INTRODUCTION

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The SOS regulatory system controls the response of Escherichia coli to treatments that damage DNA or inhibit DNA replication (12, 30). During normal cell growth, LexA protein represses a set of about 20 genes. Inducing treatments generate an inducing signal that activates another regulatory protein, RecA. Activated RecA in turn mediates the cleavage of LexA, inactivating it and leading to derepression of the SOS regulon. In vitro, RecA can be activated by forming a ternary complex with single-stranded DNA and a nucleoside triphosphate such as ATP, dATP, or ATP(S). In this complex, RecA forms a helical filament along the single-stranded DNA. It is likely that this complex also represents the activated in vivo form of RecA.

Although interaction of LexA with activated RecA triggers the cleavage reaction, many lines of evidence indicate that RecA does not act as a true protease but instead causes LexA to cleave itself (28). LexA can undergo self-cleavage in vitro in a reaction termed autodigestion (28). This reaction cuts the same bond as in RecA-mediated cleavage; moreover, mutations that inhibit RecA-mediated cleavage also prevent autodigestion. Hence, we believe that the actual chemistry of catalysis is carried out by groups in LexA, not in RecA, and we term activated RecA a "coprotease" to emphasize its indirect role in promoting cleavage.

Activated RecA can also mediate the cleavage of two other groups of proteins. The first is a group of temperate phage repressors, exemplified by  CI repressor, which are cleaved in lysogens upon SOS-inducing treatments (43). Cleavage of CI is far slower than that of LexA. If DNA damage is severe, CI cleavage leads to prophage induction. The second set of substrates is a set of mutagenesis proteins, exemplified by the host UmuD protein, that are activated by specific cleavage to perform specific roles in SOS mutagenesis. Again, UmuD cleavage is slower than that of LexA, so that mutagenesis only takes place in severely damaged cells. The cleavage reactions of  CI and UmuD appear to be entirely parallel mechanistically to that of LexA. Both proteins undergo self-cleavage, and the residues involved in cleavage are conserved in CI and UmuD. Hence, it is believed that RecA acts indirectly to stimulate these reactions as well.

It is not yet clear how RecA stimulates cleavage. Our evidence with LexA favors a conformational model in which RecA stabilizes a reactive conformation of LexA (44). However, it remains possible that RecA makes a more direct contribution to the chemistry of bond breakage. One analogy can be made with GTPase-activating proteins (GAPs), which greatly stimulate the GTPase activity of Ras and other small G proteins by contributing groups to the active site of this reaction (47). One approach to distinguishing these models is to identify the binding sites for LexA and other cleavable proteins on the RecA protein, and the work described here was carried out with this goal.

Two previous lines of evidence have suggested that LexA, CI, and UmuD interact at different sites in RecA. First, several recA mutant proteins appear to exhibit specific defects for cleaving some but not all substrates (see below), suggesting that these alleles affect residues that contact some substrates but not others. Second, many  CI mutations that block RecA-mediated cleavage in vivo were isolated (13, 14); biochemical analysis showed that 9 of 15 mutant proteins were not impaired for autodigestion. These findings are consistent with the model in which these nine mutations affect residues that interact with RecA, although this has not been shown directly. Strikingly, these mutations do not affect residues that are conserved in other cleavable proteins, suggesting that the RecA-binding site on  CI is not conserved in these other proteins.

In this work, we have sought to identify the LexA binding site in RecA by site-directed mutagenesis. Although several recA alleles show specific defects for cleavage of certain substrates, none of these is specifically defective for LexA cleavage; that is, no recA mutant protein yet characterized is defective for promoting LexA cleavage and proficient for the other functions of RecA. Our approach was to use the crystal structure of RecA as a guide to identify potential residues and to test a particular model for the location of the binding site for cleavable substrates.

Story et al. (53) determined the structure of a helical filament of RecA and proposed that the cleavable proteins bind to a cleft located between two adjacent RecA monomers in this filament (Fig. 1). This model was based on the fact that two particular recA alleles change residues located in this cleft and affect cleavage reactions. These alleles are recA1734, which changes Arg243 to Leu (8), and recA91, which changes Gly229 to Ser (39). recA1734 and two other recA alleles at Arg243 (recA433 and recA435 [9]) confer defects for cleavage of UmuD and phage 80 repressor but allow cleavage of LexA and  CI. The recA91 allele is also reportedly defective for cleavage of 80 repressor and normal for  CI (39), although no data have been published for this mutant allele. The findings that these mutant proteins are specifically defective for cleavage of some but not all substrates suggests, first, that the mutant RecA protein can be activated; second, that the mutations are likely to affect a protein-protein interaction directly; and third, that not all cleavable proteins bind at the same site, as discussed above. In any case, the location of recA1734 and recA91 in the cleft between two monomers suggests that UmuD and 80 repressor, at least, bind in this cleft.

View larger version (90K): [in this window] [in a new window]   FIG. 1.   Location of site-directed changes in the RecA structure. Shown are two adjacent monomers from the RecA filament (see the text), colored dark and light blue. In the view in panel D, the helix axis lies directly behind the interface between the two monomers and is oriented vertically (not shown). The views in panels A and B are that of panel D rotated by 30° or 30° along the y axis; that in panel C is the view of panel D rotated 90° along the y axis so that the cleft is viewed from beneath; the helix axis is shown. (A to C) Mutational changes in the cleft. Residues Arg243 and Gly229 are shown in red; residues changed in the cleft (identified in Table 3) are shown in yellow. (D) Locations of other mutations and changes on the outer surface of the filament. Mutations destroying RecA function (listed in Table 2) are shown in pink; those that leave LexA cleavage intact (Table 2) are shown in green; changes made in the present work (Table 3) are shown in orange; yellow and red are as in panels A to C. RasMol scripts generating these views are available at http://www.biochem.arizona.edu/Little/Littlelab.htm.

Another supportive line of evidence for this model came from image reconstructions of electron micrographs of LexA bound to a RecA filament (62). In these images, extra electron density, attributed to LexA, was found in an area that corresponds roughly to the locations of Arg243 and Gly229. In addition, LexA appeared to contact two adjacent RecA monomers and possibly made contact across the groove with a RecA subunit in the next turn of the helix.

In this work, we have tested the model in which LexA, UmuD, and  CI bind in the cleft by using site-directed mutagenesis to change most of the polar residues in the cleft to alanine. In addition, we changed many other residues located on the surface of the crystal structure. Mutant proteins were then tested in vivo for the ability to support specific cleavage and to carry out other functions of RecA.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. Chemicals were from Sigma, Fisher Chemicals, and U.S. Biochemicals. The restriction enzymes, T4 DNA polymerase, and T4 DNA ligase were from New England Biolabs, Promega, and Boehringer Mannheim. Protran nitrocellulose was from Schleicher & Schuell. Anti-rabbit immunoglobulin G-horseradish peroxidase (IgG-POD) was from Boehringer Mannheim, and the ECL Western blotting detection system was from Amersham. Rabbit antibodies against LexA were described previously (26); rabbit antibodies against RecA and  CI were made by similar methods. Affinity-purified rabbit antibody against UmuD was a generous gift from Roger Woodgate. Oligonucleotides for site-directed mutagenesis and DNA sequencing were from the Midland Certified Reagent Company or the Division of Biotechnology at the University of Arizona. The Altered Sites site-directed mutagenesis kit was from Promega. [³⁵S]methionine was from ICN.

Computer modeling of the RecA crystal structure. Coordinates for the RecA monomer were obtained from the Brookhaven Protein Data Base (PDB file 2REB). A model of the RecA filament was produced by using the sixfold helical symmetry of the monomers in the filament; we are grateful to Sue Roberts for doing this transformation. This RecA filament model was then examined by using the Insight II and RasMol version 2.5 programs (46).

Bacterial and phage growth. Luria broth and tryptone growth media were prepared as described previously (34) with antibiotic concentrations as described elsewhere (33). Phage  growth was as described previously (48). M9 minimal medium for pulse labeling was also described previously (26).

Bacterial strains. The derivatives of E. coli K-12 used are listed in Table 1.

Construction of plasmids. pFG600 was made by Fred Gimble (personal communication); it contains a lacp/o:: cI fusion from pKB280 (2), a silent SphI site at the codons for residues 92 and 93 of cI, and 264 bp of  DNA downstream of cI, including a PstI site and ending at a ClaI site, cloned into the EcoRI and ClaI sites of pBR322. pJAM13 carried a lacP/O:: cI operon fusion on pGB2, a low-copy-number vector compatible with pBR322, and was made by cloning a 1,033-bp EcoRI-PstI fragment from pFG600 into pGB2. pJAM92 carried a lacP/O::lexAEK45 fusion and was made by cloning a 1,152-bp EcoRI-HindIII fragment of pATT49 into pGB2. pJWL319 was made by R. Ramage by cloning EcoRI-HincII and HincII-MspI fragments from pMC9 (35) into pBR322 cut with EcoRI and ClaI; the resulting insert contained the wild-type lacI promoter and lacI gene, starting at a HincII site at 50 relative to the lacI transcript and ending at an MspI site within the lacZYA promoter. pJAM93 carried lacI⁺ on pGB2 and was constructed by subcloning the EcoRI-HindIII region containing lacI⁺ from pJWL319. Plasmids for site-directed mutagenesis were made by cloning regions of recA from pTrecA220 into pAlter1. pJAM20 carried a 546-bp PstI-EcoRI fragment (amino acids 79 to 260). pJAM30 carried a 960-bp PstI-NruI fragment (amino acids 79 to 352) cloned into pAlter1 digested with SmaI and PstI. pJAM50 contained a 273-bp HindIII-PstI fragment (amino acids 1 to 76).

Site-directed mutagenesis. Site-directed mutagenesis was done as described in the Promega Altered Sites II kit technical manual (revised August 1994). Regions of the recA gene from pTRecA220 were subcloned into pAlter1 for mutagenesis (see above). Mutagenic oligonucleotides were designed to create or destroy restriction sites so that the presence of the mutations could be detected using restriction digests (details available upon request). Plasmids with the desired change were sequenced to confirm the presence of the mutation. The region carrying the desired mutation was then subcloned into pTrecA220. The entire subcloned region was sequenced to ensure that no other changes were present. Mutations are referred to by the one-letter amino acid code of the mutated residue followed by its residue number. Since each mutant has more than one mutation, the changes are combined into one "word." Thus, the mutation R105K106 has Arg105 and Lys106 changed to Ala.

Expression of RecA. Wild-type and mutant RecA proteins were expressed from the pTrecA220 plasmid and derivatives. The level of RecA expressed from pTrecA220 is about equal to that of RecA expressed from the wild-type gene on the chromosome after 15 min of growth in the presence of the SOS-inducing drug nalidixic acid (data not shown). Levels of RecA for each mutant strain were determined by Western analysis in derivatives of JL2460.

Analysis of cleavage using Western blots. To analyze LexA cleavage, we used a mutant LexA protein, LexAEK45, in order to uncouple LexA cleavage from potential effects due to differing levels of other SOS genes. This mutant does not bind the normal LexA DNA binding site (55); hence, differences in levels of cleavage do not affect expression of the SOS genes. A low-copy-number plasmid carrying a lacP::lexAEK45 fusion, pJAM92, was transformed into a lexA-defective or lexA(Def), strain, JL2460, and this strain, JAM444, was used for the LexA cleavage assays. Strains derived from JL2460 and JAM267 were used for analysis of UmuD and CI cleavage, respectively. The Western blotting protocol was as described previously with minor changes (52). Anti-rabbit immunoglobulin G-POD from Boehringer Mannheim was used as a secondary antibody. Chemiluminescence detection was done with the Amersham ECL Western blotting detection system according to the instructions provided.

UV sensitivities of cells containing recA mutations. Two different assays were done to examine the UV sensitivities of the different recA mutations in JL2460-derived strains. For a semiquantitative assay, cells were grown to mid-exponential phase (2 × 10⁸ cells/ml) in L broth. Five microliters of culture was spread on tryptone plates in a streak running the length of the plate. Zones of the streak were subjected to varying doses of UV radiation by shadowing them with an opaque card. After incubation overnight at 37°C, the relative sensitivities of the recA mutants were gauged by the amount of growth in areas that received different doses of UV radiation. To measure quantitative survival curves (40), cultures were grown to mid-exponential phase (2 × 10⁸ cells/ml) in tryptone broth. The cultures were diluted 1:100 in cold 10 mM MgSO₄. All steps after UV exposure were done in dim light. Five milliliters of diluted culture was placed in a glass petri dish and exposed to UV light. After various doses, aliquots were removed from the culture. The aliquots were then diluted in 10 mM MgSO₄, and 100 µl of the appropriate dilutions was spread on  plates. After incubation at 37°C for 48 h, colonies were counted, and the surviving fraction was calculated.

Recombination assays. Two different assays were used to assess recombination defects. In the first assay,  red gam mutants are dependent on functional RecA for packaging their DNA, and consequently for the formation of plaques (51). Derivatives of JL2460 containing plasmids with wild-type recA, no recA, or the recA mutations were grown to mid-exponential phase (2 × 10⁸ cells/ml) in tryptone broth. The cultures were spun down and resuspended in an equal volume of 10 mM MgSO₄. Cells (300 µl) were mixed with 3 ml of BBL top agar and plated on tryptone plates. Several different dilutions of the red gam phage, MMS885, were spotted onto the lawns. The plates were incubated overnight at 37°C, and phage growth was assessed. In the second assay, intrachromosomal recombination was examined as described previously (17), except that the plates were incubated at 37°C for approximately 4 days. Plasmids containing recA⁺, the mutant recA alleles, or no recA gene, were transformed into JAM485, a derivative of GY7066, which contains two nonoverlapping lac deletions on the chromosome. The number of lac⁺ papillae per colony for 10 colonies about 0.5 cm in diameter was determined. The average number of lac⁺ papillae per colony is reported below.

Analysis of LexA cleavage by RecA mutant proteins using ³⁵S pulse labeling. Analysis of LexA cleavage by ³⁵S pulse labeling was done as described previously (26) except that nalidixic acid (50 µg/ml) was used as an inducing treatment instead of UV light and the distribution of radioactivity was determined using a PhosphorImager. In addition, the plasmid carrying the LexAEK45 gene used for Western analysis did not produce enough protein to be seen in this assay (data not shown). Instead, derivatives of strain JL783 were used. This strain carries the wild-type lexA gene under the lexA promoter on the chromosome. Since LexA represses its own promoter in this situation, cells with RecA mutants that cannot cleave LexA well will produce less LexA during labeling than recA⁺ cells.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Choice of residues in recA for mutagenesis. At the outset of this work, we made several assumptions in designing our approach to the analysis of RecA interaction with its substrates. First, we assumed that the residues directly involved in the interaction could be changed without affecting other functions of RecA. Second, although the crystal structure is of a filament that is probably not the active form (61), we assumed that most of the residues on the surface of this structure would also be on the surface of the active RecA filament. Third, we assumed that the binding site is located on a part of the structure that is visible rather than in a disordered region that is not seen in the structure (subsequent work by others [37] suggests that this assumption may be flawed). Finally, we assumed that weaker binding would result in slower cleavage and that this would result in differences in LexA levels that could be detected by our assays.

Mutagenesis of recA. For each mutation, two, or in one case three, residues that are grouped spatially in the crystal structure were changed to alanine. We reasoned that if the RecA interaction with its coprotease substrates is dependent on several relatively weak interactions, multiple changes would more likely yield a cleavage-defective phenotype. In general, polar residues were chosen for mutagenesis. In one case, two hydrophobic residues in the cleft were changed. Residues targeted for mutagenesis are shown in Fig. 1 and listed in Table 3. Targeted residues were changed (see Materials and Methods) to alanine, a residue whose small side chain should reduce contacts without perturbing the overall folding of the protein. Similar approaches have been used to examine other protein-protein interactions (60).

Cellular levels of RecA mutant proteins. To uncouple levels of RecA from SOS induction, the RecA mutant proteins were expressed from a multicopy plasmid under the tac promoter. We compared the levels of mutant RecA proteins with that of the wild type using Western analysis (data not shown; summarized in Table 3). The levels of most mutant proteins were similar to those of the wild type. Several mutants had lower levels of RecA. K294E318 and E285K286 had very low levels; however, K294E318 was like the wild type for almost all the tested functions of RecA described below, so even this low protein level did not appear to impair RecA function in these assays. Q16K19 had elevated levels of protein.

UV sensitivity and recombination of mutants. To test whether the mutations affected other functions of RecA, mutants were examined for defects in DNA repair and homologous recombination. Because some of these mutants might be defective for cleavage of LexA, and therefore unable to derepress the DNA repair genes of the SOS system, UV light sensitivity was tested in strains without functional LexA to avoid complications due to SOS regulation, using a semiquantitative test (see Materials and Methods) for UV sensitivity (summarized in Table 3). Most of the mutants were similar to the wild type. Two, I102Y103 and R176K177, were completely defective, and T242R243 was also very sensitive.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   UV survival curves of wild type and selected mutants. Derivatives of JL2460 carrying plasmids with the indicated recA alleles are shown; the recA host carried pBR322. Cells were grown and irradiated with various doses of UV light as described in Materials and Methods; the fraction surviving was determined as described. The legend inset in the graph identifies the recA allele carried by each strain.

red gam

red gam

red gam

red gam

CI cleavage. Mutants were assessed for cleavage of  CI protein. Host cells lacked LexA function to avoid complications due to variations in induction of SOS function and were treated with nalidixic acid to induce the SOS system. The ability of the mutant RecA proteins to cleave CI was based on the amount of intact protein after 40 min of growth in nalidixic acid. Cells with or without treatment were harvested and analyzed by Western analysis. Typical Western blots are shown in Fig. 3, and the data are summarized in Table 3.

View larger version (65K): [in this window] [in a new window]   FIG. 3.   In vivo cleavage of  CI repressor by mutant RecA proteins. Derivatives of JAM267 carrying pTrecA220 or mutant derivatives were treated as described in Materials and Methods. The strain marked recA carried pBR322. Each panel represents the results of a representative experiment. For each mutant strain, a time course is shown. The lanes marked "0" contain samples taken without treatment; those marked "20" and "40" were taken 20 and 40 min after the addition of nalidixic acid (50 µg/ml). The samples were analyzed by Western blotting with antibody against  CI (see Materials and Methods). The name of the mutation is shown above each set of samples. The location of intact CI is shown; the band just above CI is a cross-reacting protein. wt, wild type.

UmuD cleavage. The ability of the RecA mutants to mediate cleavage of UmuD to UmuD' was examined. UmuD was expressed from the chromosomal umuD gene, which was derepressed due to the presence of a lexA(Def) mutation. The cleavage product, UmuD', was visible in the Western blots, and the ratio of UmuD to UmuD' was useful for assessing cleavage. Since UmuD cleavage is slower than that of CI, cleavage was assessed at later time points. Typical results are shown in Fig. 4 and summarized in Table 3.

View larger version (61K): [in this window] [in a new window]   FIG. 4.   In vivo cleavage of UmuD by mutant RecA proteins. Derivatives of JL2460 carrying pTrecA220 or mutant derivatives were grown and treated as described in Materials and Methods; the strain marked recA carried pBR322. Each panel represents the results of a representative experiment. For each mutant strain, a time course is shown. Lanes marked "D" and "D'" are standards (Stds) from cells overproducing UmuD and UmuD', respectively, and serve as markers. The lanes marked "0" contain samples taken without treatment; those marked "60" and "90" were taken 60 and 90 min after addition of nalidixic acid (50 µg/ml). The samples were analyzed by Western blotting with antibody to UmuD, as described in Materials and Methods. The name of the mutation is shown above each set of samples. wt, wild type.

LexA cleavage. The ability of the mutant RecA proteins to support cleavage of LexA was examined by Western analysis, using a mutant LexA protein, LexAEK45. Because LexA cleavage is rapid, samples were taken 5 and 15 min after the addition of nalidixic acid. Unlike the cases of  CI and UmuD, analysis of LexA cleavage involves a complication. In cells with the recA⁺ plasmid, some of the LexAEK45 was cleaved even in the absence of inducing treatment, as can be seen by comparing the amount of intact LexA in the recA strain with the amount in the wild-type recA strain at time zero in Fig. 5. This is referred to as "basal cleavage" and has been seen in many other studies (19, 26, 29, 45, 50, 52). For wild-type recA, after the addition of the inducing agent, the rate of LexAEK45 cleavage increased substantially (Fig. 5); we refer to this increased rate of cleavage as "induced cleavage." We do not know what signals activate RecA for basal cleavage or if they are the same as those in induced cells.

View larger version (46K): [in this window] [in a new window]   FIG. 5.   In vivo cleavage of LexA by mutant RecA proteins. Derivatives of strain JAM444 carrying pTrecA220 or mutant derivatives were grown and treated as described in Materials and Methods; the strain marked recA carried pBR322. Each panel represents the results of a representative experiment. For each mutant strain, a time course is shown. The lanes marked "0" contain samples taken without treatment; those marked "5" and "15" were taken 5 and 15 min after the addition of nalidixic acid (50 µg/ml). Samples were analyzed by Western blotting with antibody to LexA, as described in Materials and Methods. The name of the mutation is shown above each set of samples. The location of intact LexA is shown; the other bands are proteins that cross-reacted with the LexA antibody. Cleavage fragments are not shown; their levels are uninformative, since LexA cleavage fragments are unstable (27) (for an example, cf. Fig. 6). As described in the text, several mutants had less basal cleavage than the wild type. The amount of basal LexA cleavage did not appear to depend on the amount of RecA mutant protein. Q16K19 had very low levels of basal cleavage yet had higher levels of mutant RecA. On the other hand, K294E318 protein was present at very low levels and yet had the same amount of basal cleavage of LexA as the wild type (wt). The fact that the amount of basal cleavage did not correlate with the amount of RecA suggests that the extent of basal cleavage was limited by the amount of signal in the cell and/or by the ability of RecA protein to become activated by it.

View larger version (49K): [in this window] [in a new window]   FIG. 6.   Pulse-chase analysis of LexA cleavage for selected mutants. Derivatives of JL783 carrying pTrecA220 or mutant derivatives were grown as described in Materials and Methods; the strain marked recA carried pBR322. The cultures were treated with nalidixic acid for 30 min, followed by the addition of [35S]methionine (50 µCi/ml) and sampling at the times indicated. The identity of each set of samples is indicated above the gel. The samples were precipitated with antibody to LexA and analyzed as described in the text. The locations of intact LexA and the C-terminal cleavage product are indicated. The other bands are cross-reacting species also precipitated by the antibody. In the recA strain, less LexA is made, because LexA negatively autoregulates its own expression.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We first discuss the effects of the recA mutations on cleavage of CI and UmuD and the implications of our findings for the model in which cleavable proteins bind in the cleft region. Next we consider mechanisms by which RecA might be activated constitutively for cleavage. Then we discuss cleavage of LexA, ask why we were unable to recover LexA-specific mutant RecA proteins, and consider the possibility that the actual rate of RecA-mediated LexA cleavage might not be the rate-determining step in the LexA cleavage pathway.

Evidence for binding of  CI and UmuD to the cleft region. Several of the recA mutations had effects upon cleavage of  CI and/or UmuD. Several mutations affecting residues in the cleft had modest defects in cleavage of  CI, consistent with the model in which CI interacts with RecA in the cleft. If this is the case, the data are most consistent with the suggestion that the CI-RecA interaction involves many weak interactions and that interruption of any one or two of these has only modest effects on the strength of binding, since none of the mutations completely abolished CI cleavage.

Coprotease-constitutive mutations. Several of our mutations, and many previously described mutations, confer constitutive activation upon RecA. For those constitutive mutations tested biochemically, this property has been found to result from a relaxed specificity for the polynucleotide and/or nucleotide cofactors that activate RecA (20, 57, 59). For a mutant protein made constitutive by this mechanism, one would expect constitutive activation for all substrates, given comparable levels of RecA (cf. reference 10). Indeed, we observed that the Q300N304 mutation had this property. By contrast, two mutations in the cleft, K256Q257 and E259Q261, were specifically constitutive for cleavage of  CI. One plausible mechanism for specific constitutivity is that the mutations create a new contact and thereby confer extremely tight binding of the mutant protein to a particular substrate. In this mechanism, since cells contain a low level of activated RecA (as judged by the basal cleavage of LexA), a much tighter interaction would result in cleavage of that substrate. This mechanism seems unlikely in our mutations, however, given that they changed polar residues to Ala. Hence, we suggest that specific constitutivity can arise from some mechanism other than creation of a new contact.

Cleavage of LexA. RecA-dependent cleavage of LexA occurs rapidly after induction of the SOS system but can also be observed at a low rate in untreated cells (26, 45), a reaction we term basal cleavage. The molecular basis of basal cleavage is poorly understoodfor instance, it is not known if the signal that activates RecA for basal cleavage differs from that which is present after SOS induction. It is also not known whether basal cleavage operates all the time in a given cell or if instead it acts sporadically. DNA replication forks in untreated cells frequently encounter sporadic damage (5, 6), some of which is repaired by RecA-dependent pathways. It is likely that RecA can be activated at such sites, resulting in basal cleavage, which might well operate only a portion of the time in any given cell.