Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382X© 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd? 2005591338349Original ArticleLow level of RecA delays of DNA DSB repair in D. radioduransE. Jolivet et al.

Molecular Microbiology (2006) 59(1), 338–349

doi:10.1111/j.1365-2958.2005.04946.x First published online 15 November 2005

Limited concentration of RecA delays DNA doublestrand break repair in Deinococcus radiodurans R1 Edmond Jolivet,1‡ François Lecointe,1†‡ Geneviève Coste,1 Katsuya Satoh,2 Issay Narumi,2 Adriana Bailone1 and Suzanne Sommer1* 1 Institut de Génétique et Microbiologie, CNRS UMR 8621, LRC CEA 42V, Bâtiment 409, Université Paris-Sud, F-91405 Orsay Cedex, France. 2 Research Group for Biotechnology Development, Department of Ion-beam-applied Biology, Japan Atomic Energy Research Institute, Takasaki 370-1292, Japan. Summary To evaluate the importance of RecA in DNA doublestrand break (DSB) repair, we examined the effect of low and high RecA concentrations such as 2500 and 100 000 molecules per cell expressed from the inducible Pspac promoter in Deinococcus radiodurans in absence or in presence of IPTG respectively. We showed that at low concentration, RecA has a negligible effect on cell survival after γ-irradiation when bacteria were immediately plated on TGY agar whereas it significantly decreased the survival to γirradiation of ∆ddrA cells while overexpression of RecA can partially compensate the loss of DdrA protein. In contrast, when cells expressing limited concentration of RecA were allowed to recover in TGY2X liquid medium, they showed a delay in mending DSB, failed to reinitiate DNA replication and were committed to die during incubation. A deletion of irrE resulted in sensitivity to γ-irradiation and mitomycin C treatment. Interestingly, constitutive high expression of RecA compensates partially the ∆irrE sensitization to mitomycin C. The cells with low RecA content also failed to cleave LexA after DNA damage. However, neither a deletion of the lexA gene nor the expression of a non-cleavable LexA(Ind–) mutant protein had an effect on survival or kinetics of DNA DSB repair compared with their lexA+ counterparts in recA+ as well as in bacteria expressing limiting concentration of RecA, suggesting an absence of relationship between the absence of LexA cleavage and Accepted 29 September, 2005. *For correspondence. E-mail [email protected]; Tel. (+33) 1 69154614; Fax (+33) 1 69157808. †Present address: Génétique Microbienne, UR895 INRA, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France. ‡Joint first authors.

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

the loss of viability or the delay in the kinetics of DSB repair. Thus, LexA protein seems to play no major role in the recovery processes after γ-irradiation in D. radiodurans. Introduction Deinococcus radiodurans belongs to a family of bacteria characterized by an exceptional capacity to withstand the lethal effects of DNA-damaging agents, including ionizing radiation, UV light and desiccation (Minton, 1994; Mattimore and Battista, 1996; Battista, 1997). The highly radiation-resistant bacterium D. radiodurans R1 exhibits an extraordinary ability to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments, whereas the genomes of most organisms are irreversibly shattered under the same conditions (Battista et al., 1999). It is believed that genome restitution in D. radiodurans’ cells is mediated by homologous recombination and requires RecA function, although DNA repair via RecA-independent pathways may also take place (Daly and Minton, 1996; Battista, 1997; Battista et al., 1999). In addition to its recombinase activity, the D. radiodurans RecA protein acts as a coprotease in LexA cleavage (Narumi et al., 2001). However, contrary to the SOS model described in Escherichia coli (Little and Mount, 1982), LexA protein does not regulate RecA induction following γ-irradiation (Narumi et al., 2001; Bonacossa de Almeida et al., 2002). A novel regulatory protein IrrE, also called PprI, has been shown to be a positive effector that maximally increases the expression of RecA protein and of PprA, a protein which interacts with DNA ends, protects them from DNA degradation and stimulates DNA ligase activity (Earl et al., 2002; Hua et al., 2003; Narumi et al., 2004).The loss of the irrE gene product increases the cell’s sensitivity to UV, ionizing radiation and mitomycin C. This protein appears to play a crucial role in regulating multiple DNA repair and protection pathways to radiation exposure and to be part of a putative signal transduction pathway in response to DNA damage in D. radiodurans (Earl et al., 2002; Hua et al., 2003). Recently the DdrA protein (ddrA gene product), which is a homologue of the eukaryotic Rad52 in D. radiodurans (Iyer et al., 2002), was shown to contribute to ionizing radiation resistance (Harris et al., 2004; Tanaka et al.,

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Fig. 1. Survival following γ-irradiation of D. radiodurans bacteria expressing a low concentration of RecA is highly dependent on the presence of DdrA. Bacteria GY12354: recA+ ddrA+/ pGY11559: Pspac (squares), GY12359: ∆recA ddrA+/pGY11562: Pspac::recA+ (diamonds), GY12363: recA+ ∆ddrA/pGY11559: Pspac (triangles) and GY12364: ∆recA ∆ddrA/ pGY11562: Pspac::recA+ (circles) were grown in TGY2X supplemented with spectinomycin in presence (panel B, closed symbols) or in absence (panel A, open symbols) of IPTG (10 mM) to an OD650 of about 2–3. The cultures were irradiated and dilutions were plated as described in Experimental procedures.

2004). The DdrA protein is highly induced in D. radiodurans immediately following γ-irradiation (Tanaka et al., 2004). This protein binds to single-stranded DNA with a significant affinity for 3′ ends, and protects those ends from exonuclease I degradation in vitro, suggesting that this protein is a component of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation (Harris et al., 2004). To gain insight into the importance of RecA following γirradiation, we removed the recA gene from its normal regulatory circuit and expressed it from the IPTG-inducible Pspac promoter (Lecointe et al., 2004). Using this system, we tested the effect of RecA concentration on the kinetics of DNA double-strand break (DSB) repair, replication reinitiation, and LexA cleavage after γ-irradiation. Results

et al., 2004). The strains used, ∆recA Pspac::recA+, contained plasmid-borne as well as chromosomal copies of the lacI regulatory gene. In these bacteria the cellular level of RecA protein is either 2500 or 100 000 RecA monomers per cell depending on the absence or presence of the IPTG in the culture (Fig. S1). In the absence of IPTG, cultures have a nearly wild-type level of resistance to γ-irradiation (Fig. 1A, diamonds) in spite of the fact that their RecA content is below the basal level of RecA expressed from its natural promoter (Fig. S1) and no longer inducible by DNA damage. In contrast, these bacteria were extremely sensitive to mitomycin C when plated on drug-containing TGY agar (Lecointe et al., 2004) (Fig. 2A, diamonds). This suggests that induced RecA synthesis becomes critical when the cells are continuously exposed to a genotoxic agent while a low level of RecA suffices to ensure cell survival after a single exposure to γ-irradiation.

Radioresistance of D. radiodurans expressing RecA protein at a low constitutive level To examine the dependence of DNA repair on the cellular RecA concentration, we took advantage of the availability of plasmid in which the recA gene is placed under the control of the IPTG-inducible Pspac promoter (Lecointe

A low constitutive level of RecA renders γ-rays cell survival highly dependent on the presence of DdrA The ionizing radiation resistant phenotype of uninduced ∆recA Pspac::recA+ bacteria indicates that they can Fig. 2. The sensitivity to mitomycin C of IrrE deficient D. radiodurans bacteria expressing a low concentration of RecA is compensated by the overexpression of RecA protein. GY12354: recA+ irrE+/pGY11559: Pspac (squares), GY12359: ∆recA irrE+/pGY11562: Pspac::recA+ (diamonds), GY12417: recA+ ∆irrE/pGY11559: Pspac (triangles) and GY12418: ∆recA ∆irrE/ pGY11562: Pspac::recA+ (circles) were grown in TGY2X supplemented with spectinomycin in presence (panel B, closed symbols) or in absence (panel A, open symbols) of IPTG (10 mM) to an OD650 of about 2–3. The cultures were plated on TGY agar (panel A) or TGY agar supplemented with 1 mM IPTG (panel B) containing mitomycin C at concentrations ranging from 0 to 60 ng ml−1 as indicated on the abscissa. Colonies were counted after 3– 5 days’ incubation at 30°C.

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 338–349

340 E. Jolivet et al. Fig. 3. The acute sensitivity to ionizing radiation of IrrE deficient D. radiodurans bacteria expressing a low concentration of RecA is not compensated by the overexpression of RecA. Bacteria GY12354: recA+ irrE+/pGY11559: Pspac (squares), GY12359: ∆recA irrE+/ pGY11562: Pspac::recA+ (diamonds), GY12417: recA+ ∆irrE/pGY11559: Pspac (triangles) and GY12418: ∆recA ∆irrE/pGY11562: Pspac::recA+ (circles) were grown in TGY2X supplemented with streptomycin in presence (panel B, closed symbols) or in absence (panel A, open symbols) of IPTG (10 mM) to an OD650 of about 2–3. The cultures were irradiated and dilutions were plated as described in Experimental procedures.

reconstitute an intact genome after γ-irradiation. However, the low RecA content of these bacteria could decrease the rate of recombinational repair of DSBs and render this process particularly dependent on the availability of a long-lived recombination substrate. Recently, it was shown that, in vitro, the DNA damage-inducible DdrA protein (Tanaka et al., 2004) provides a DNA end-protection mechanism that prevents extensive genome degradation after DNA damage (Harris et al., 2004). We surmised that the radioresistance of uninduced ∆recA Pspac::recA+ strains would be highly dependent on the presence of DdrA. To test this hypothesis, we constructed a ∆ddrA ∆recA Pspac::recA+ mutant and determined its level of resistance to γ-irradiation. As previously shown (Harris et al., 2004), lack of DdrA protein increased the ionizing radiation sensitivity of recA+ bacteria at doses in excess of 5000 Gy (Fig. 1A, triangles). This effect was dramatically amplified in uninduced ∆ddrA ∆recA Pspac::recA+ strain (Fig. 1A, circles). In contrast, increasing RecA expression by adding IPTG increases the resistance of ∆ddrA ∆recA Pspac::recA+ strain relative to their ∆ddrA recA+ counterpart (Fig. 1B), suggesting that elevated RecA concentration can partially compensate for the loss of DdrA protein. Constitutive high expression of RecA does not compensate the absence of IrrE for the resistance to γ-irradiation Following DNA damage, synthesis of RecA protein is induced as part of a general cellular response through the activation of the key positive regulatory protein IrrE (Earl et al., 2002; Hua et al., 2003). To further investigate if other genes upregulated by IrrE are required in ∆recA Pspac::recA+ bacteria following γ-irradiation, we constructed a ∆irrE ∆recA Pspac::recA+ mutant and measured its level of resistance to γ-irradiation. Earl et al. (2002) showed that disruption of irrE resulted in severe sensitivity to ionizing radiation of recA+ D. radiodurans bacteria. Our results show that ∆irrE ∆recA Pspac::recA+ bacteria expressing constitutively high con-

centration of RecA remain sensitive to γ-irradiation but they appeared slightly less sensitive to doses ranging from 3400 to 6800 Gy relative to their ∆irrE recA+ counterpart, suggesting that the increased concentration of RecA protein could only partially compensate the IrrE deficiency (Fig. 3A and B). In contrast at doses up to 6800 Gy, ∆irrE ∆recA Pspac::recA+ and ∆irrE recA+ bacteria showed a comparable survival. This suggests that IrrE-mediated induction of crucial genes other than recA is required for resistance to ionizing radiation. Interestingly, although D. radiodurans’ cells lacking the IrrE function were highly sensitive to mitomycin C, the IPTGinduced overexpression of RecA can fully restore the resistance to mitomycin C (40 ng ml−1) of ∆irrE ∆recA Pspac::recA+ bacteria (Fig. 2A and B). Low level expression of RecA results in a delay in mending DSB, failure to reinitiate DNA replication, and commits bacteria to die during incubation in liquid medium after γ-irradiation We measured the kinetics of genome restitution in uninduced ∆recA Pspac::recA+ exposed to 6800 Gy γirradiation to test directly the hypothesis that the low RecA content slows down the rate of DNA DSB repair. This dose introduces approximately 200 DSBs per equivalent genome in a D. radiodurans cell (Battista, 1997) but does not affect cell viability at least as measured in a conventional plating assay (Fig. 1A). Recovery from DNA damage was monitored by the appearance of the complete pattern of the 11 resolvable fragments generated by NotI digestion of D. radiodurans genomic DNA (Kikuchi et al., 1999). As seen in Fig. 4, the intact genome was reconstituted within 3 h post irradiation in wild-type R1 and in IPTGinduced ∆recA Pspac::recA+ cells (Fig. 4A, B and D). Further incubation of these cells resulted in an increase in the amount of genomic DNA. In contrast, uninduced ∆recA Pspac::recA+ strains mended DSB slowly relative to the wild-type or the IPTG-induced cells and failed to reinitiate replication after completion of repair. Indeed, in

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Fig. 4. Delays in the kinetics of DNA DSB repair and inhibition of replication reinitiation after γ-irradiation in cells expressing RecA at a low constitutive level. Bacteria, GY12354: recA+/pGY11559: Pspac (panels A and B) and GY12359: ∆recA/pGY11562: Pspac::recA+ (panels C and D) were grown in TGY2X supplemented with spectinomycin in absence or in presence of IPTG (10 mM) to an OD650 of about 2–3, concentrated 10 times, exposed to 6800 Gy γ-irradiation, diluted in TGY2X without or with IPTG (10 mM) to an OD650 of 0.2 and then incubated at 30°C with agitation (150 rpm) for recovery. DNA agarose plugs were prepared at the post-irradiation times indicated on the abscissa and digested with NotI before being analysed by PFGE. The approximate number of RecA molecules per cell is indicated.

these cells, an intact genome was reconstituted only after 6 h post-irradiation incubation and no increase in the amount of genomic DNA was observed during the next 3 h (Fig. 4C). Consistent with their failure to reinitiate replication, the irradiated uninduced strain also showed a progressive decrease in cell viability during post-irradiation incubation (Fig. 5A, diamonds). The number of colony-forming units (cfus) was close to that of the non-irradiated control when the cells were plated immediately after irradiation while it decreased approximately 20-fold (Fig. 5A and B, diamonds) and 100-fold (Fig. 5B, plus signs) after 9 h postirradiation incubation in TGY broth if cells were exposed to 3.4 and 6.8 kGy respectively. An examination of the culture with a light microscope showed that the cells had a normal diplo-tetracoccal morphology and no clumps were observed that could explain the reduction in cfus (data not shown). The decrease in plating efficiency was dose-dependent (Fig. 5B) but even after an exposure to a dose as low as 300 Gy, the irradiated cells failed to resume growth within 9 h incubation period (Fig. 5B, circles).

The enhanced cell survival of uninduced ∆recA Pspac::recA+ bacteria recovering from DNA damage on plates could be ascribed to a transient inhibition of the cell cycle allowing time for excision repair of DNA damage. To test this hypothesis, the irradiated cells were plated on TGY agar or incubated for 3 h in MgSO4 buffer prior to transfer and incubation in TGY2X liquid medium. As shown in Fig. 5C, these treatments did not protect the cells from death in TGY2X liquid medium. Moreover, it seems that cell viability decreased more rapidly in TGY2X when incubated for 3 h in MgSO4 prior to transfer to TGY2X liquid medium (Fig. 5C, triangles). These results suggest that the DSBs that remain when cells are transferred in TGY2X liquid medium require induced levels of RecA protein to be rapidly repaired and allow cell survival. We also measured the RecA concentration in cells incubated during 3 h on TGY agar or in TGY2X liquid medium to test if the Pspac::recA+ construct could be induced upon plating even in the absence of IPTG. As shown in Fig. S3, we did not observe any significant increase in RecA protein concentration which could explain the radiation resis-

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Fig. 5. Cells containing limiting concentration of RecA are committed to die during post-irradiation incubation in liquid medium. A. Loss of viability during incubation in TGY2X following 3400 Gy γ-irradiation. Bacteria GY10974: ∆recA/pGY11536: Pspac::recA+ (diamonds), GY10973 recA+/pGY11530: Pspac (squares) were grown in TGY2X supplemented with chloramphenicol in absence (open symbols) or in presence (closed symbols) of IPTG (10 mM) to an OD650 of 2–3. The cultures were concentrated 10 times in TGY2X and γ-irradiated at a dose of 3400 Gy. Following irradiation, samples were diluted in TGY2X supplemented (closed symbols) or not (open symbols) with IPTG (10 mM). At times indicated on the abscissa, diluted samples were plated on TGY agar, or TGY agar supplemented with 1 mM IPTG. B. The decreased viability in liquid medium of cells expressing RecA at a low constitutive level was dose-dependent. Bacteria GY10974: ∆recA/ pGY11536: Pspac::recA+ were grown in TGY2X supplemented with chloramphenicol to an OD650 of 2–3. The cultures were concentrated 10 times in TGY2X and γ-irradiated at different doses as indicated. Following irradiation, samples were diluted in TGY2X to an OD650 of 0.2. At times indicated on the abscissa, diluted samples were plated on TGY plates and the colonies were counted after 3–5 days of incubation at 30°C. C. The decreased viability in liquid medium of cells expressing RecA at a low constitutive level was also observed if cells were incubated in MgSO4 buffer or plated on TGY agar for 3 h prior transfer to TGY2X liquid medium. Bacteria GY10974: ∆recA/pGY11536: Pspac::recA+ were grown in TGY2X supplemented with chloramphenicol to an OD650 of 2–3, washed twice with 10 mM MgSO4, concentrated 10 times in the same buffer and γ-irradiated at 6.8 kGy. Following irradiation, samples were diluted in TGY2X to an OD650 of 0.2 (diamonds) and incubated at 30°C, or maintained for 3 h at 30°C in MgSO4 buffer prior dilution in TGY2X liquid medium to an OD650 of 0.2 (vertical arrow) and further incubation at 30°C (triangles), or plated on TGY agar (approximately 107 cells per plate), incubated for 3 h at 30°C prior transfer to TGY2X liquid medium (vertical arrow) (squares). At the indicated times, diluted samples were plated on TGY agar and colonies were counted after 4 days incubation at 30°C.

tance of cells plated on TGY agar immediately after γ-ray irradiation (Fig. 1, diamonds). It has been proposed that D. radiodurans possesses mechanisms (analogous to eukaryotic DNA-damage induced checkpoints) for sensing the completion of DNA repair and relaying this information to the replication machinery (Battista, 1997; Battista et al., 1999). Our result suggests that RecA may play an important role in these regulatory processes. It appears that the uninduced irradiated strain does not synthesize sufficient RecA to ensure a rapid DNA repair, committing cells to die even though they were able to reconstitute a complete genome. How these cells can overcome this apparent regulatory checkpoint when directly plated onto solid media remains to be elucidated. Absence of LexA cleavage in ∆recA Pspac::recA+ strains Studies with mutant RecA proteins indicate that the coprotease function of RecA can play a key role in radioresistance (Satoh et al., 2002). We tested whether LexA cleavage occurs in uninduced ∆recA Pspac::recA+ strain after γ-irradiation. As seen in Fig. 6, the cellular amount of LexA protein in ∆recA Pspac::recA+ bacteria exposed to 6800 Gy γ-irradiation remained constant during 2 h

post-irradiation incubation. In contrast, when Pspac::recA+ was IPTG-induced, the LexA concentration decreased approximately by 10-fold, as observed in the wild-type (Fig. 6). This result suggests that the cellular level of RecA protein in uninduced bacteria was too low to efficiently promote LexA cleavage. At variance with the E. coli LexA protein, the deinococcal LexA protein plays no role in induction of RecA after DNA damage (Narumi et al., 2001; Bonacossa de Almeida et al., 2002). Nevertheless, the absence of LexA cleavage could prevent the induction of some critical unknown repair protein and inhibits the recovery of these cells after γ-irradiation. To establish a role for the LexA regulation in radioresistance, we constructed mutants encoding non-cleavable LexA proteins. Alignment of the D. radiodurans LexA with the E. coli and Bacillus subtilis LexA proteins indicates that the Ala-Gly cleavage site (amino acids 83 and 84 in the deinococcal LexA) and the two residues Ser119 and Lys158, located in the catalytic site, are conserved among these three proteins (Narumi et al., 2001). To mutate the putative cleavage or catalytic sites, we introduced a Gly84→Asp or a Lys158→Arg change into the deinococcal LexA protein. The two mutant proteins were purified and shown to be refractory to RecA-stimulated cleavage

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Fig. 6. Absence of LexA cleavage after γ-irradiation in cells expressing RecA at a low constitutive level. RecA-mediated cleavage of purified LexA protein was visualized by Western analysis with D. radiodurans LexA antiserum (diluted 1:10 000). Bacteria GY12354: recA+/pGY11559: Pspac and GY12359: ∆recA/pGY11562: Pspac::recA+ were grown in TGY2X supplemented with spectinomycin to an OD650 of 2–3 in presence or in absence of IPTG 10 mM. The cultures were concentrated 10 times in TGY2X, γ-irradiated at 6.8 kGy and then allowed to recover for 2 h post irradiation before preparation of cell extracts. LexA and RecA proteins were visualized by Western analysis with D. radiodurans LexA and RecA antiserum respectively.

in vitro (Fig. 7). Cells expressing the two non-cleavable LexA proteins behaved as the wild-type with respect to the kinetics of DSB repair as well as reinitiation of replication after γ-irradiation (Fig. 8). Moreover, a deletion of the lexA gene in bacteria expressing limited concentration of RecA protein was not sufficient to restore wild-type kinetics of DNA DSB repair (Fig. 8F). Thus, LexA protein seems to play no major role in these recovery processes. Discussion Deinococcus radiodurans displays an exceptional ability to withstand the lethal effect of ionizing radiations and desiccation (Mattimore and Battista, 1996; Battista, 1997). This property is related to efficient DNA DSB repair. Among the genes which were identified as involved in D. radiodurans radioresistance, only polA and recA, and to a lesser extent ddrA, irrE and pprA (Gutman et al., 1993;

Daly et al., 1994; Daly and Minton, 1995; Earl et al., 2002; Harris et al., 2004; Narumi et al., 2004), seem to play a major role. Restitution of an intact genome from a myriad of DNA fragments requires recA-dependent recombinational repair and other recA-independent pathways involved in early assembly of DNA fragments (Daly and Minton, 1996). Among these mechanisms, non-homologous end-joining (NHEJ) could be facilitated by a compact structure of the nucleoid, maintaining, even after γirradiation, the broken ends at proximity (Levin-Zaidman et al., 2003; Zimmerman and Battista, 2005). Singlestrand annealing (SSA) could also participate to assembling of DNA fragments by strand invasion of broken recessed ends on an intact homologous chromosomal region (Daly and Minton, 1996). The precise role of RecA protein in this process is not known. To investigate the role of RecA in radioresistance and to determine whether a limiting concentration of RecA

Fig. 7. A. Purification of D. radiodurans LexAG84D and LexAK158R proteins. Samples were subjected to sodium dodecyl sulphate – 15% PAGE and stained with Coomassie brilliant blue. Left panel, Lanes: 1 and 6, 10 kDa protein ladder (Invitrogen); 2, total cellular proteins from E. coli BL21(DE3)/pLysS/pET3lexAG84D induced by IPTG; 3, resuspension from 30% ammonium sulphate precipitation; 4, pooled LexAG84D fractions from HiTrap Heparin HP column; 5, pooled LexAG84D fractions from a Mono S column. The position of the 25 kDa band of LexA is indicated on the right. Right panel: purification of the LexAK158R protein from E. coli BL21(DE3)/pLysS/pET3lexAK158R induced by IPTG. All the lanes ranging from 1 to 6 are similar to the left panel except that LexAK158R replace LexAG84D. B. Purified LexAG84D and LexAK158R were uncleavable while LexA protein from wild-type R1 is cleavable. Purified LexA(wt), LexAG84D and LexAK158R proteins (0.4 µM) were incubated in Tris-HCl buffer as mentioned in Experimental procedures section in absence (–) or in presence (+) of RecA protein (4.2 µM) for 3 h at 37°C. LexA proteins were analysed by Western analysis with D. radiodurans LexA antiserum (diluted 1:10 000). The arrows on the left indicate the positions of LexA protein and its breakdown products. © 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 338–349

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Fig. 8. Kinetics of DNA DSB repair following γ-irradiation of lexA deficient mutants. A. Bacteria GY12207: recA+ ∆lexA/pI8; B. GY12208: recA+ ∆lexA/pGY11408: pI8 lexA+ C. GY12209 recA+ ∆lexA/pGY12106: pI8 lexAG84D; D. GY12210 recA+ ∆lexA/pGY12107: pI8 lexAK158R; E. GY12422: ∆lexA ∆recA/pGY11562: Pspac::recA+ (IPTG induced); F. GY12422: ∆lexA ∆recA/ pGY11562: Pspac::recA+ (non-induced) were grown to an OD650 of about 2–3, exposed to γ-irradiation at a dose of 6800 Gy, diluted in TGY2X to an OD650 of 0.2 and then incubated at 30°C for recovery. DNA agarose plugs were prepared at the indicated post-irradiation times on the abscissa and digested with NotI before being analysed by PFGE.

protein could influence the cell’s capacity to repair the radiation-induced DNA damage, we examined the effect of low and high RecA concentration. We showed that, when plated on TGY agar immediately after γ-irradiation, bacteria expressing limiting concentration of RecA (2500 molecules per cell) were as radioresistant as the wild-type bacteria expressing radiation-induced concentration of RecA (44 000 molecules per cell). This suggests that a limited number of RecA molecules are sufficient to repair several hundred DNA DSBs. However, limiting the concentration of RecA protein increased radiation sensitivity of ddrA deficient cells suggesting that, under these conditions, a functional ddrA gene product is required for survival. DdrA is involved in protection of single-strand DNA with 3′ end and presumably ensures long-lived recombinational substrates and recycling of RecA protein. The additive effect of a defect in ddrA gene and of a limiting concentration of RecA also suggests that DdrA is involved in a recA-independent repair mechanism. As DdrA is distantly but specifically related to the Rad52 family of eukaryotic proteins, it was suggested that DdrA could be a component of a SSA system that functions in conjunction with RecA-dependent homologous recombination (Harris et al., 2004). High concentrations of RecA protein can partially suppress radiation sensitivity of ddrA deficient cells suggesting that (i) by polymerization on ssDNA, RecA can protect from degradation single-strand tails, or (ii) more rapid recombinational repair could partially compensate for defects in alternative DSB repair mechanisms.

Although, IrrE deficient bacteria were sensitive to irradiation and mitomycin C, the sensitivity to mitomycin C was suppressed by the high constitutive expression of RecA protein (100 000 molecules per cell). This result suggests that some induction of irrE-dependent genes, different from RecA, were required for DNA repair after γirradiation whereas induction of RecA protein was sufficient for DNA repair of mitomycin-promoted DNA lesions. This can be explained by the variety of structures generated at DNA ends by the γ-irradiation requiring maturation steps before an efficient repair by a RecA-dependent or a RecA-independent pathway. We propose that some genes under the control of IrrE could be involved in the processing of damaged DNA ends after γ-irradiation. To determine the kinetics of DNA DSB repair, cells expressing limiting concentration of RecA protein were incubated post irradiation in TGY broth. Surprisingly, the viability of cells after γ-irradiation decreased with time of incubation and cells were unable to restart growth in TGY2X liquid medium. This effect was observed even at doses as low as 300 Gy. However, when exposed to 6800 Gy γ-irradiation, an intact genome was reconstituted with a delay of about 3 h compared with wild-type R1, the delay observed when the concentration of RecA protein at low level is insufficient for repairing DNA DSBs rapidly. Strikingly, no increase in the cellular DNA content was observed during the 3 h following reconstitution of an intact genome, suggesting a defect in replication reinitiation in cells containing limited concentration of RecA pro-

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 338–349

Low level of RecA delays of DNA DSB repair in D. radiodurans tein. The isolation by Mattimore et al. (1995) of a mutant presenting no defect in DSB repair but a delay in restarting growth suggests that D. radiodurans possesses mechanisms for sensing the completion of DNA repair, a DNA damage checkpoint to allow replication reinitiation of cell division (Battista, 1997; Battista et al., 1999). Cells, in which RecA was expressed at low level, allow repair but are committed to die. The reasons for this phenomenon are not understood. By analysing recA mutants, Satoh et al. (2002) proposed that the coprotease activity of RecA was as important as its recombinase activity for an efficient repair of DNA DSB. Here we showed that at low concentration RecA was not sufficient to promote LexA cleavage. By measuring the kinetics of DSB repair in lexA deficient mutants expressing limiting concentrations of RecA protein or in lexA(Ind–) mutants in a recA+ background, we show that a defect in LexA cleavage was not responsible for the delay in DSB repair, for the absence of replication reinitiation and for cell death. Thus, LexA protein (DR0344) seems to play no major role in the recovery processes after γ-irradiation. However, we cannot rule out the possibility that expression of some critical genes is under dual regulation. For example, it has been previously shown that, in B. subtilis, expression of a second effector, ComK, can force expression of LexA-controlled genes in the presence of intact LexA repressor, resulting in induction of recA gene expression during the development of competence (Hamoen et al., 2001). In D. radiodurans, RecA protein acts as a coprotease in DR0074 (a second LexA homologue) cleavage. Results from Sheng et al. (2004) showed that DRA0074 is not involved in the radiation-induced DNA damage response but seems to act as a regulator in some other metabolic pathway (Sheng et al., 2004). However, we cannot exclude that the coprotease activity of RecA is essential in D. radiodurans for maturation of a protein, different from LexA, involved in tolerance of DNA lesions, as in E. coli in which RecAdependent maturation of UmuD is required for translesion synthesis in SOS mutagenesis (Nohmi et al., 1988). Experimental procedures Cultures, media and transformation Cultures of D. radiodurans and E. coli, media, and transformation of D. radiodurans with plasmid or genomic DNA were done as described by Bonacossa de Almeida et al. (2002). When necessary, media were supplemented with the appropriate antibiotics used at the following final concentrations: ampicillin, 100 µg ml−1 for E. coli; chloramphenicol, 20 µg ml−1 for E. coli or 3 µg ml−1 for D. radiodurans; tetracycline 2.5 µg ml−1 for D. radiodurans; kanamycin 6 µg ml−1 for D. radiodurans; spectinomycin, 40 µg ml−1 for E. coli or 75 µg ml−1 for D. radiodurans.

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Bacterial strains and plasmids Bacterial strains are listed in Table 1. All D. radiodurans strains used were derivatives of the wild-type strain R1 ATCC13939. The E. coli DH5α strain was used routinely for cloning whereas SCS110 was used to propagate plasmids prior to transformation of D. radiodurans, because this process is sensitive to the methylation status of the transforming DNA (Meima et al., 2001). Alleles: ∆(ddrA)Ωcat (∆DR0423), ∆(lexA)Ωkan (∆DRA0344), ∆(lexA)Ωcat (∆DRA0344) and ∆(irrE)Ωcat (∆DR0167) were constructed in vitro by the tripartite ligation method described by Lecointe et al. (2004) using the primer pairs indicated in Table S1. In these constructs the cat gene was under the control of the deinococcal PtufA promoter (Earl et al., 2002) and the kan gene under the control of the deinococcal plasmid pI8 Pout promoter (Bonacossa de Almeida et al., 2002). The ∆(ddrA)Ωcat, the ∆(lexA)Ωcat and the ∆(irrE)Ωcat alleles were introduced into GY12354 and GY12359 to give rise to ddrA deficient GY12363 and GY12364, to lexA deficient GY12421 and GY12422, and to irrE deficient GY12417 and GY12418 respectively (Table 1). The ∆(lexA)Ωkan allele was introduced into D. radiodurans R1 to give rise to strain GY12201 (Table 1). The genetic structure and purity of GY12363, GY12364, GY12421, GY12422, GY12417, GY12418 and GY12201 were checked by polymerase chain reaction (PCR) using primers described in Table S1 (data not shown). Plasmids are listed in Table 2. To give rise to plasmid pGY11096, the lexA gene was amplified from D. radiodurans genomic DNA using primers lexA15 and lexA16. After cleavage with XhoI and Eco47III, the amplicon was cloned between the XhoI and NruI sites of the vector pBCSKP.

Analysis of expression of RecADr protein by immunoblotting Cell extracts were prepared from a known amount of cells as described by Bonacossa de Almeida et al. (2002). Cell concentrations were determined by counting bacteria in a Malassez chamber using a light microscope. Protein concentrations were measured with a protein assay kit (Bio-Rad) using the manufacturer’s micro assay procedure. Portions of the cell extracts were subjected to electrophoresis through a 12% SDS-polyacrylamide gel, along with known amounts of purified RecA protein (a gift of Michael Cox) as standard. The proteins were transferred to a nitrocellulose membrane (BA85 0.45 mm Schleicher and Schuell) and treated with a 1:1000 dilution of antibodies raised against RecA protein from E. coli. Antigen–antibody complexes were visualized using goat antirabbit IgG coupled to alkaline phosphatase and quantified using Image-Quant software (Molecular Dynamics). RecADr cell concentrations were calculated from the RecADr concentration in the cell extract, the molecular mass of a RecA Dr monomer (38 144 Da), and the bacterial concentration of cultures from which cell extracts were prepared.

Survival after γ-irradiation Bacteria were grown to an OD650 of 2–3 in TGY2X supplemented with 10 mM IPTG or without IPTG and appropriate

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 338–349

346 E. Jolivet et al. Table 1. Bacterial strains. Name

Relevant markersa

Source/reference(s)

E. coli strains DH5α SCS110 BL21(DE3)

hsdR17 recA1 endA1 lacZ∆M15 endA1 dam dcm (F′ lacI q lacZ∆M15) Host for gene expression

Invitrogen Stratagene Novagen

D. radiodurans strains R1 GY10973 GY10974 GY12235 GY12330 GY12354 GY12359 GY12362 GY12363 GY12364 GY12201 GY12207 GY12208 GY12209 GY12210 GY12417 GY12418 GY12421 GY12422

ATCC 13939 as R1 but amyE Ω (PtufA::lacI kan) as GY10973 but ∆(cinA ligT recA)Ωtet GY10973 (pGY11530) GY10974 (pGY11536 Pspac::recA+) GY10973 (pGY11559) GY10974 (pGY11562 Pspac::recA+) as GY10973 but ∆(ddrA)Ωcat as GY12354 but ∆(ddrA)Ωcat as GY12359 but ∆(ddrA)Ωcat as R1 but ∆(lexA)Ωkan GY12201 ∆(lexA)Ωkan (pI8) GY12201 ∆(lexA)Ωkan (pGY11408 = pI8 lexA+) GY12201 ∆(lexA)Ωkan (pGY12106 = pI8 lexAG84D) GY12201 ∆(lexA)Ωkan (pGY12107 = pI8 lexAK158R) as GY12354 but ∆(irrE)Ωcat as GY12359 but ∆(irrE)Ωcat as GY12354 but ∆(lexA)Ωcat as GY12359 but ∆(lexA)Ωcat

Anderson et al. (1956) Lecointe et al. (2004) Lecointe et al. (2004) 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

a. Genes of D. radiodurans are named for their E. coli orthologues (Makarova et al., 2001). Ω indicates an insertion; :: indicates a novel joint.

antibiotics when bacteria contained a plasmid. The cultures were concentrated 10 times in TGY2X and irradiated on ice with a 137Cs irradiation system (Institut Curie, Orsay, France) at a dose rate of 56.6 Gy min−1. Following irradiation, diluted samples were plated on TGY agar or TGY agar supplemented with 1 mM IPTG and incubated for 3–4 days at 30°C before the colonies were counted.

Pulsed field gel electrophoresis Irradiated cultures (6800 Gy) and unirradiated control (0 Gy) were diluted to an OD650 of 0.2 in TGY2X supplemented with

10 mM IPTG or without IPTG and incubated at 30°C. At different post-irradiation incubation times, culture aliquots (5 ml) were taken to prepare DNA plugs as described by Harris et al. (2004). The DNA in the plugs was digested for 16 h at 37°C with 10 units of NotI restriction enzyme. After digestion, the plugs were subjected to pulsed field gel electrophoresis as described by Harris et al. (2004). In parallel, the aliquots were diluted and plated on TGY agar supplemented or not with 1 mM IPTG; colonies were counted after 3–4 days incubation at 30°C to estimate survival in liquid medium and reinitiation of cell division during post-irradiation incubation.

Table 2. Plasmids. Designation

Relevant descriptiona

Source or reference

Plasmids that replicate pGY11096 pGY12102 pGY12101 pET3lexAwt pET3lexAG84D pET3lexAK158R

in E. coli pBCSKP with a fragment encoding LexADr+ pBCSKP with a fragment encoding LexADrG84D (site-directed mutagenesis) pBCSKP with a fragment encoding LexADrK158R (site-directed mutagenesis) pET3a with a fragment encoding LexADr+ pET3a NdeI-BamHI::635-bp PCR product from pGY12102 pET3a NdeI-BamHI::635-bp PCR product from pGY12101

This work This work This work Narumi et al. (2001) This work This work

Plasmids that replicate in E. coli and D. radiodurans pI8 Shuttle vector AmpR in E. coli, CamR in D. radiodurans pGY11530 Expression vector; Pspac, PtufA::lacI, CamR in E. coli and in D. radiodurans pGY11536 pGY11530 with a fragment encoding RecADr; Pspac::recADr pGY11559 Expression vector; Pspac, PtufA::lacI, SpcR in E. coli and in D. radiodurans pGY11562 pGY11559 with a fragment encoding RecADr; Pspac::recADr pGY11408 pI8 with a fragment encoding LexADr+ pGY12106 pI8 with a fragment encoding LexADrG84D pGY12107 pI8 with a fragment encoding LexADrK158R

Meima and Lidstrom (2000) Lecointe et al. (2004) Lecointe et al. (2004) Mennecier et al. (2004) This work Bonacossa de Almeida et al. (2002) This work This work

a. Gene denomination and genetic symbols are as in Table 1. © 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 338–349

Low level of RecA delays of DNA DSB repair in D. radiodurans Measurement of in vivo LexA cleavage LexA cleavage was measured by the decrease in the cellular level of the LexADr protein in cells exposed to 6800 Gy γirradiation. Non-irradiated and irradiated cells were diluted in TGY2X to an OD650 of 0.2 and incubated for 2 h at 30°C before cell extracts were prepared as described in Bonacossa de Almeida et al. (2002). Protein concentrations were measured with a protein assay kit (Bio-Rad) using the manufacturer’s micro assay procedure. Portions of the cell extracts were run on a 12% SDS-polyacrylamide gel along with purified LexADr protein as standard and subjected to immunoblotting using anti-deinococcal LexA antibodies (Narumi et al., 2001). Antigen–antibody complexes were visualized using goat anti-rabbit IgG coupled to alkaline phosphatase.

Construction of lexADrG84D and lexADrK158R mutants by directed mutagenesis The plasmid-borne lexA [Ind–] alleles, lexADrG84D and lexADrK158R, were constructed by site-directed mutagenesis using an Ex-Site PCR-based kit (Stratagene). The mutations lexADrG84D and lexADrK158R were introduced into plasmid pGY11096: lexA+ (Bonacossa de Almeida et al., 2002) using the mutated primer pairs LexAG84Dplus/LexAG84Dminus and LexAK158Rplus/LexAK158Rminus (see Table S1). The resulting plasmids were designated pGY12102 and pGY12101 respectively (Table 2; Table S1). The mutated genes were then subcloned as a 1.3 kb HincII-BamHI fragment into the shuttle vector pI8 between the BamHI and EcoICRI sites (see Fig. S2 for the details of the construction). The presence of the desired lexA mutations in the resulting plasmids pGY12106 and pGY12107 were verified by sequencing. Sequencing was performed by GENOME Express (Grenoble, France). The mutated genes were subcloned as an amplified NdeI-BamHI fragment from pGY12102 and pGY12101 and replacing the NdeI-BamHI fragment containing the wild-type lexA (lexAwt) coding region in pET3lexAwt (Narumi et al., 2001) given rise to pET3lexAG84D and pET3lexAK158R (see Fig. S2 for the details of the construction). The DNA sequence of the expression plasmids was checked to confirm the lack of introduction of errors by PCR.

Purification of the wild-type and mutated LexA proteins The wild-type and mutant lexA genes were induced in E. coli strain BL21(DE3) containing pLysS and plasmids pET3lexAwt, pET3lexAG84D or pET3lexAK158R, and proteins were purified as described by Narumi et al. (2001) except that a Mono S column was used instead of a Resource S column in the last purification step.

In vitro LexA cleavage assay A buffer consisting of 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 12.5 nM oligonucleotide (35-mer), 1 mM ATPγS and 4.2 µM RecA protein was incubated for 10 min at 37°C. Then LexA (0.4 µM) was added and the mixture was further incubated

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for 3 h at 37°C. Samples were then used for Western analysis with D. radiodurans LexA antiserum (diluted 1:10 000).

Acknowledgements We thank M. M. Cox for the gift of RecADr purified protein, P. Servant, J.R. Battista and M. Radman for their helpful discussion, and R. Devoret for his constant interest. We thank the Institut Curie for the use of the 137Cs irradiation system, and V. Favaudon for his help in gamma irradiation. This work was supported by Electricité de France (N°RB2003), ARC (N°4360), the Centre National de la Recherche Scientifique (GEOMEX) and the Commissariat à l’Energie Atomique (LRC CEA 42V). E. Jolivet gratefully acknowledges the Centre National de la Recherche Scientifique for its support in this work (CNRS postdoctoral fellowship N°1002121). This work was carried out in compliance with the current laws governing genetic experimentation in France.

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348 E. Jolivet et al. tional inhibition without physically displacing LexA. J Biol Chem 276: 42901–42907. Harris, D.R., Tanaka, M., Saveliev, S.V., Jolivet, E., Earl, A.M., Cox, M.M., and Battista, J.R. (2004) Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1. PLoS Biol 2: e304. Hua, Y., Narumi, I., Gao, G., Tian, B., Satoh, K., Kitayama, S., and Shen, B. (2003) PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans. Biochem Biophys Res Commun 306: 354–360. Iyer, L.M., Koonin, E.V., and Aravind, L. (2002) Classification and evolutionary history of the single-strand annealing proteins, RecT, Redbeta, ERF and RAD52. BMC Genomics 3: 8. Kikuchi, M.N., Narumi, I., Kitayama, S., Watanabe, H., and Yamamoto, K. (1999) Genomic organization of the radioresistant bacterium Deinococcus radiodurans: physical map and evidence for multiple replicons. FEMS Microbiol Lett 174: 151–157. Lecointe, F., Coste, G., Sommer, S., and Bailone, A. (2004) Vectors for regulated gene expression in the radioresistant bacterium Deinococcus radiodurans. Gene 336: 25–35. Levin-Zaidman, S., Englander, J., Shimoni, E., Sharma, A.K., Minton, K.W., and Minski, A. (2003) Ringlike structure of the Deinococcus radiodurans genome: a key to radioresistance? Science 299: 254–256. Little, J.W., and Mount, D.W. (1982) The SOS regulatory system of Escherichia coli. Cell 29: 11–22. Makarova, K.S., Aravind, L., Wolf, Y.I., Tatusov, R.L., Minton, K.W., Koonin, E.V., and Daly, M.J. (2001) Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiol Mol Biol Rev 65: 44–79. Mattimore, V., and Battista, J.R. (1996) Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation. J Bacteriol 178: 633–637. Mattimore, V., Udupa, K.S., Berne, G.A., and Battista, J.R. (1995) Genetic characterization of forty ionizing radiationsensitive strains of Deinococcus radiodurans: linkage information from transformation. J Bacteriol 177: 5232– 5237. Meima, R., and Lidstrom, M.E. (2000) Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors. Appl Environ Microbiol 66: 3856–3867. Meima, R., Rothfuss, H.M., Gewin, L., and Lidstrom, M.E. (2001) Promoter cloning in the radioresistant bacterium Deinococcus radiodurans. J Bacteriol 183: 3169–3175. Mennecier, S., Coste, G., Servant, P., Bailone, A., and Sommer, S. (2004) Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism Deinococcus radiodurans. Mol Genet Genomics 272: 460– 469. Minton, K.W. (1994) DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans. Mol Microbiol 13: 9–15. Narumi, I., Satoh, K., Kikuchi, M., Funayama, T., Yanagisawa, T., Kobayashi, Y., et al. (2001) The LexA protein from Deinococcus radiodurans is not involved in RecA

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Supplementary material The following supplementary material is available for this article online: Fig. S1. Quantification of RecA protein in cells containing an additional lacI gene and expressing recADr gene from the Pspac promoter. Cellular levels of RecA protein were analysed in strain GY10973: recA+ PtufA::lacI containing the expression vector pGY11530: Pspac PtufA::lacI and in strain GY10974: ∆recA PtufA::lacI containing the expression vector pGY11530: Pspac PtufA::lacI or its derivative pGY11536: Pspac::recA+ PtufA::lacI. GY10973 bacteria were grown in TGY2X, concentrated 10 times by centrifugation, exposed to γ-rays (6800 Gy) or not (0 Gy) as indicated, diluted 100 times and incubated 2 h before cell extracts were prepared. GY10974 derivatives were grown in TGY2X without (–) or with (+) IPTG at a final concentration of 10 mM before cell extracts were prepared. Samples of cell extracts, equivalent to 15 µg or 5 µg of protein as indicated, were loaded in each lane. RecA protein was visualized by Western analysis using E. coli RecA antiserum. Fig. S2. Diagram for construction of pI8 and pET3a derivatives encoding lexADrK158R and lexADrG84D. The constructions of pGY12106: pI8 lexADrG84D and pET3lexADrG84D were performed as described for pGY12107 and pET3lexADrK158R, except that the plasmid pGY12102 was used instead of pGY12101. Fig. S3. RecA protein concentration did not increase when cells were incubated on TGY agar. Cellular levels of RecA protein were analysed in GY12330: ∆recA/pGY11536: Pspac::recA+ and in GY12235: recA+/pGY11530: Pspac. Bacteria were grown overnight in TGY2X supplemented with chloramphenicol to an OD650nm = 2.5. Then, the cells were diluted 10 times in TGY2X and incubated for 3 h, or were

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 338–349

Low level of RecA delays of DNA DSB repair in D. radiodurans plated on TGY agar, incubated for 3 h and subsequently resuspended in TGY2X. Cell extracts were prepared from the overnight cultures (lanes 1 and 4), from the cultures incubated for 3 h in TGY2X (lanes 2 and 5) and from cells incubated for 3 h on TGY agar (lanes 3 and 6). Samples of cell extracts, equivalent to 20 µg of protein, were loaded in each lane along with known amounts of purified RecA protein as

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standard (lane 7, 50 ng; lane 8, 100 ng). RecA protein was visualized by Western analysis using E. coli RecA antiserum. Table S1. Overview of primers used for cloning and diagnostic PCR experiments. This material is available as part of the online article from http://www.blackwell-synergy.com

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 338–349