Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair
- Judita Mascarenhas†1, 3,
- Humberto Sanchez†2,
- Serkalem Tadesse1,
- Dawit Kidane1,
- Mahalakshmi Krisnamurthy1,
- Juan C Alonso2 and
- Peter L Graumann1Email author
© Mascarenhas et al; licensee BioMed Central Ltd. 2006
Received: 06 January 2006
Accepted: 16 June 2006
Published: 16 June 2006
Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.
A deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC) or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD), but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC.
Our experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC), most likely through HR, and suggest that NHEJ via Ku serves as a backup DNA repair system. The cell biological experiments show that SbcC functions in close proximity to the replication machinery, suggesting that SbcC may act on stalled or collapsed replication forks. Our results show that different patterns of localization exist for DNA repair proteins, and that the B. subtilis SMC proteins RecN and SbcC play distinct roles in the repair of DNA damage.
All organisms need to ensure the integrity of their genome. A major threat are inter-strand cross-links and double strand breaks (DSBs) in the DNA that can cause cell death or cellular transformation. The repair of inter-strand cross-links appears to be achieved via two pathways, including proteins involved in nucleotide excision repair, and DNA polymerase II or proteins mediating homologous recombination (HR) . At least two pathways also exist for the repair of DSBs. These can be repaired through direct end joining (non homologous end joining, NHEJ), but this process creates the danger of connecting the wrong ends, and of losing genetic information. In eukaryotic and in some prokaryotic cells, Ku protein binds to broken DNA ends, protects them from exonucleolytic degradation and specifically recruits a dedicated DNA ligase that seals the ends [2, 3]. In Bacillus subtilis, deletion of the genes encoding for Ku and DNA ligase (ykoV and ykoU) leads to sensitivity to DSBs during stationary phase , showing that this pathway has an important function in non-growing cells. A much less error prone pathway is DNA repair through HR, in which a variety of proteins uses the intact sister chromosome to fix a DSB [5–7]. Central to this pathway is strand exchange protein RecA (Rad51 in eukaryotes) that promotes the annealing of the 3'-single-stranded (ss) DNA from the broken chromosome with the homologous sister copy and thereby catalyzes HR. In B. subtilis, the AddAB (nuclease/helicase) complex (RecBCD in Escherichia coli) or alternatively, RecQ/RecS/RecJ act upstream of RecA. These enzymes generate a ssDNA region at the DSB, which is used for strand exchange . AddAB or RecO/RecR load or help to load RecA onto the 3' ssDNA end . In eukaryotes, the Rad50/Mre11/Xrs2 (Nbs1 in human cells) complex is thought to perform the degradation of one DNA strand to generate a 3' ssDNA overhang. Downstream of RecA, different DNA helicases, such as RecG, and the RuvABC complex are involved in the formation of Holliday junctions and in the resolution of crossovers , while DNA polymerase I is required to fill ssDNA gaps.
The eukaryotic Rad50 complex plays an important role in DNA repair in eukaryotes through HR [10, 11], and is apparently also involved in NHEJ through bridging of the broken DNA ends [12, 13]. Mre11 has endo- and exonuclease activity in the presence of Rad50 . Rad50 has been shown to bind to DNA in an ATP-dependent manner , although the Rad50 complex was found to be able to bind to DNA in the absence of ATP ; DNA end-bridging is ATP independent . Rad50 belongs to the family of SMC proteins that share a similar modular structure. These proteins are composed of conserved N- and C- terminal domains that are separated by two long coiled coil domains and a central hinge domain. Two monomers invariably form a dimer through specific interaction of the hinge domains, creating a symmetrical dimer with a central hinge, two long coiled coil arms and two ATPase head domains [18, 19]. In Rad50, the coiled coil arms lack a hinge domain. Instead, the coiled coils can connect in a dynamic manner via a Zn bridge formed by an invariable CxxC motif, which may mediate end bridging . SMC proteins are central components of several essential protein complexes in pro- and eukaryotes, and mediate a wide range of chromosome dynamics . In bacteria, the SMC complex localizes as two foci, one within each cell half, and actively compacts and organises chromosomes from these sites . The chromosome is replicated in and moves through the centrally located DNA polymerase complex . Thus, bacterial chromosomes are segregated during ongoing replication, while both processes occur at different times during the cell cycle in eukaryotic cells.
In E. coli, the Rad50 ortholog, SbcC, forms a complex with the Mre11-homologous SbcD nuclease , which has been shown to be involved in cleavage of DNA hairpins that can occur during replication . Many other bacteria possess sbcC homologous genes, but no reports on SbcC in other bacteria are available, and it is unclear if SbcC plays a similar role in DNA repair as Rad50 in eukaryotes.
In recent years, repair of DNA DSBs has been visualized in live eukaryotic cells, giving vital insight into the function of proteins. The Rad50 complex, Rad51, Rad52 and other repair proteins have been shown to form discrete foci on damaged chromosomes, and to dynamically interact within the repair structures, or to form distinct repair foci [26–30]. In bacteria, RecA, RecN, RecO and RecF have been shown to co-localize to discrete centres on the nucleoids after induction of DNA damage, showing that bacteria also form DNA repair centres after DNA damage [31, 32]. RecN is also a member of the SMC protein family, plays an important function in DNA repair via HR, and has ssDNA-binding activity in vitro [8, 33].
In this work, we have investigated the role of prokaryotic SbcC in DNA repair, and show it has an important function in this process, which is different from that of RecN. We have visualized the dynamic formation of defined SbcC foci on the DNA in response to DNA damage. The foci were generally coincident with the position of the replication machinery, and therefore most likely represent stalled replication forks, whose restart may be facilitated by the action of SbcC.
B. subtilis SbcC is involved in DNA repair
Genetic interactions of sbcC
To gain further insight into the involvement of SbcC in DNA repair, double mutant strains were constructed in the prophage-free YB886 background, and their survival rates were compared with those of wild type cells, of a recN deletion (ΔrecN) strain, of a ykoV (encodes the B. subtilis Ku protein) deletion strain or of ΔrecA cells. The deletion of sbcC, ykoV or sbcC and ykoV did not alter the growth rate of the culture compared with wild type cells, similar to sbcC recN or ykoV recN mutant cells. Contrarily, recA mutant cells show a strong reduction in growth and plating efficiency . When exponentially growing wild type or mutant cells were exposed to MMC, the strains could be grouped into three different classes, moderately/very/extremely sensitive to MMC.
Figure 3 shows the concentrations of MMC that affected the plating efficiency of sbcC, recN, or recA mutant cells, respectively. SbcC or ykoV mutant cells and the double sbcC ykoV mutant strain were moderately sensitive to MMC, with a growth defect at 150 ng/ml and a moderate sensitivity in the presence of 125 ng/ml of MMC. In contrast, the growth of the wild type was unaffected by the presence of 150 ng/ml of MMC (Fig. 3), but survival was impaired in the presence of 175 ng/ml (data not shown, ). The ΔrecN strain was sensitive to 40 ng/ml of MMC, and the double ΔsbcC ΔrecN or ΔykoV Δr ecN strains were considerably more sensitive than the single ΔrecN mutant (Fig. 3). The ΔrecN strain showed confluent growth at the 10-2 dilution (2nd row) and about 50 microcolonies at the 10-3 dilution (3rd row), whereas the double ΔsbcC ΔrecN or ΔykoV ΔrecN strains neither showed confluent growth at the 10-2 dilution, nor microcolonies at the higher dilution. The higher sensitivity of the double mutant strains can be considered a non-epistatic interaction, suggesting that SbcC acts on a DNA repair pathway different from RecN.
One pathway of DNA repair via HR in E. coli involves the RecBCD complex, whose analog in B. subtilis is the AddAB complex [40, 41]. The survival rate after MMC treatment was reduced in addAB mutant cells (29% or 12%, 50 or 100 ng/ml of MMC), compared to wild type cells (Fig. 2). However, addAB sbcC mutant cells were more severely affected in the survival test compared with the single mutants (Fig. 2). Therefore, SbcC is also not epistatic with AddAB. We also performed similar experiments using bleomycin that induces DSBs, which yielded similar results (data not shown).
To substantiate these interactions, and to test for sensitivity to DNA damage using another method, stationary phase cultures of wild type or of mutant cells were exposed to ionizing radiation. Wild type, ΔsbcC, ΔykoV, or ΔsbcC ΔykoV cells exhibited significant resistance to irradiation up to a dose of 100 Gy (Fig. 4A). At higher doses, the lethal dose leaving 50% survivors (LD50) was ~147 Gy for wild type cells, ~140 Gy for ΔykoV cells, ~128 Gy for ΔsbcC cells, and ~130 Gy for ΔsbcC ΔykoV double mutant cells. This finding differs from previous work reporting that the LD50 is ~180 Gy for wild type and ~73 Gy for the ΔykoV strain . It is possible and even likely that this discrepancy is due to different strain backgrounds, i.e. the presence of naturally inducible prophages in the strains used in the cited work. Additionally, differing numbers of spores formed by the two different parent strains could account for the discrepancy, because Bs-Ku plays an important role in DNA repair in spores .
The ΔrecN strain was very sensitive to γ-radiation, with a LD50 of ~92 Gy. However, the LD50 was ~61 Gy for the ΔsbcC ΔrecN strain, and ~40 Gy for the ΔykoV ΔrecN strain (Fig. 4B), representing a 1.5-fold and 2.3-fold increase in the sensitivity of the double mutant strains compared to the ΔrecN strain. These results confirm that scbC and recN or ykoV and recN show a non-epistatic interaction.
The ΔrecA strain was highly sensitive to γ-radiation, with a LD50 of ~20 Gy (Fig. 4B), however, the LD50 of the double ΔykoV ΔrecA mutant strain was ~16 Gy. The sensitivity of the double mutant strain was clearly higher than that of the already sensitive single mutant and can be considered a non-epistatic interaction, consistent with a previous report . In contrast, the ΔsbcC ΔrecA double mutant strain was as sensitive to gamma irradiation as the recA mutant strain (data not shown, see ΔrecA in Fig. 4).
The data show that SbcC plays an important role in DNA inter-strand cross-link repair, and acts in a different pathway than AddAB or RecN, while it is epistatic with RecA. Its importance becomes clearer in the ΔrecN background, which is also true for the function of Bs-Ku protein. The data also suggest that HR is the main repair system for DNA inter-strand cross-links and DSBs in B. subtilis, while NHEJ constitutes a backup system that is required in the absence of HR repair.
SbcC forms defined foci on the nucleoids upon induction of DNA DSBs
In exponentially growing cells, extremely faint fluorescence above background was detectable for SbcC-YFP, which appeared to be present throughout the cells (Fig. 5A). However, in 2% of the cells, discrete SbcC-YFP foci were visible (Fig. 5B). Interestingly, the number of SbcC-YFP foci increased as soon as 30 min after addition of MMC (Fig. 5C, in 5% of the cells, >250 cells analyzed), and was highest 2 hours after addition of MMC (40% of the cells containing foci, >300 cells counted, Fig. 5D). Exposure times of 3 s were necessary to visualize SbcC-YFP foci because signal intensity was very weak (exposure of more than 3 s did not increase signal intensity, as is the case for any GFP fusion analyzed so far with our technical setup). About 90% of the SbcC-YFP foci were located on the nucleoids, showing that the foci were generally associated with DNA. To investigate if SbcC-YFP foci are only induced through inter-strand cross links, or also through DSBs, exponentially growing cells were irradiated with 12 Gy of hard X-rays, which is a dose that induces DSBs but does not reduce viability of B. subtilis cells. Similar to the addition of MMC, hard X-rays induced fluorescent foci in 30 to 35% of the cells (data not shown), showing that SbcC foci are also formed after generation of radiation-induced DSBs.
After addition of 50 ng/ml of MMC, only one SbcC-YFP focus was detectable in 34% of the cells, while 6% of the cells contained 2 foci, and 60% no visible focus. Interestingly, the number of cells containing foci and the number of foci per cell did not vary significantly between sublethal and lethal doses of MMC (50 to 200 ng/ml, compare Fig. 5D and 5E).
SbcC centers mostly co-localize with the replication machinery
Inter-strand cross-links block the progression of the replication fork. Also, DSBs can arise at the replication fork, when DNA lesions hinder the progression of the replicative polymerase complex. To test whether SbcC acts at the replication fork, or also at other sites within the cell, we created a dually labelled strain, expressing DnaX-CFP (the tau subunit of DNA polymerase III) and SbcC-YFP. It was technically challenging to obtain clear CFP and YFP signals within the same cell, mostly due to low SbcC-YFP signal intensity. However, we were able to obtain clear signals in both channels for 32 cells (out of 300 cells analyzed). Interestingly, when inter strand cross links were induced, 22 of the SbcC-YFP foci co-localized with DnaX-CFP (Fig. 5F–G), and 4 foci were in close proximity (< 0.3 μm), while 6 of the foci were clearly separate (> 0.3 μm). Thus, SbcC foci mostly co-localize with the replication machinery.
It is interesting to note that the pattern of localization of the replication machinery was altered upon induction of DNA damage. 85% of exponentially growing cells contained one single or two bipolar DnaX-CFP foci (and 15% no signal), whereas after addition of MMC, only 62% of the cells contained this proper localization of the replisome, and 23% of the cells contained signals at various places in the cell (15% did not show any signal, data not shown). Additionally, 66% of exponentially growing cells contained one central DnaX-CFP focus, and 19% two bipolar foci (15% did not contain a signal), whereas after induction of damage, a higher proportion of the cells (40%) contained two or more foci, rather than one (45%, 15% did not contain any focus). Thus, the specific localization of the replication factory becomes more random during DNA repair, and, apparently, replication forks separate (and/or replication reinitiates in the absence of cell division) in a considerable fraction of the cells. Our finding that SbcC forms no more than 1 or 2 foci after induction of DNA damage agrees with the finding that SbcC generally colocalizes with the replication machinery.
The AddAB complex localizes throughout the cells
Our work shows that the bacterial counterpart of the eukaryotic Rad50 protein, SbcC, confers an important function in the repair of inter strand cross links in DNA caused by MMC, and plays a role in the repair of DSBs (caused by gamma irradiation), most likely in the context of repair of collapsed replication forks. The deletion of sbcC led to a considerable sensitivity to DNA damage. However, the loss of SbcC in a recN or in an addAB mutant background increased the sensitivity to DNA damage of the very sensitive recN or addAB single mutant strains. Contrarily, the loss of SbcC in recA mutant cells did not increase the extreme sensitivity of the single recA mutant strain, showing that SbcC is epistatic with RecA, but not with RecN or with AddAB. These experiments suggest that SbcC acts on DNA repair via homologous recombination (HR), and show that it confers a function distinct from RecN or from AddAB.
Interestingly, like SbcC, Bs-Ku protein was non-epistatic with RecN or AddAB, and the loss of BS-Ku in recN mutant cells led to a similar increase in sensitivity towards DNA damage than the loss of SbcC in this mutant background. Because the loss of Bs-Ku protein in a sbcC mutant strain did not increase the mild sensitivity of the sbcC single mutant strain, and vice versa, it is possible that SbcC may also play a role in NHEJ. Possibly, upon encountering DNA ends, the SbcC alone, or in concert with the Bs-Ku protein, might tether DNA ends together, which are finally joint by a DNA ligase . Indeed, several reports have supported a dual function of the eukaryotic Rad50 complex in DNA repair via HR as well as in NHEJ. However, many more experiments are required to establish a connection between SbcC and NHEJ, and the finding that Bs-Ku is non epistatic with RecA, in contrast to SbcC, shows that the main function of SbcC does not lie in NHEJ, but rather in HR.
It is clear from our work that repair of DSBs and of inter-strand cross-links via HR plays a major role in B. subtilis cells, because the loss of proteins participating in HR [e.g. RecN, AddAB] caused severe sensitivity to radiation-induced DNA damage, while the loss of Bs-Ku led to moderate sensitivity. The activity of Bs-Ku became important in cells defective in repair via HR, indicating that NHEJ serves as a back up system to repair DSBs that can not be fixed via HR, e.g. non-duplicated chromosome regions, for which no sister chromosome is present to set up HR. Interestingly, it has recently been shown that Bs-Ku and DNA ligase encoded by ykoU play an important role in resistance of spores to killing through dry heat, a treatment that introduces breaks into DNA . Because the spore contains only a single chromosome, DSBs can not be repaired via HR, but only through NHEJ, which is also important for DSB repair during cell cycle exit in all other types of cells. However, our results show that Bs-Ku also performs a function in DNA repair in exponentially growing cells, because the absence of Bs-Ku leads to mild but noticeable sensitivity towards MMC.
A further major finding of this work that provides a clue as to the function of SbcC is the observation that SbcC transiently assembles into discrete subcellular centers on the nucleoids after the induction of inter-strand cross-links and of DSBs. In growing cells, SbcC was mostly dispersed throughout the cells, while the accumulation of SbcC was observed as early as 30 min after induction of DNA damage, its main accumulation occurring between 1 and 2 hours post induction. Thereafter, the SbcC-GFP foci dissipated, concomitant with the resumption of growth. Intriguingly, SbcC assembled most frequently at the replication machinery, suggesting that SbcC acts on DNA damage occurring at the replication forks, which can block replication (e.g. cross links or base adducts), or lead to fork collapse (e.g. when forks run into ssDNA gaps) . However, at low frequency, replication forks collapse in non-stressed exponentially growing cells . Consistent with this, we also observed SbcC foci at low frequency in exponentially growing cells in the absence of externally induced damage. The E. coli SbcCD complex, which shows some similar in vitro activities than the Rad50 complex , has been shown to be involved in the cleavage of DNA hairpins that can occur at the replication forks . Possibly, this is one of the specific roles SbcC confers during replication, and at stalled or collapsed replication forks, SbcC could be involved in replication restart through HR, and/or removal of inter-strand cross-links.
Recently, we have shown that RecN assembles at defined DSBs or at random DNA lesions as early as 15 min, while RecO and RecA are recruited 30–60 min after induction of DSBs [31, 32]. DSB repair appears to last for about 3 hours, and RecF protein is recruited to DSBs after 90 min, thus representing a late-recruited protein. RecN binds to ssDNA at an internal site, and gathers the 3'-hydroxyl ssDNA ends in vitro . These finding are consistent with the idea that RecN, like eukaryotic Rad50 , might be an early sensor of sites of DNA damage on replicating and non-replicating chromosomes . The cytological data presented here provide a clear distinction between the functions of SbcC and of RecN, because RecN is generally not associated with the replication machinery , and appears to act as a nucleation factor of DNA repair centers away from the replication machinery, while SbcC appears to assemble at DNA damage occurring at the replication factory. A different function for SbcC and RecN – dependent on whether DNA damage occurs at the replication fork or elsewhere on the nucleoid – can explain why SbcC-YFP foci are only observed in about 40% of the cells after induction of random DNA damage (this work), while RecN-YFP foci occur in about 70% of the cells (but not in all cells) under these conditions .
SbcC and RecN belong to the SMC protein family, whose members are key players in various chromosome dynamics. Our report establishes that these two B. subtilis proteins SMC like proteins confer important yet distinct functions in the repair of DNA base modifications, of DNA inter-strand cross-links and of DSBs. It will be interesting to elucidate the molecular basis of their different modes of action.
Bacterial strains and media
Strains used in this work
trpC 2 metB 5 amyE sigB 37 xin-1 att SPβ
Construction of plasmids and strains
All CFP, GFP or YFP fusions were done by PCR amplification of the 3' regions of each gene, and cloning into a vector containing a C-terminal cfp, gfp or yfp gene. All fusion vectors contained inducible promoters ensuring transcription of downstream genes. To create a C-terminal fusions of AddA and of SbcD to GFP, the corresponding 3' regions (about 500 bp) were PCR amplified (AddA-GFP 5'-ATTCGGGCCCAGCTGAGCTGGACCTAC-3' and 5'-TCGGAATTCACCACCGCCT-AATGTCAGAATGTGCCC-3', SbcD-GFP 5'-TCGGAATTCACCGCCTTTCGCA-TCCTCCTCTTCAAC-3' and 5'-ATTCGGGCCCTTTGCATCGCCCGCAAACG-3') and were cloned into Apa I and Eco RI sites of pSG1164 , giving rise to plasmids pA1164 and pD1164, respectively. The 3' region of yirY (sbcC) (primers 5'-ATTCAAGCTTGCAAA-CTTGAAAACGAG-3' and 5'- TCGGAATTCACCACCGCCCATCAACTCAAGTGATAC-CCG- 3') was cloned into pSG1187 (C-terminal yfp) using Hind III and Eco RI sites, giving rise to plasmid pC87. This plasmid could not be integrated into the B. subtilis chromosome because of the downstream yisB gene. An Eco RI Spe I fragment from pC87 containing the 3'yirY-yfp fusion was cloned into pSG1164, giving rise to pC87xyl. The 3' region of addB (primers 5'-CCATCGATACCGCCTCCGGAATGTTCATTGCCATC -3' and 5'-TTGATTTATCGATTACACATTC -3') was cloned into pMutinYFP (containing an IPTG inducible promoter for downstream genes) using Cla I, giving rise to plasmid pBy. All plasmids were transformed into wild type (PY79) B. subtilis cells selecting for chloramphenicol (Cm) resistance (5 μg/ml). All fluorescent tag vectors were integrated into the B. subtilis chromosome via single crossover integration, which was verified by PCR and Western Blot analysis.
For disruption of sbcC, a 1000 bp internal fragment of sbcC was PCR amplified (primers 5'-ACATGCATGCAATCTGCCCTTCGCCTCTTG-3' and 5'-TCATAAGCTT-CAAACAGGAACAGCTTTCACG-3') and was cloned into Hind III and Sph I sites of pJQ43 (containing a constitutive promoter for downstream genes) , giving rise to plasmid pSbcKO. Single crossover integration of pSbcKO lead to disruption of the sbcC gene (sbcC::cm) and to the synthesis of a C-terminal truncated SbcC. For the disruption of ykoV, a 480 bp internal fragment from ykoV was cloned into pSG1164, and the resulting plasmid pykoVKO was transformed into PY79, generating strain JH1 (ykoV::cm).
The addAB sbcC triple mutant strain was generated by transforming addA5 addB72 (collectively termed addAB mutant cells) mutant cells  with chromosomal DNA from strain JM42, selecting for Cm resistance. The inactivation of the recN and recA genes by a double crossover event was previously described [41, 55]. To study genetic interactions in a strain freed of inducible prophages (which complicate analysis of DSBs), all deletions were moved into strain YB886 (rec+ control) . The ΔsbcC and ΔykoV strains were generated by transferring the sbcC::cm or ykoV::cm allele onto the YB886 strain by a single crossover event. To generate double mutants, the Cm resistance cassette in ΔsbcC or ΔrecN cells was replaced by tetracycline resistance in vivo by transformation with plasmid pCm::tet (BGSC, Wisconsin, USA). Double mutants were produced by transformation of ΔrecN, ΔrecA, ΔsbcC and ΔykoV mutant strains with the appropriate chromosomal DNA from other mutant strains.
DNA repair sensitivity studies
DNA modifications generated by Mitomycin C (MMC) result in the formation of base adducts and inter-strand cross-links, which generate single-strand breaks and DSBs [35–37]. Ionizing radiation (γ-rays) leads to the formation of apurinic/apyrimidinic sites and generates nicks and DSB. All survival studies were performed at least 3 times, and were performed in the absence of antibiotics. For sensitivity to MMC assays (Fig. 3), exponentially growing B. subtilis cells were obtained by inoculating overnight cultures in fresh LB media and by growing to an A560nm of 0.4 at 37°C. Appropriate dilutions (10 μl of serial 10-fold dilutions, 1 × 10-2 to 1 × 10-5) were spotted onto LB plates supplemented with the indicated concentration of MMC, and were incubated overnight at 37°C. MMC and all plates used for survival tests were kept in the dark at all times, in order to avoid complications generated by photoadducts.
For ionizing radiation assays (Fig. 4), stationary phase cultures of B. subtilis cells were obtained by inoculating overnight cultures in fresh LB media and by growing to an A560nm of 2 at 37°C. Irradiation was carried out with 137Cs γ-rays using a Mark I irradiator (JL Shepard & Associates) at a dose-rate of 3.7 Gy/min, without shaking at room temperature, using 50 μl of cells from a 100-fold dilution. Appropriate dilutions of irradiated cells were plated, incubated overnight at 37°C and colonies were counted from at least three independent experiments.
Western blot analysis
Western blotting was performed using same amounts of cell extracts, as determined by Bradford tests, using antiserum generated against purified GFP-6-his protein (kind gift from David Rudner, Harvard Medical School).
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 MircoMax CCD camera; signal intensities and cell length were measured using the Metamorph 4.6 program. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI; final concentration 0.2 ng/ml) and membranes were stained with FM4-64 (Molecular Probes, USA, final concentration 1 nM).
We thank Nicole Scherr and Castillo Cózar for valuable technical assistance. This work was partially supported by grants BMC2003-00150 and BIO2001-4342-E from DGI-MEC to J.C.A and from the Deutsche Forschungsgemeinschaft (Emmy Noether Programm) to P.L.G.
- Berardini M, Foster PL, Loechler EL: DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli. J Bacteriol. 1999, 181: 2878-2882.PubMed CentralPubMedGoogle Scholar
- Lieber MR, Ma Y, Pannicke U, Schwarz K: Mechanism and regulation of human non-homologous DNA end-joining. Nat Rev Mol Cell Biol. 2003, 4: 712-720. 10.1038/nrm1202View ArticlePubMedGoogle Scholar
- Downs JA, Jackson SP: A means to a DNA end: the many roles of Ku. Nat Rev Mol Cell Biol. 2004, 5: 367-378. 10.1038/nrm1367View ArticlePubMedGoogle Scholar
- Weller GR, Kysela B, Roy R, Tonkin LM, Scanlan E, Della M, Devine SK, Day JP, Wilkinson A, d'Adda di Fagagna F, Devine KM, Bowater RP, Jeggo PA, Jackson SP, Doherty AJ: Identification of a DNA nonhomologous end-joining complex in bacteria. Science. 2002, 297: 1686-1689. 10.1126/science.1074584View ArticlePubMedGoogle Scholar
- Fernandez S, Ayora S, Alonso JC: Bacillus subtilis homologous recombination: genes and products. Res Microbiol. 2000, 151: 481-486. 10.1016/S0923-2508(00)00165-0View ArticlePubMedGoogle Scholar
- Cromie GA, Connelly JC, Leach DR: Recombination at double-strand breaks and DNA ends: conserved mechanisms from phage to humans. Mol Cell. 2001, 8: 1163-1174. 10.1016/S1097-2765(01)00419-1View ArticlePubMedGoogle Scholar
- Paques F, Haber JE: Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1999, 63: 349-404.PubMed CentralPubMedGoogle Scholar
- Sanchez H, Alonso JC: Bacillus subtilis RecN binds and protects 3'-single-stranded DNA extensions in the presence of ATP. Nucleic Acids Res. 2005, 33: 2343-2350. 10.1093/nar/gki533PubMed CentralView ArticlePubMedGoogle Scholar
- Kowalczykowski SC, Dixon DA, Eggleston AK, Lauder SD, Rehrauer WM: Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev. 1994, 58: 401-465.PubMed CentralPubMedGoogle Scholar
- Bressan DA, Baxter BK, Petrini JH: The Mre11-Rad50-Xrs2 protein complex facilitates homologous recombination-based double-strand break repair in Saccharomyces cerevisiae. Mol Cell Biol. 1999, 19: 7681-7687.PubMed CentralView ArticlePubMedGoogle Scholar
- D'Amours D, Jackson SP: The yeast Xrs2 complex functions in S phase checkpoint regulation. Genes Dev. 2001, 15: 2238-2249. 10.1101/gad.208701PubMed CentralView ArticlePubMedGoogle Scholar
- Chen L, Trujillo K, Ramos W, Sung P, Tomkinson AE: Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. Mol Cell. 2001, 8: 1105-1115. 10.1016/S1097-2765(01)00388-4View ArticlePubMedGoogle Scholar
- de Jager M, van Noort J, van Gent DC, Dekker C, Kanaar R, Wyman C: Human Rad50/Mre11 is a flexible complex that can tether DNA ends. Mol Cell. 2001, 8: 1129-1135. 10.1016/S1097-2765(01)00381-1View ArticlePubMedGoogle Scholar
- Trujillo KM, Sung P: DNA structure-specific nuclease activities in the Saccharomyces cerevisiae Rad50*Mre11 complex. J Biol Chem. 2001, 276: 35458-35464. 10.1074/jbc.M105482200View ArticlePubMedGoogle Scholar
- Lee JH, Ghirlando R, Bhaskara V, Hoffmeyer MR, Gu J, Paull TT: Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with Ataxia-Telangiectasie-like disorder mutantz complexes. J Biol Chem. 2003, 278: 45171-45181. 10.1074/jbc.M308705200View ArticlePubMedGoogle Scholar
- de Jager M, Wyman C, van Gent DC, Kanaar R: DNA end-binding specificity of human Rad50/Mre11 is influenced by ATP. Nucl Acids Res. 2002, 30: 4425-4431. 10.1093/nar/gkf574PubMed CentralView ArticlePubMedGoogle Scholar
- Chen L, Trujillo KM, Van Komen S, Roh DH, Krejci L, Lewis LK, Resnick MA, Sung P, Tomkinson AE: Effect of Amino Acid Substitutions in the Rad50 ATP Binding Domain on DNA Double Strand Break Repair in Yeast. J Biol Chem. 2005, 280: 2620-2627. 10.1074/jbc.M410192200View ArticlePubMedGoogle Scholar
- Hopfner KP, Karcher A, Shin DS, Craig L, Arthur LM, Carney JP, Tainer JA: Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell. 2000, 101: 789-800. 10.1016/S0092-8674(00)80890-9View ArticlePubMedGoogle Scholar
- Lowe J, Cordell SC, van den Ent F: Crystal structure of the SMC head domain: an ABC ATPase with 900 residues antiparallel coiled-coil inserted. J Mol Biol. 2001, 306: 25-35. 10.1006/jmbi.2000.4379View ArticlePubMedGoogle Scholar
- Hopfner KP, Craig L, Moncalian G, Zinkel RA, Usui T, Owen BA, Karcher A, Henderson B, Bodmer JL, McMurray CT, Carney JP, Petrini JH, Tainer JA: The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature. 2002, 418: 562-566. 10.1038/nature00922View ArticlePubMedGoogle Scholar
- Strunnikov AV, Jessberger R: Structural maintenance of chromosomes (SMC) proteins: conserved molecular properties for multiple biological functions. Eur J Biochem. 1999, 263: 6-13. 10.1046/j.1432-1327.1999.00509.xView ArticlePubMedGoogle Scholar
- Mascarenhas J, Soppa J, Strunnikov A, Grauman PL: Cell cycle dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein. EMBO J. 2002, 21: 3108-3118. 10.1093/emboj/cdf314PubMed CentralView ArticlePubMedGoogle Scholar
- Lemon KP, Grossman AD: Localization of bacterial DNA polymerase: evidence for a factory model of replication. Science. 1998, 282: 1516-1519. 10.1126/science.282.5393.1516View ArticlePubMedGoogle Scholar
- Connelly JC, de Leau ES, Leach DR: DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli. Nucleic Acids Res. 1999, 27: 1039-1046. 10.1093/nar/27.4.1039PubMed CentralView ArticlePubMedGoogle Scholar
- Connelly JC, Kirkham LA, Leach DR: The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA. Proc Natl Acad Sci U S A. 1998, 95: 7969-7974. 10.1073/pnas.95.14.7969PubMed CentralView ArticlePubMedGoogle Scholar
- Liu Y, Li M, Lee EY, Maizels N: Localization and dynamic relocalization of mammalian Rad52 during the cell cycle and in response to DNA damage. Curr Biol. 1999, 9: 975-978. 10.1016/S0960-9822(99)80427-8View ArticlePubMedGoogle Scholar
- Nelms BE, Maser RS, MacKay JF, Lagally MG, Petrini JH: In situ visualization of DNA double-strand break repair in human fibroblasts. Science. 1998, 280: 590-592. 10.1126/science.280.5363.590View ArticlePubMedGoogle Scholar
- Maser RS, Monsen KJ, Nelms BE, Petrini JH: hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks. Mol Cell Biol. 1997, 17: 6087-6096.PubMed CentralView ArticlePubMedGoogle Scholar
- Haaf T, Golu EI, Reddy G, Radding CM, Ward DC: Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes. Proc Natl Acad Sci U S A. 1995, 92: 2298-2302. 10.1073/pnas.92.6.2298PubMed CentralView ArticlePubMedGoogle Scholar
- Lisby N, Mortensen UH, Rothstein R: Colocalization of multiple DNA double-strand breaks at single Rad52 repair centre. Nat Cell Biol. 2003, 5: 572-577. 10.1038/ncb997View ArticlePubMedGoogle Scholar
- Kidane D, Sanchez H, Alonso JC, Graumann PL: 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. Mol Microbiol. 2004, 52: 1627-1639. 10.1111/j.1365-2958.2004.04102.xView ArticlePubMedGoogle Scholar
- Kidane D, Graumann PL: Dynamic formation of RecA filaments at DNA double strand break repair centres in live cells. J Cell Biol. 2005, 170: 357-366. 10.1083/jcb.200412090PubMed CentralView ArticlePubMedGoogle Scholar
- Kidane D, Graumann PL: Intracellular protein and DNA dynamics in competent Bacillus subtilis cells. Cell. 2005, 122: 73-84. 10.1016/j.cell.2005.04.036View ArticlePubMedGoogle Scholar
- Palom Y, Suresh Kumar G, Tang LQ, Paz MM, Musser SM, Rockwell S, Tomasz M: Relative toxicities of DNA cross-links and monoadducts: new insights from studies of decarbamoyl mitomycin C and mitomycin C. Chem Res Toxicol. 2002, 15: 1398-1406. 10.1021/tx020044gView ArticlePubMedGoogle Scholar
- Jonnalagadda VS, Matsuguchi T, Engelward BP: Interstrand crosslink-induced homologous recombination carries an increased risk of deletions and insertions. DNA Repair (Amst). 2005, 4: 594-605. 10.1016/j.dnarep.2005.02.002View ArticleGoogle Scholar
- Frankenberg-Schwager M, Kirchermeier D, Greif G, Baer K, Becker M, Frankenberg D: Cisplatin-mediated DNA double-strand breaks in replicating but not in quiescent cells of the yeast Saccharomyces cerevisiae. Toxicology. 2005, 212: 175-184. 10.1016/j.tox.2005.04.015View ArticlePubMedGoogle Scholar
- Dusre L, Covey JM, Collins C, Sinha BK: DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells. Chem Biol Interact. 1989, 71: 63-78. 10.1016/0009-2797(89)90090-2View ArticlePubMedGoogle Scholar
- Yasbin RE, Fields PI, Andersen BJ: Properties of Bacillus subtilis 168 derivatives freed of their natural prophages. Gene. 1980, 12: 155-159. 10.1016/0378-1119(80)90026-8View ArticlePubMedGoogle Scholar
- Sciochetti SA, Blakely GW, Piggot PJ: Growth phase variation in cell and nucleoid morphology in a Bacillus subtilis recA mutant. J Bacteriol. 2001, 183: 2963-2968. 10.1128/JB.183.9.2963-2968.2001PubMed CentralView ArticlePubMedGoogle Scholar
- Chedin F, Ehrlich SD, Kowalczykowski SC: The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro. J Mol Biol. 2000, 298: 7-20. 10.1006/jmbi.2000.3556View ArticlePubMedGoogle Scholar
- Alonso JC, Stiege AC, Luder G: Genetic recombination in Bacillus subtilis 168: effect of recN, recF, recH and addAB mutations on DNA repair and recombination. Mol Gen Genet. 1993, 239: 129-136.PubMedGoogle Scholar
- Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, Setlow P, Losick R, Eichenberger P: The Forespore Line of Gene Expression in Bacillus subtilis. J Mol Biol. 2006, 358: 16-37. 10.1016/j.jmb.2006.01.059View ArticlePubMedGoogle Scholar
- Lin DCH, Levin PA, Grossman AD: Bipolar localization of a chromosome partition protein to Bacillus subtilis. Proc Natl Acad Sci USA. 1997, 94: 4721-4726. 10.1073/pnas.94.9.4721PubMed CentralView ArticlePubMedGoogle Scholar
- Lindow JC, Kuwano M, Moriya S, Grossman AD: Subcellular localization of the Bacillus subtilis structural maintenance of chromosomes (SMC) protein. Mol Microbiol. 2002, 46: 997-1009. 10.1046/j.1365-2958.2002.03235.xView ArticlePubMedGoogle Scholar
- Della M, Palmbos PL, Tseng HM, Tonkin LM, Daley JM, Topper LM, Pitcher RS, Tomkinson AE, Wilson TE, Doherty AJ: Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair machine. Science. 2004, 306: 683-685. 10.1126/science.1099824View ArticlePubMedGoogle Scholar
- Michel B, Grompone G, Flores MJ, Bidnenko V: Multiple pathways process stalled replication forks. Proc Natl Acad Sci U S A. 2004, 101: 12783-12788. 10.1073/pnas.0401586101PubMed CentralView ArticlePubMedGoogle Scholar
- Haber JE: Partners and pathways repairing a double-strand break. Trends Genet. 2000, 16: 259-264. 10.1016/S0168-9525(00)02022-9View ArticlePubMedGoogle Scholar
- Hopfner KP, Putnam CD, Tainer JA: DNA double-strand break repair from head to tail. Curr Opin Struct Biol. 2002, 12: 115-122. 10.1016/S0959-440X(02)00297-XView ArticlePubMedGoogle Scholar
- Lisby M, Barlow JH, Burgess RC, Rothstein R: Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell. 2004, 118: 699-713. 10.1016/j.cell.2004.08.015View ArticlePubMedGoogle Scholar
- Sanchez H, Kidane D, Castillo Cozar M, Graumann PL, Alonso JC: Recruitment of Bacillus subtilis RecN to DNA Double-Strand Breaks in the Absence of DNA End Processing. J Bacteriol. 2006, 188: 353-360. 10.1128/JB.188.2.353-360.2006PubMed CentralView ArticlePubMedGoogle Scholar
- Jaacks KJ, Healy J, Losick R, Grossman AD: Identification and characterization of genes controlled by the sporulation regulatory gene spo0H in Bacillus subtilis. J Bacteriol. 1989, 171: 4121-4129.PubMed CentralPubMedGoogle Scholar
- Lewis PJ, Marston AL: GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene. 1999, 227: 101-110. 10.1016/S0378-1119(98)00580-0View ArticlePubMedGoogle Scholar
- Quisel JD, Burkholder WF, Grossman AD: In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis. J Bacteriol. 2001, 183: 6573-6578. 10.1128/JB.183.22.6573-6578.2001PubMed CentralView ArticlePubMedGoogle Scholar
- Alonso JC, Tailor RH, Luder G: Characterization of recombination-defcient mutants of Bacillus subtilis. J Bacteriol. 1988, 170: 3001-3007.PubMed CentralPubMedGoogle Scholar
- Ceglowski P, Luder G, Alonso JC: Genetic analysis of rec E activities in Bacillus subtilis. Mol Gen Genet. 1990, 222: 441-445. 10.1007/BF00633853View ArticlePubMedGoogle Scholar
- Hopfner KP, Karcher A, Craig L, Woo TT, Carney JP, Tainer JA: Structural biochemistry and interaction architecture of the DNA double- strand break repair Mre11 nuclease and Rad50-ATPase. Cell. 2001, 105: 473-485. 10.1016/S0092-8674(01)00335-XView ArticlePubMedGoogle Scholar
- de Jager M, Trujillo KM, Sung P, Hopfner KP, Carney JP, Tainer JA, Connelly JC, Leach DR, Kanaar R, Wyman C: Differential arrangements of conserved building blocks among homologs of the Rad50/Mre11 DNA repair protein complex. J Mol Biol. 2004, 339: 937-949. 10.1016/j.jmb.2004.04.014View ArticlePubMedGoogle Scholar
- Graumann PL, Losick R, Strunnikov AV: Subcellular localization of Bacillus subtilis SMC, a protein involved in chromosome condensation and segregation. J Bacteriol. 1998, 180: 5749-5755.PubMed CentralPubMedGoogle Scholar
- Alonso JC, Luder G, Tailor RH: Characterization of Bacillus subtilis recombinational pathways. J Bacteriol. 1991, 173: 3977-3980.PubMed CentralPubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.