous wild-type cells (i.e. well partitioned nucleoids). This indicates that the accumulation of intermediates of HR-mediated DSB repair results in chromosome nondisjunction and the production of anucleate cells. MMC-treated ⌬recN and recA Q300R  cells were as filamentous as wild-type cells, but had an abnormal morphology characterized by multiple, short, diffuse nucleoids (Fig. 3A). In wild-type cells, the number of nucleoids per cell was largely unaffected by exposure to MMC, whereas the number of nucleoids per cell increased when ⌬recN and recA Q300R cells were exposed to MMC (Fig. 3B). Furthermore, abnormal nucleoid morphology was not generally observed in UV-irradiated ⌬recN and recA Q300R cells (supplemental Fig.  S1). These results support the conclusion that recA Q300R is a phenocopy of ⌬recN. Our interpretation of this result is that RecN is dysfunctional in the recA Q300R mutant.
We hypothesized that the abnormal nucleoid morphology of MMC-treated ⌬recN cells might reflect the presence of unrepaired DSBs and chromosome fragmentation. Therefore, a fluorescence-based method was used to visualize chromosome fragments. For this purpose, oriC was labeled indirectly, via LacI-ECFP (enhanced cyan fluorescent protein) bound to an ectopic tandem array of Lac repressor-binding sites (240 ϫ lacO) at the ori1 locus (15 kb counterclockwise of oriC) (40). LacI-ECFP was expressed from the chromosomally integrated gene under the control of the PBAD promoter. Wild-type and ⌬recN cells were treated or not with MMC and visualized using fluorescence microscopy. The results revealed 2-4 ori1 foci per cell in the majority of wild-type and ⌬recN cells in the absence of MMC. In these cells, all nucleoids contained at least one ori1 focus (Fig. 3C). When treated with MMC, the number of ori1 foci per nucleoid increased significantly in wild-type and ⌬recN cells, and the foci were distributed throughout the elongated nucleoid (Fig. 3C). Notably, Ͼ15% of ⌬recN cells carried nucleoids lacking ori1 foci, whereas such nucleoids were infrequent in wild-type cells (Ͻ1.4%) (Fig. 3D). These results demonstrate the presence of aberrant nucleoids lacking oriC in MMCtreated ⌬recN cells, which likely represent subchromosomal fragments.
RecA Q300R Is Defective in Recruiting RecN to Nucleoids in MMC-treated Cells-The results described above suggest that RecA Q300R does not recruit RecN to the nucleoid, under conditions where wild-type RecA does so (i.e. in MMC-treated wild-type cells). To explore this further, GFP-RecN foci were quantified in MMC-treated ⌬recA ⌬recN cells expressing SOSinducible GFP-recN and either wild-type recA or recA Q300R . After exposure to MMC for 60 min, Ͼ90% of wild-type cells contained nucleoid-associated GFP-RecN foci (Fig. 4, A and B). By contrast, Ͻ5% of cells expressing recA Q300R had nucleoidassociated GFP-RecN foci, whereas the number and fraction of cells with cytoplasmic GFP-RecN foci was higher in cells expressing recA Q300R than that in cells expressing wild-type recA (Fig. 4, A and B). These results indicate that RecA is required for the formation of MMC-induced, nucleoid-associated RecN foci and that RecA Q300R has a specific defect in this function/role.
RecA Is Required to Recruit RecN to sites of DSBs-To examine the recruitment of RecN to a unique DSB site in RecAproficient cells, I-SceI was used to introduce a site-specific DSB into a strain that carries the P BAD I-SceI cassette on the chromosome and a single I-SceI recognition site on the FЈ episome (39). Appropriately engineered cells were transformed with a plasmid expressing GFP-recN from its native SOS-inducible promoter, grown to early log phase and exposed to arabinose to induce I-SceI. Control cells were grown in medium lacking arabinose. VspI endonuclease digestion resulted in a 1.8-kb fragment containing the I-SceI cleavage site. I-SceI digestion produced two fragments, one of which with a size of 1.2 kb hybridized to the site 1 probe (Fig. 5A). The kinetics of DSB formation was monitored by Southern blot analysis of VspIdigested DNA isolated from samples taken at different times after the addition of 0.2% arabinose or glucose to the culture. A 1.2-kb fragment was not detected when cells were maintained in glucose-containing medium, whereas it was detected within 30 min in wild-type cells proficient for RecBCD after the addition of arabinose (Fig. 5, A and B). The intensity of the 1.2-kb fragments increased with time, reaching a maximum intensity ϳ1 h after the addition of arabinose. Similar results were obtained when the site 2 probe was used to detect the I-SceI cleavage site (Fig. 5A and supplemental Fig. S2). One possible explanation for the kinetics of DSB formation is that I-SceI digestion is not synchronous in the entire population, and the breaks may be repaired very efficiently. Thus, the only breaks generated just before samples were taken might be detected by Southern blotting. This is consistent with previous studies using chromosomally integrated I-SceI site, where DSB products are readily detected in wild-type cells even after 1 h of I-SceI induction (48). Fig. 5C shows that nucleoid-associated GFP-RecN foci were detected in cells that expressed I-SceI and carried an FЈ episome with an I-SceI cleavage site. By contrast, GFP-RecN foci were not observed when the same cells were grown in glucose-containing medium (to repress I-SceI) or if the cells did not carry an I-SceI-sensitive FЈ episome (Fig. 5C). Furthermore, the number of nucleoid-associated GFP-RecN foci was much lower in recA Q300R mutant cells (Ͻ1%) than in cells expressing wild-type recA (18%) (Fig. 5D). These results indicate that, in wild-type cells, a single I-SceI-induced DSB induces an SOS response and promotes the formation of nucleoid-associated GFP-RecN foci in a RecA-dependent manner.
RecN ATPase Activity Is Required for Release from Growth Arrest in Cells with DNA Damage-RecN has a typical SMC family protein domain structure, including an extensive, centrally located coiled-coil domain and globular N-and C-terminal domains with Walker A and Walker B nucleotide-binding motifs, respectively (49). A previous study showed that substitution of Lys-35 with alanine in the Walker A motif resulted in a complete loss of RecN DNA repair activity in vivo (50). Biochemical characterization of Deinococcus radiodurans RecN showed that RecN K67A (an lysine-to-alanine substitution at position 67, which corresponds to E. coli RecN Lys-35) abolished ATPase activity, but did not impair ATP binding in vitro (49). Fig. 6A shows that expression of recN K35A in a ⌬recN background conferred sensitivity to MMC that was equivalent to that of ⌬recN. The overproduction of RecN K35A rendered wildtype cells sensitive to MMC (Fig. 6A), demonstrating that recN K35A is a dominant-negative allele of recN. GFP-RecN K35A formed nucleoid-associated foci in Ͼ80% of MMC-treated cells, and these foci failed to form in cells expressing recA Q300R (Fig. 6B). This result indicates that the ATPase activity of RecN is not required for formation of nucleoid-associated RecN foci. However, ⌬recN cells expressing wild-type GFP-recN resumed normal cell growth, and nucleoid-associated GFP-RecN foci dissociated after exposure to MMC was terminated (Fig. 6C). By contrast, ⌬recN cells expressing GFP-recN K35A became highly filamented, acquired fragmented nucleoid structures, and retained GFP-RecN K35A foci for 2 h after exposure to MMC was terminated (Fig. 6C). These results demonstrate that ATPase-defective RecN K35A is recruited to sites of DNA damage, but may not be properly released because of the defects in HR-mediated repair of MMC-induced DSBs.

DISCUSSION
Previous studies demonstrate that RecN protein forms both nucleoid-associated and cytoplasmic foci in cells exposed to DSB-inducing agents and that cytoplasmic RecN aggregates are degraded by the ClpXP protease (27). Here, we demonstrate that RecN is recruited to nucleoids in a RecA-dependent manner. We characterize a novel recA allele, recA Q300R , which promotes expression of SOS-inducible genes but does not promote formation of nucleoid-associated RecN foci. RecN accumulates at a unique I-SceI-induced DSB in wild-type recA cells but not in recA Q300R cells. Thus, we conclude that RecA plays an essential role in DNA damage-induced expression of recN and the assembly of RecN foci at the sites of DSBs. ATPase-deficient recN K35A mutants are proficient in forming nucleoid-associated foci at DSBs, but fail to resume growth after release from MMC-induced cell-cycle arrest. This results in highly filamented cells with nucleoid-associated GFP-RecN K35A foci and fragmented nucleoid structures. One possible explanation for the presence of persistent foci associated with damaged DNA in recN K35A cells is that RecN K35A is recruited to DSBs, but is not released from DSB sites because it lacks ATPase activity; under such conditions, mutant RecN K35A DNA damage foci persist and accumulate, which interferes with RecA-mediated synaptic steps in the HR pathway.
This study also reveals that ⌬recN and recA Q300R cells are hypersensitive to MMC but not to UV. A previous study showed that cells expressing recA⌬C17 (a deletion mutant lacking residues 336 -352) are hypersensitive to MMC but not to UV (51). Here, we confirm that recA⌬C17 cells are sensitive to MMC, although they are less sensitive than ⌬recN and recA Q300R cells (supplemental Fig. S3). We found that nucleoidassociated GFP-RecN foci form normally in MMC-treated recA⌬C17 cells (supplemental Fig. S3), indicating that the defects in the response to MMC in recA Q300R cells are not a result of a dysfunctional RecA C-terminal domain. However, it still remains possible that the C-terminal region of RecA plays a role in modulating RecN function at a later step in the repair/ response to MMC-induced DSBs.
Previous studies suggest that the SOS response plays a critical role in DSB repair in E. coli but not in Bacillus subtilis (52). Indeed, unlike in E. coli, the expression of B. subtilis RecN appears to be SOS-independent (53), and GFP-B. subtilis RecN foci associate with DSBs before RecA is recruited to the DNA lesion (10). By contrast, E. coli recN is typical of SOS-regulated genes in that its expression is tightly repressed in unstressed cells. This suggests that E. coli RecN participates in HR repair of DSBs after RecA senses DNA damage. Consistent with this, the present study indicates that RecA actively recruits RecN to DSBs. These results may reflect species-specific attributes of E. coli and B. subtilis HR pathways. The purified B. subtilis RecN (and also D. radiodurans RecN) binds to DNA and has DNA-stimulated ATPase activity in vitro (54,55). Unfortunately, it has been difficult to purify E. coli RecN because it is relatively insoluble and highly susceptible to degradation (data not shown). A recent study showed that Haemophilus influenzae RecN can be purified to near homogeneity and is fully functional in E. coli (50). Purified Haemophilus influenzae RecN does not bind DNA, and DNA had no significant effect on Haemophilus influenzae RecN ATPase activity in vitro, which contrasts with the activity of B. subtilis RecN. This observation supports our conclusion that E. coli RecN is recruited to DSBs through its interaction with RecA. Thus, the difference in the DNA binding specificities of RecN orthologs may explain their different affinities for their respective bacterial nucleoids. How- ever, our data do not exclude the possibility that E. coli RecN has DNA binding activity. It is also conceivable that RecA facilitates the binding and/or retention of RecN on damaged DNA. SMC proteins are highly conserved ATPases whose role in higher order chromosome organization and dynamics is conserved from bacteria to humans. DSBs are one of the most cytotoxic forms of DNA damage, and therefore, the repair of DSBs is crucial for cell survival and for maintaining the integrity of the genome. In this study, we provide evidence that the recruitment of RecN to DSBs requires interaction with RecA. Any defect in the interaction results in chromosomal fragmentation, such as that observed in cells exposed to the DSB-inducing agent MMC. Based on these results and implications, we propose a mechanism by which RecN promotes RecA-dependent DSB repair. The initial presynaptic step of the DNA strand exchange reaction is formation of a RecA-ssDNA nucleoprotein filament. RecA-dependent recruitment of SMC-like RecN to DSBs follows, serving a scaffolding function to facilitate subsequent search by RecA for homologous templates in the segregated sister chromatids. Lastly, RecA mediates strand exchange. This model might be compatible with the recN studies in B. subtilis; here, we allow for the fact that RecN plays a role in an early step of DSB repair and that the mechanism by which RecN is recruited to DSBs differs.
In future studies, it will be interesting to investigate how RecN SMC complexes actually promote RecA-dependent DSB repair. Therefore, novel integrated biochemical and structural approaches to examine this and other questions concerning the roles of RecN and RecA will be required. The results of such studies should advance our understanding of the mechanism of DSB repair in prokaryotic and eukaryotic cells.