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J Bacteriol. Jan 2002; 184(2): 488–493.
PMCID: PMC139587

Localization of UvrA and Effect of DNA Damage on the Chromosome of Bacillus subtilis


We found that the nucleotide excision repair protein UvrA, which is involved in DNA damage recognition, localizes to the entire chromosome both before and after damage in living Bacillus subtilis cells. We suggest that the UvrA2B damage recognition complex is constantly scanning the genome, searching for lesions in the DNA. We also found that DNA damage induces a dramatic reconfiguration of the chromosome such that it no longer fills the entire cell as it does during normal growth. This reconfiguration is reversible after low doses of damage and is dependent on the damage-induced SOS response. We suggest that this reconfiguration of the chromosome after damage may be either a reflection of ongoing DNA repair or an active mechanism to protect the cell’s genome. Similar observations have been made in Escherichia coli, indicating that the alteration of chromosome structure after DNA damage may be a widespread phenomenon.

All organisms face constant challenges to their genomes from both endogenous and exogenous DNA-damaging agents. To repair damage, most, if not all, organisms possess an array of DNA repair systems (15). One of the most important repair systems is nucleotide excision repair (NER), which is an error-free repair system that is capable of recognizing and repairing virtually any type of chemically damaged DNA base (reviewed in reference 43). In addition to this global mode in which NER can act anywhere in the genome, there is also a transcription-coupled repair mode by which the NER machinery is specifically targeted to RNA polymerases that are stalled at lesions in the template DNA strand (43).

The importance of NER is underscored by the fact that it is functionally conserved from bacteria to humans. The specific proteins that carry out NER are highly conserved within prokaryotes and eukaryotes, but they are not conserved between the two groups (11). In humans, defects in NER are associated with a variety of diseases, the most notable being xeroderma pigmentosum. Xeroderma pigmentosum is a syndrome associated with an extremely high risk of skin cancer, which is caused, at a cellular level, by an increased sensitivity to DNA-damaging agents such as UV light (10, 15). In bacteria, much of the resistance to UV light and other damaging agents is provided by NER, which is accomplished by the highly conserved UvrA, -B, and -C proteins (11, 43).

Biochemical studies of NER using purified proteins primarily from Escherichia coli have found that recognition of DNA damage is carried out by the UvrA2B complex (22, 40, 46, 49), which detects lesions in DNA by sensing the disruptions they cause in the double helix (43, 51, 53). The UvrA2B complex then separates the two DNA strands at the site of the lesion and loads UvrB onto the DNA (16, 17, 58). After the UvrA dimer dissociates, UvrC binds the UvrB-DNA complex and the two proteins excise a short (~13 nucleotides) piece of single-stranded DNA (ssDNA) encompassing the damaged base (32, 33, 42, 48). This DNA fragment is removed by DNA helicase II (also called UvrD) (3) and the gap is filled in by DNA polymerase I, although other polymerases may be able to participate as well (15, 48).

In E. coli, uvrA+ and uvrB+, but not uvrC+, are members of the SOS regulon (8, 12, 13, 24, 44), a group of more than 40 unlinked genes that are induced after the cell suffers DNA damage (54, 55). Most of the SOS genes encode proteins involved in the repair of, or recovery from, DNA damage. These SOS genes are transcriptionally repressed by LexA dimers, which bind DNA sites called SOS boxes that are usually located in the promoter regions of SOS genes (1, 37). Induction of the SOS response is mediated by RecA (41), which also plays a central role in homologous recombination (26). After the cell suffers DNA damage, RecA binds to ssDNA that is thought to be generated by the cell’s failed attempts to replicate its damaged chromosome (9, 45). After forming a nucleoprotein filament with ssDNA, RecA becomes competent to activate the latent autodigestive activity of LexA. When LexA interacts with the RecA-ssDNA complex, it cleaves itself in two, thereby relieving the repression of the SOS regulon (35, 36).

Bacillus subtilis also possesses uvrA+, uvrB+, and uvrC+ genes (4, 5, 27). It has been found that the uvrBA operon is part of the B. subtilis SOS response (5) and that purified B. subtilis UvrC will function in vitro with E. coli UvrA and UvrB to carry out NER (34), which indicates that the NER systems in the two organisms are highly conserved. We have used a fusion of green fluorescent protein (GFP) to localize UvrA, the DNA damage recognition protein, in living B. subtilis cells. We found that UvrA is induced after DNA damage in a recA+-dependent manner and that the vast majority of the protein is associated with the chromosome both before and after damage. Based on this observation, we suggest that UvrA is constantly scanning the DNA searching for lesions. We also observed that DNA damage causes a dramatic, recA+-dependent alteration of nucleoid morphology. Following damage, the nucleoid appears to occupy less space in the cell than during normal growth. However, after low levels of DNA damage, the morphology returns to normal within 3 h after the damaging treatment. We suggest that the reconfiguration of the nucleoid is a controlled response to DNA damage and may be either a consequence of ongoing repair or could function directly as a protective mechanism.


Bacterial strains, media, and growth conditions.

All cultures were grown at 30°C in S7 defined minimal medium (23, 52) supplemented with 1% glucose, 0.1% glutamate, and the amino acids tryptophan (40 μg/ml) and phenylalanine (40 μg/ml). Where necessary, the following antibiotics were used: spectinomycin (spc), 40 μg/ml; chloramphenicol (cat), 5 μg/ml; mitomycin C (MC; Sigma), 0.25 μg/ml; and erythromycin (0.5 μg/ml) and lincomycin (12.5 μg/ml) in combination (mls).

All B. subtilis strains were based on the SPβ prototroph PY79 (57). Construction of the uvrA-gfp translational fusion strain, BTS4, was accomplished by using PCR to amplify the last 500 bp of uvrA+, adding an EcoRI site to the 5′ end of the PCR product and replacing the stop codon with an XhoI restriction site. This PCR product was then cloned into pKL147 (29) upstream of the gfpmut2 gene (7) via the EcoRI and XhoI restriction sites to generate plasmid pBS213. This plasmid was then transformed into PY79 using standard techniques (19) to generate a strain (BTS4) containing the uvrA-gfp fusion gene in single copy at the native position in the chromosome downstream of the uvrB+ gene. A complete uvrA deletion was constructed by cloning DNA immediately upstream and downstream of uvrA+ (generated using PCR) into pJL74 (28) on either side of the spc cassette to generate plasmid pBS229. PY79 was then transformed with pBS229 to generate the ΔuvrA::spc deletion strain BTS30. Construction of BTS49, a recA derivative of BTS4, was accomplished by transforming chromosomal DNA from strain YB300 (6), which contains the recA260 allele (an Eryr Cmr single-crossover disruption of recA) into BTS4.

UV light survival curves.

Five milliliters of exponential-phase cultures of PY79, BTS4, and BTS30 growing in S7 medium were placed in glass petri dishes (10-cm diameter) and irradiated using a standard germicidal fluorescent tube (254 nm) at 1 J/m2 · s. (BTS30 was irradiated at 0.25 J/m2 · s.) Cells were removed from the dish just prior to irradiation (for 0-J/m2 dose) and at the appropriate times during irradiation. The cells were serially diluted in 1× Tbase-1 mM MgSO4 (19), and appropriate dilutions were plated on Luria-Bertani agar plates to determine CFU per milliliter of the unirradiated and irradiated cells.

Live cell microscopy.

Microscopy of live cells was performed essentially as described previously (29). Briefly, an exponentially growing culture of cells was split equally, and half was UV irradiated or treated with MC or chloramphenicol, depending on the experiment. The parallel cultures were then allowed to continue growing for 1 or 3 h. Aliquots of cells were then stained with the membrane dye FM4-64 (240 ng/ml to 1 μg/ml; Molecular Probes) and the DNA stain 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml). Cells were then placed on a pad of 1% agarose in a solution of 1× Tbase-1 mM MgSO4 and covered with a coverslip. The following Chroma filter sets were used: 41002C for FM4-64, 41001 for GFP, and 31000 for DAPI. Images were acquired using a Nikon E800 microscope with a charge-coupled device camera (Hamamatsu; model C4742-95) and OpenLab software (Improvision). The exposure time for capturing UvrA-GFP images was 3 s. Images were colorized in OpenLab and then transferred to Photoshop (Adobe) and Canvas (Deneba) for figure assembly.


UvrA-GFP is associated with the nucleoid, even in the absence of DNA damage.

To determine the subcellular localization of the UvrA protein, which is involved in the initial DNA damage recognition step of NER (43), we constructed a strain expressing a UvrA-GFP fusion protein such that uvrA-gfp is the only version of the uvrA gene in the cell. It is located at the uvrA gene’s normal position in the chromosome and is driven by the uvrBA operon’s native promoter. The sensitivity of the strain to killing by UV light was essentially the same as that of its uvrA+ parent and significantly less than a ΔuvrA::spc strain (Fig. (Fig.1),1), indicating that the UvrA-GFP fusion protein retains wild-type function in vivo.

FIG. 1.
UV survival curves of wild-type, uvrA-gfp, and ΔuvrA::spc strains. Cells growing exponentially at 30°C in S7 medium were irradiated with UV light (254 nm) at 1 J/m2 · s (0.25 J/m2 · s for ΔuvrA) and aliquots were ...

In untreated, exponentially growing cells, there was a low basal level of fluorescence from UvrA-GFP throughout the cell (Fig. (Fig.2A).2A). The fluorescence in untreated cells was of a somewhat heterogeneous nature, being lower near the edges of the cells and at septa (Fig. (Fig.2A).2A). This suggested that the UvrA-GFP protein might be associated with the bacterial chromosome even prior to treatment with DNA damaging agents. Because the nucleoid (the chromosome and its associated proteins) normally fills almost all of a cell’s volume (Fig. (Fig.2A),2A), UvrA-GFP that is associating with the chromosome cannot be easily distinguished from protein that is free in the cytoplasm. To address this issue, cells were treated with the antibiotic chloramphenicol, which inhibits protein synthesis and causes a condensation of the nucleoid (59), but does not induce the SOS response (20). After treatment with chloramphenicol, the nucleoids became condensed, and the majority of the UvrA-GFP signal was entirely coincident with the condensed nucleoids (compare Fig. Fig.2B2B to to2A2A).

FIG. 2.
Fluorescence microscopy images of live uvrA-gfp (BTS4) and uvrA-gfp recA260 (BTS49) cells. Cultures growing exponentially at 30°C in S7 medium were split equally, and half was UV irradiated or treated with MC or chloramphenicol (Cm), depending ...

The effects of DNA damage on UvrA localization and on the nucleoid.

To examine whether there was an effect of DNA damage on the localization of UvrA-GFP, the cells were treated with UV light (25 J/m2). When the cells were viewed after 1 h, we observed an increase in the level of UvrA-GFP fluorescence (compare Fig. Fig.2C2C with Fig. Fig.2A).2A). Immunoblotting indicated that UV irradiation increased the steady-state levels of UvrA-GFP approximately threefold (data not shown). The uvrBA operon in B. subtilis has previously been identified as a member of the DNA damage-induced SOS regulon, and transcription of the operon was found to increase approximately fivefold after DNA damage (5), which is consistent with our results.

In addition to the induction of UvrA-GFP after UV irradiation, we also observed a striking concentration of UvrA-GFP fluorescence in a large central region of the cell (Fig. (Fig.2C).2C). Since we found that UvrA is associated with the entire chromosome in the absence of damage, it was likely that the UvrA-GFP fluorescence after damage marked the position of the chromosome as well. We therefore visualized the location of the nucleoid in these UvrA-GFP-expressing cells using DAPI staining. In live, untreated cells, the bacterial nucleoid filled almost the entire cell volume (Fig. (Fig.2A).2A). However, by 1 h after irradiation with 25 J/m2, we found that the nucleoid filled only a portion of the cell volume, the same region occupied by UvrA-GFP (Fig. (Fig.2C).2C). It is important to note at this point that the unusual nucleoid morphology that we observe after DNA damage, which is reminiscent of the effect of chloramphenicol treatment, is unlikely to be caused by an inhibition of protein synthesis since the doses of UV light that we are using do not inhibit bulk protein synthesis (2).

In addition to developing the unusual nucleoid morphology, the cells appeared to become somewhat longer after UV irradiation (compare Fig. Fig.2C2C to to2A),2A), and there was an increase from <1% to 25% (145 and 163 cells counted, respectively) of cells with a “cut” phenotype in which a septum has come down on a nucleoid (see cell in upper right corner of Fig. Fig.2C).2C). Fifteen minutes after treatment with 25 J/m2, similar, although less dramatic, effects on UvrA-GFP induction, nucleoid morphology, cell lengthening, and cut nucleoids were observed (data not shown).

Three hours after treatment with 25 J/m2, the cells looked quite different than they did after 1 h (Fig. (Fig.2D).2D). Many of the cells were extremely long. This is consistent with the DNA damage-induced filamentation that has been previously observed in B. subtilis (18, 38). However, it should be noted that the previous study of UV-induced filamentation found that significant filamentation was observed 90 min after a 5-J/m2 dose (38). We only observed filamentation after a 25-J/m2 dose. This discrepancy could be due to differences in strain backgrounds or perhaps to growth conditions. E. coli cells also filament after DNA damage, but this filamentation is dependent on the SOS-regulated sulA+, which B. subtilis does not possess (27). We also observed that, after 3 h, some of the cells became anucleated. The DNA in cells with nucleoids still filled only a portion of the entire cell volume, and in those nucleated cells, UvrA-GFP was still present at high levels and was still associated with the nucleoids (Fig. (Fig.2D).2D). A few cells were observed that did not exhibit these extreme morphological effects, and they may have represented the small number of cells that will ultimately survive the 25-J/m2 UV dose (Fig. (Fig.11).

To investigate whether these results were specific to UV irradiation, we similarly treated UvrA-GFP-expressing cells with a 250-ng/ml concentration of MC, a DNA strand cross-linking agent that damages DNA in a manner distinct from that of UV light (15, 50). Similar results (i.e., induction of UvrA-GFP, colocalization of UvrA-GFP with the nucleoid, altered nucleoid morphology, and increased cell length) to those seen after 25 J/m2 were observed by 1 h after MC treatment (Fig. (Fig.2E).2E). Treatment with this dose of MC results in a level of cell killing somewhat higher than that seen after a 25-J/m2 UV dose (data not shown).

These data indicate that most of the UvrA-GFP protein is associated with the chromosome both before and after DNA damage, consistent with its role in damage recognition. While the final steps in DNA damage recognition by UvrA2B have been studied in considerable detail in vitro, what is less well understood is the proposed low-resolution mode of UvrA2B in which it is able to scan the large regions of DNA for damage (17, 25, 43). Our observations support the notion that the UvrA2B complex is continually associated with the DNA, scanning the genome for damage. However, these studies cannot determine if this low-resolution scanning process is processive or the result of rapid association-dissociation events.

A previous study, using cell fractionation and immuno-electron microscopy techniques, localized the UvrA, -B, and -C proteins in E. coli (31). Consistent with our results, cell fractionation experiments showed that, prior to damage, UvrA was present in the DNA fraction. However, after UV irradiation, a significant portion (40%) of the UvrA was present in the membrane fraction. Immuno-electron microscopy also indicated that the distribution of UvrA in the cell shifted towards the inner membrane after UV irradiation. In our studies of living B. subtilis cells, we did not observe a migration of UvrA-GFP from the DNA to the membrane after UV irradiation. The vast majority of UvrA-GFP remained associated with the chromosome. We do not know the reason for this apparent discrepancy in the localization of UvrA between B. subtilis and E. coli.

The localization of NER proteins in living cells has also been studied in a eukaryotic system (21). A GFP fusion to ERCC1, the factor that, in combination with XPF, makes the incision 5′ to the site of damage, was visualized in living Chinese hamster ovary cells. From these studies, the authors concluded that eukaryotic NER acts by the assembly of individual NER factors at the site of damage, and that ERCC1/XPF acts to repair DNA damage in a distributive fashion, not by scanning the DNA (21). However, since UvrA and ERCC1/XPF are involved in different steps of NER, a direct comparison between their localizations cannot be made. Considering the high degree of functional similarity in vitro between prokaryotic and eukaryotic NER, direct comparisons of the localizations of NER proteins in prokaryotic and eukaryotic cells may reveal if this functional conservation is also reflected in the way the proteins act in vivo.

The morphological effects of DNA damage are reversible after a low dose of UV irradiation.

The 25-J/m2 dose of UV light used above results in a plating efficiency of ~15% (meaning that ~85% of the irradiated cells will eventually die; Fig. Fig.1).1). To determine if the unusual nucleoid morphology and increased cell length that was observed after a 25-J/m2 dose of UV light is the result of a controlled response to DNA damage or whether it is simply an event that takes place in dying cells, we repeated the above experiment using a lower dose of UV light, 5 J/m2, that results in a plating efficiency of ≈80% (Fig. (Fig.1).1). One hour after a culture was irradiated with 5 J/m2, induction and localization of UvrA-GFP was quite similar to a culture irradiated with 25 J/m2. The cells exhibited altered nucleoid morphology and cell lengthening, but these phenotypes were less extensive than what was observed after cells were treated with the higher, 25-J/m2 dose of UV light (compare Fig. Fig.2F2F to to2C2C).

In stark contrast to what is observed 3 h after the 25-J/m2 dose (Fig. (Fig.2D),2D), 3 h after the 5-J/m2 dose, most of the irradiated cells had returned to a state quite similar to that of the unirradiated control cells, although the UvrA-GFP levels might be somewhat higher (compare Fig. 2G to 2A). At the 3-h time point, there were some cells in the 5-J/m2 culture that resembled the cells in the 25-J/m2 culture, and it seems likely that these cells represented the portion of the population that are destined to die (Fig. (Fig.1).1). Thus, these data indicate that after low doses of DNA damage, the reversible morphological effects (i.e., altered nucleoid morphology and cell lengthening) are the result of a controlled cellular response to DNA damage.

The SOS response is required for the DNA damage-induced alteration of nucleoid morphology.

To determine if the recA+-regulated SOS response to DNA damage was required for the striking damage-induced alteration of nucleoid morphology that we observed, we UV irradiated a recA derivative of the UvrA-GFP strain. As expected, when observed 1 h after irradiation with 25 J/m2, this strain did not exhibit an induction of UvrA-GFP (compare Fig. Fig.2I2I to to2H).2H). We did not observe a dramatic change in the overall nucleoid morphologies in recA cells 1 h after UV irradiation (compare Fig. Fig.2I2I to to2H).2H). This indicates that RecA itself or a component of the recA+-regulated SOS response is responsible for the alteration of nucleoid morphology that occurs after damage.

In addition to its role in the regulation of the SOS response, RecA plays a central role in recombination (26). If RecA is directly involved in the reconfiguration of the nucleoids through its role in recombinational repair, recombinational intermediates linking sister chromosomes during repair could prevent the nucleoid from filling the entire cell volume after damage. If this is the case, then the reversibility of the unusual nucleoid morphology after low doses of UV light could be due to the completion of repair, which would release the constraints on the nucleoid. However, since RecA is required for the induction of SOS-regulated genes, we cannot distinguish this model from an alternate one in which the induction of another SOS gene is responsible for the altered nucleoid morphology.

The unusual nucleoid morphology that we observe in living B. subtilis cells after DNA damage is reminiscent of a recent electron microscopic study of fixed E. coli cells which found that DNA damage results in the aggregation of the chromosome into a lattice-like assembly that fills approximately one-fifth of the cell (30). Significantly, RecA is required for the formation of these DNA assemblies and is an integral component of them as well. These assemblies begin to form by 15 min after the damaging treatment, and in the case of UV-treated cells, the assemblies were reversible. All of these characteristics are quite similar to the alteration of chromosomal morphology that we observe in B. subtilis. Since E. coli and B. subtilis are such distantly related organisms, the fact that they exhibit similar nucleoid morphologies after DNA damage suggests that this response may be widespread among bacteria.

Interestingly, the E. coli Dps protein, which protects DNA from oxidative damage and is induced in response to oxidative stress or starvation, also promotes the formation of condensed DNA assemblies in vivo (14, 39, 56). Another example of a modification of chromosomal structure in response to stress is the coating of B. subtilis spore DNA by the SASP proteins, which contributes to the high resistance of spores to UV light (47). Our findings, in combination with these examples, suggest that the reconfiguration of the chromosome in response to stress may be a general mechanism to maintain the integrity of the cell’s genome.


We thank K. P. Lemon and P. A. Levin for providing considerable technical and scientific assistance during the course of this work. We also thank all the members of the Walker and Grossman labs for their advice and support.

This work was supported by Public Health Service grants CA21615 to G.C.W. and GM41934 to A.D.G. B.T.S. was partially supported by an NIH predoctoral training grant.


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