Replication is required for the RecA localization response to DNA damage in Bacillus subtilis

Simmons et al. 10.1073/pnas.0607123104.

Supporting Information

Files in this Data Supplement:

SI Figure 5
SI Results
SI Table 2
SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Table 3




SI Figure 5

Fig. 5. RecA-GFP confers survival to wild-type levels at lower doses of IR. Survival curves were generated as described in Material and Methods. (A) Survival curves of exponentially growing strains bearing the recA+, recA-gfp, or recA::neo (1) alleles and their respective sensitivities to a range of IR between 0 and 20 Gy. (B) Sensitivities of the strains to an increased level of IR ranging between 20 and 60 Gy. The data points plotted for 0-20-Gy dosages are the same for both graphs. The error bars reflect the standard deviation between independent experiments.

1. Sciochetti SA, Piggot PJ, Sherratt DJ, Blakely G (1999) J Bacteriol 181:6053-6062.

SI Figure 6

Fig. 6. RecA-GFP foci are inducible with an increasing dosage of IR. Strain LAS40 (recA-gfp) was grown to mid-log phase in S7₅₀ minimal medium and treated with the indicated amount of IR. (A) Time course induction of RecA-GFP focus formation in cells treated with 5 Gy. Filled circles, cells treated with IR; filled squares, untreated control. The number of cells scored for each untreated time point are: T = 0 (n = 270), T = 5 min (n = 150), T = 10 (n = 409), T = 20 (n = 423), T = 40 (n = 508). The number of cells scored for each time point after exposure to 5 Gy are as follows: T = 0 (n = 535), T = 5 min (n = 157), T = 10 (n = 280), T = 20 (n = 282), T = 40 (n = 360). (B) Number of RecA-GFP foci per cell with an increasing dosage of IR. The number of cells (n) that were scored for each treatment are indicated. Whole-cell fluorescence (WCF) indicates the percentage of cells that were filled with an elevated level RecA-GFP fluorescence that was not discernable as individual foci.

SI Figure 7

Fig. 7. Visualization of replisome foci in damaged cells. Cells were grown in S7₅₀ minimal medium at 30°C, followed by challenge with the indicated DNA-damaging agent: (A) untreated; (B) 5 J/m² of UV; (C) 20 ng/ml MMC; (D) 5 Gy of IR. DnaX-GFP foci are shown in green, and the membrane stain (FM4-64) is in red. (Scale bar: 2 mm.)

SI Figure 8

Fig. 8. Visualization of cells with Spo0J-GFP and DnaX-GFP foci before and after DnaAN depletion. Cells were grown in minimal medium at 30°C in the presence or absence of IPTG, as follows: (A) Spo0J-GFP + IPTG; (B) DnaX-GFP + IPTG; (C) Spo0J-GFP - IPTG; (D) DnaX-GFP - IPTG. It should be noted that the number of Spo0J-GFP foci per cell is reduced in the absence of IPTG and replication initiation (C). Spo0J-GFP serves as a marker for the number of replication origins per cell. We observe one Spo0J-GFP focus in »97% (n > 250) of cells scored after DnaAN depletion. This result supports the conclusion that DnaAN-depleted cells are blocked for initiation of replication.

SI Figure 9

Fig. 9. Existing RecA-GFP protein is sufficient for focus formation. B. subtilis cells (recA-gfp; lexA⁺) were grown in LB, split, and treated with the indicated levels (1 or 40 J/m²) of UV, followed by immunoblot analysis. (A) Level of RecA-GFP from cells treated with 1 J/m² of UV and from the untreated control. For each sample, a dilution series was loaded in 2-fold increasing amounts. The loading control is an arbitrary section of a duplicate Coomassie-stained SDS/PAGE. (B) Immunoblot analysis of RecA-GFP levels in cells treated with 40 J/m² of UV. The numerical values shown in A and B represent the percentage of cells (n > 400) with RecA-GFP foci after treatment with the indicated level of UV.

SI Figure 10

Fig. 10. Visualization of RecA-GFP threads bridging two nucleoids. Strain LAS40 (recA-gfp) was grown in S7₅₀ minimal medium and treated with 10 Gy of IR. (A) Visualization of RecA-GFP (green) only. (B) Merge of RecA-GFP and the cell membrane (gray) stained with FM4-64. (C) Merge of DNA (blue) stained with DAPI and the cell membrane stained with FM4-64.

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Table 2. RecA-GFP forms foci in response to exogenous DNA damage

*Cultures of LAS40 (recA-gfp) were grown to mid-log phase (OD₆₀₀ = 0.3-0.6) in S7₅₀ minimal medium at 30^oC prior to microscopy. The cells treated with mitomycin C (20 ng/ml) or 2-aminopurine (600 µg/ml) were incubated at 30^oC for 45 minutes prior to preparation of the samples for microscopy. For UV and IR treatment, a 5-ml aliquot of the culture was extracted and exposed to 1 J/m² of UV or 5 Gy of IR, followed by immediate preparation of the sample for microscopy. Each of the treatments were based on an equitoxic dosage corresponding to >90% survival. The concentrations of 2-AP and mitomycin C were based on survival curves determined from exposing the cells for 1 hr followed by washing and plating on LB to determine viable counts. For DSB formation, the I-SceI endonuclease was expressed in cells with the addition of 0.5% xylose for 30 min, followed by visualization. The number of cells scored is indicated in the rightmost columns.

^†Whole-cell fluorescence indicates that the entire cell was filled with an elevated level of RecA-GFP fluorescence that could not be distinguished as countable foci or patches. This particular localization pattern is not inducible by exogenous sources of DNA damage.

Table 3. Strain list

 

Strain	Genotype	Reference
PY79	Prototroph SPb-	(1)
BTS8	PY79 dnaX-gfpmut2 (spc)	(2)
LAS24	PY79 recA::neo (neo)	This work
LAS26	PY79 spo0J-gfpmut2 (spc)	This work
LAS40	PY79 recA-mgfpmut2A206K (spc)	This work
LAS60	PY79 recA-mcfp(w7)A206K (spc)	This work
LAS66	PY79 dnaA::PspacdnaA (cat), recA-mgfpmut2A206K (spc)	This work
LAS72	PY79 recA-yfpmut2 (cat)	This work
LAS141	PY79 dnaA::PspacdnaA (cat), dnaX-gfpmut2 (spc)	This work
LAS179	PY79 recA-mcfp(w7)A206K (spc), dnaX-myfpmut2A206K (cat)	This work
JJJ39	trpC2, Dupp	(3)
LAS184	JJJ39, recA-mgfpmut2A206K (spc)	This work
LAS185	JJJ39 dinR3, recA-mgfpmut2A206K (spc)	This work
LAS186	PY79 recA-mgfpmut2A206K (spc), dnaB134(ts), zhb83::Tn 917 (mls)	This work
LAS195	PY79 amyE::PxylI-SceI (cat)	This work
LAS202	PY79 cgeD::I-SceI recognition site (cat)	This work
LAS204	PY79 amyE::PxylI-SceI (mls)	This work
LAS208	PY79 amyE::PxylI-SceI (mls), cgeD::DI-SceI recognition site (cat)	This work
LAS210	PY79 amyE::PxylI-SceI (mls), cgeD::I-SceI recognition site (cat)	This work
LAS220	PY79 amyE::PxylI-SceI (mls), cgeD::I-SceI recognition site (tet)	This work
LAS213	PY79 amyE::PxylI-SceI (mls), cgeD::I-SceI recognition site (cat). ), recA-mgfpmut2A206K (spc)	This work
LAS214	PY79 amyE::PxylI-SceI (mls), cgeD::DI-SceI recognition site (cat), recA-mgfpmut2A206K (spc)	This work
LAS223	PY79 dnaA:: PspacdnaA (tet)	This work
LAS237	PY79 amyE::PxylI-SceI (mls), cgeD::I-SceI recognition site (cat), dnaA::PspacdnaA (tet)	This work
LAS254	PY79 spo0J-gfpmut2 (spc), dnaA::PspacdnaA (tet)	This work
LAS261	PY79 amyE::PxylI-SceI (mls), cgeD::I-SceI recognition site (cat), dnaA::PspacdnaA (tet), recA-mgfpmut2A206K (spc)	This work
LAS301	PY79 lacA::Pxyl recA-mgfpmut2A206K (tet)	This work
LAS305	PY79 amyE::PxylI-SceI (mls), cgeD::I-SceI recognition site (tet), recA-mgfpmut2A206K (spc), dnaB134(ts), zhb83::Tn 917 (mls)	This work

1. Youngman P, Perkins JB, Losick R (1984) Plasmid 12:1-9

2. Lemon KP, Grossman AD (1998) Science 282:1516-1519.

3. Fabret C, Ehrlich SD, Noirot P (2002) Mol Microbiol 46:25-36.

SI Results

Exogenous DNA-Damaging Agents Stimulate RecA-GFP Foci. RecA-GFP forms a single focus in »11% (n = 1,454) of untreated B. subtilis cells (Fig. 1A and SI Table 2) grown at 30°C in minimal medium. These results are similar to those in E. coli (1) and are consistent with estimates for the frequency at which replication forks encounter DNA damage requiring recombinational repair during normal growth in E. coli (2). Considering these observations, the frequency of RecA-GFP focus formation in untreated cells suggests that the foci likely result from endogenous DNA damage.

The percentage of cells with RecA-GFP foci increased following damage with an exogenous agent, and time course microscopy experiments revealed that RecA-GFP foci are established within minutes following IR exposure (SI Fig. 6). IR creates several types of DNA damage that include single-strand breaks (SSBs), DSBs, and base damage sites [for review (3)]. In addition to IR, we examined RecA-GFP focus formation following exposure of cells to MMC and UV. MMC can generate interstrand cross-links; however, the majority of MMC-generated lesions are monoadducts at the N2 and N7 positions of guanine [for review (4)]. The predominant UV-generated lesions are cyclobutane pyrimidine dimers and 6-4 photoproducts [for review (5)].

We used equitoxic doses (>90% survival) of IR, MMC, and UV irradiation and found that the percentage of cells with RecA-GFP foci increased following exposure to these exogenous agents (SI Table 2 and Fig. 1A). After visualizing RecA-GFP foci following challenge with the indicated damaging agents, we found that the size of the foci formed and the percentage of cells with foci are different, even at equitoxic doses (see Fig. 1A and SI Table 2, and see below). This suggests that the number of lesions per kb is moderately different, even though the level of toxicity is the same for the different damage treatments. We also tested the mutagen 2-AP. This agent induces mismatches and has been shown to increase the percentage of cells with mismatch repair protein foci in B. subtilis (6). We did not observe an increase in RecA foci after treatment with this agent relative to the untreated control (SI Table 2). These experiments demonstrate that RecA-GFP foci are induced following treatment with agents that generate base damage sites (UV, MMC, IR) but not a mutagen, consistent with the idea that RecA is not involved in repair of mismatches.

We noticed that filament-like elements referred to as "threads" extend from some RecA-GFP foci (Fig. 1A and SI Fig. 10), confirming previous observations (7). We scored the percentage of foci with threads and determined that >60% of RecA-GFP foci have threads following MMC (n = 533) and IR (n = 411) treatment. In contrast, UV-treated cells (n = 392) only show threads extending from »15% of the foci.

RecA-GFP Foci Form Through a Redistribution of Existing Protein. Our previous experiments (Fig. 1A) showed that a low dose of UV (1 J/m²) was sufficient to induce RecA-GFP foci in most cells (»87%) (Fig. 1A). We used immunoblot analysis to monitor RecA-GFP protein levels in cells following a low dose of UV relative to the untreated control. It was previously shown that 30 min is sufficient to induce the SOS response in B. subtilis (8). We found that RecA-GFP protein levels in cells damaged with a low dose of UV were no different from the level of RecA-GFP in our untreated sample (SI Fig. 9A). As an additional control, we also damaged cells with a high level of UV that should result in SOS induction and increased RecA-GFP protein levels (SI Fig. 9B). Indeed, cells irradiated with 40 J/m² of UV show a >3-fold increase in RecA-GFP protein levels (SI Fig. 9B). We conclude that RecA foci are assembled from preexisting protein.

SI Materials and Methods

Bacteriological Methods. Unless indicated, strains are derivatives of PY79 (9) and were grown in LB or S7₅₀ minimal medium at 30°C, as described previously (6). The recA::neo allele used in these experiments has been described (10). Concentrations of antibiotics and other additives were also performed as described (6).

Construction of RecA-GFP-Expressing Strains. A translational fusion between the recA gene and gfp was constructed by integrating the gfp fusion at the 3' end of the recA gene using plasmid pKL147 (11) generating plasmid pLS28. Plasmid pLS28 was used as a template for site-directed mutagenesis (Quikchange; Stratagene, La Jolla, CA) with primers encoding the A206K missense mutation generating pLS30. Plasmid pLS30 was sequenced to verify the missense mutation in gfp and to verify proper cloning of the recA gene fragment before use. We determined that RecA-GFP predominantly localizes as foci following DNA damage. It should be noted that a subpopulation of RecA-GFP cells exhibit intense fluorescence that fills the entire cell (termed "whole-cell fluorescence") (SI Table 2 and SI Fig. 6B). Only 5-10% of the cells exhibit this type of localization, and unlike in RecA-GFP foci this localization pattern does not increase after exogenous DNA damage (SI Table 2 and SI Fig. 6B). We suspected that this localization pattern might reflect the ineffectiveness of the fusion protein to repair a portion of endogenously occurring damage resulting in elevated SOS expression. Indeed, we do not observe this localization pattern in cells in which RecA-GFP is expressed from an ectopic locus (LAS301) and the native recA allele is intact (data not shown). All primer sequences used in this study are available upon request.

UV and IR Treatment. 5 ml of mid-log-phase cultures were removed and placed in a Petri dish (UV) or a 25-ml Erlenmeyer flask (IR) and treated with a 10-cm-diameter standard germicidal fluorescent tube (UV) or a ⁶⁰Co gamma irradiator (for IR). For time course experiments, samples were irradiated while on a microscope slide. For all other experiments, slides were assembled following treatment. Irradiation of 1 ml exponentially growing culture, followed by sequential extraction of 100 ml of cells at each dosage, was done to generate the survival curves following IR treatment. Cells were then serially diluted and plated on LB agar plates, followed by overnight incubation at 30°C to determine the number of viable cells.

Live-Cell Microscopy. Live-cell microscopy was done essentially as described in ref. 6. For RecA-GFP images, exposure time was between 0.5 and 1.0 s.

DnaA and DnaN Depletion. We used an IPTG-regulated promoter integrated upstream of the dnaA locus (dnaA::P_spacdnaAN⁺) for controlled expression of the dnaA and the downstream dnaN gene (11). Strains bearing the dnaA::P_spacdnaAN allele were grown in S7₅₀ medium in the presence on 40 mg/ml IPTG and treated with the indicated DNA-damaging agents (Figs. 3 and 4). For DnaA and DnaN depletion, a 1.5-ml portion of the culture was removed and harvested by centrifugation. The cells were resuspended in fresh S7₅₀ medium lacking IPTG. This procedure was repeated twice to remove traces of IPTG from the medium. The cells were then used to seed a culture of S7₅₀ medium lacking IPTG and allowed to grow for one to three generations. The strains were treated with the indicated DNA-damaging agents and visualized by microscopy, as indicated in the figure legends. Strains bearing the recA-gfp allele showed a slightly higher occurrence (»15-20% of cells) of whole-cell fluorescence following DnaA and DnaN depletion. These cells were not included in our calculation of cells with foci because individual foci were not observed. As described before, this population of cells was not inducible by exogenous DNA damage.

Southern Blot Analysis of a Site-Specific DSB. Strain LAS261 was grown as described above, except the medium was supplemented with 1% arabinose, 40 mg/ml IPTG, and 6.25 mg/ml tetracycline. Expression of the I-SceI endonuclease was induced with the addition of 0.5% xylose (Sigma) to cultures growing normally or after DnaA and DnaN depletion. Chromosomal DNA was prepared using the DNeasy (Qiagen, Valencia, CA) method following recommendations from the manufacturer. 1 mg of DNA from each sample was digested with SalI and electrophoresed on a 0.75% agarose gel. Digestion with SalI will release a 1.4-kb fragment if the I-SceI recognition site has been cleaved in vivo. Chromosomal DNA was viewed using ethidium bromide fluorescence to verify proper digestion before transfer of the DNA to a membrane (Ambion). The membrane was processed using the DIG high prime procedure (Roche) according to the manufactures recommendations. A probe corresponding to the DNA segment of interest was labeled with digoxigenin-11-dUTP (DIG-dUTP) for detection with anti-DIG-dUTP antibodies (Roche). A 1:5,000 dilution of anti-DIG-dUTP antibodies was used for detection, followed by exposure to x-ray film (Kodak).

Cell Synchronization. Strains bearing the dnaB134 allele were used to synchronize the replication cycle in B. subtilis. The proper strain was grown in S7₅₀ minimal medium with 1% arabinose. After the culture reached mid-log phase (OD₆₀₀, 0.6), the culture was split and a portion was incubated at 42°C for 30 min (12). This inhibited new rounds of replication initiation and synchronized the cell population. The remaining cells continued asynchronous growth at 30°C. The asynchronous cells (30°C) were treated with MMC for 30 min or treated with 0.5% xylose for 60 min to induce the I-SceI restriction cut. The synchronize cells were incubated at 42°C for 30 min followed by treatment with 20 ng/ml MMC for an additional 30 min or 0.5% xylose for 60 min to induce I-SceI expression catalyzing a DSB. Incubation of recA-gfp strains at 42°C does cause RecA-GFP to localize in a damage-independent pattern in a subset of cells (»10%). Because this pattern is temperature shift-dependent, damage-independent, and morphologically distinct from damage-induced foci at 42°C (data not shown), these cells were excluded from our calculations. Cells were removed from 42°C to release from arrest by incubation at 30°C. These cells were allowed to grow for 30 min for MMC-treated cultures and 60 min for xylose-treated cultures. At the end of each time point, cells were treated with 0.004% formaldehyde to fix the cells and preserve each time point. The fixing procedure has no affect on RecA-GFP focus formation (data not shown). Fixed cells were analyzed by microscopy and scored relative to the DIC image for each cell.

We thank Melanie Berkmen, Mary Ellen Wiltrout, Bryan Davies, Katherine Gibson, Michi Taga, Jade Wang, C. Lee, and Alexi Goranov for critical reading of the manuscript and members of both the Walker and Grossman laboratories and two anonymous reviewers for their comments that have strengthened this work.

1. Renzette N, Gumlaw N, Nordman JT, Krieger M, Yeh SP, Long E, Centore R, Boonsombat R, Sandler SJ (2005) Mol Microbiol 57:1074-1085.

2. Steiner WW, Kuempel PL (1998) J Bacteriol 180:6269-6275.

3. Wallace SS (1998) Radiat Res 150:S60-S79.

4. Dronkert ML, Kanaar R (2001) Mutat Res 486:217-247.

5. Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T (2006) DNA Repair and Mutagenesis (Am Soc Microbiol, Washington, DC), pp 493-696.

6. Smith BT, Grossman AD, Walker GC (2001) Mol Cell 8:1197-1206.

7. Kidane D, Graumann PL (2005) J Cell Biol 170:357-366.

8. Au N, Kuester-Schoeck E, Mandava V, Bothwell LE, Canny SP, Chachu K, Colavito SA, Fuller SN, Groban ES, Hensley LA, et al. (2005) J Bacteriol 187:7655-7666.

9. Youngman P, Perkins JB, Losick R (1984) Plasmid 12:1-9.

10. Sciochetti SA, Piggot PJ, Sherratt DJ, Blakely G (1999) J Bacteriol 181:6053-6062.

11. Lemon KP, Grossman AD (1998) Science 282:1516-1519.

12. Lemon KP, Grossman AD (2000) Mol Cell 6:1321-1330.

13. Fabret C, Ehrlich SD, Noirot P (2002) Mol Microbiol 46:25-36.

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