We are interested in the pathway(s) that leads to DNA breaks after treatment with inhibitors that stabilize topoisomerase cleavage complexes. Such DNA breaks are thought to be involved in cytotoxicity and DNA rearrangements caused by inhibitors of type II topoisomerases (see Introduction). Elucidation of the detailed mechanism of DNA break formation may reveal important aspects of replication fork dynamics, and might also have implications for antibacterial and anticancer chemotherapy with topoisomerase inhibitors. We approached this issue experimentally by taking advantage of the SOS response, isolating mutants with reduced SOS induction upon nalidixic acid treatment.
Mutational inactivation of recB
blocks SOS induction by nalidixic acid but not by UV (McPartland et al., 1980
), so we focused our attention on mutants with this phenotype. Out of 19,000 transposon insertion mutants, we found 18 that were defective for induction by nalidixic acid but normal for induction by UV. Sixteen of these had a transposon insertion in the recB
gene, which confirms the validity of the genetic screen. Another two had insertions elsewhere, but we found that these two strains had an approximately 1-kb insertion in the recB
gene, which explains the induction defect. E. coli
has numerous insertion sequences that are roughly 1-kb in size (Galas and Chandler, 1989
), so we surmise that these mutants had undergone a transposition event that inactivated the recB
gene at some time prior to our genetic screen. Since the competent cells for generating the 19,000 transposon insertion mutants were grown as a single batch, these two strains may have started out as siblings with the insertion sequence in recB
prior to the transformation of the transposon.
Thus, we found a total of 18 mutants with the desired phenotype, but all 18 were defective in recB
. Since we screened 19,000 colonies and isolated so many recBC
mutants, the screen was quite thorough, and it is unlikely that any novel gene would be uncovered by screening additional mutants using this method and these criteria. Because this was a transposon mutagenesis strategy, one possibility is that RecBCD is the only non-essential protein involved in SOS induction by nalidixic acid but not by UV. Only the helicase activity of RecBCD is necessary for SOS induction after nalidixic acid treatment (Chaudhury and Smith, 1985
). Therefore, the role of RecBCD is probably limited to generation of single-stranded DNA from the break, and the enzyme is not likely involved in break formation.
These results are consistent with the possibility that essential cellular proteins are involved in DNA break formation from the cleavage complex. For example, transcription and/or DNA replication could be necessary. One specific model is the replication fork run-off model, in which the replisome directly causes DNA breaks upon collision with the cleavage complex (see Introduction). If transcription and/or replication are necessary for break formation, then some of our mutants might have reduced SOS induction simply because of slower growth. We measured the growth rate of one insertion mutant for each of the genes uncovered here, and found that several did indeed have significantly decreased growth rates: cyaA, icdA, mcrA/elbA, crp, and tufA (data not shown; we did not test tufB, but it would presumably grow similarly to the tufA mutant). While any of these mutants could have reduced SOS induction due to poor growth, we note that the other mutants we isolated had little or no growth defect, and we would have expected to isolate many other poor-growth mutants if that was all that was necessary to reduce SOS induction.
A second possible explanation for finding only recBC
mutants with the desired phenotype is that multiple pathways process the cleavage complex into an SOS-inducing signal. Some candidates for genes that might be involved in redundant pathways were uncovered in mutants that were modestly defective for SOS induction after nalidixic acid but seemingly normal after UV, namely icdA
(). The only mutants in this group with altered sensitivity to nalidixic acid were those in trkH
, which were modestly resistant to the drug (). The trkH
gene encodes an integral membrane protein involved in potassium uptake (Silver, 1996
) and trkH
mutations might somehow impact nalidixic acid uptake or efflux. Thus, the decreased SOS response of trkH
mutants could simply reflect a lower effective level of inhibitor inside the cell. None of the other genes mentioned above has a function that clearly suggests an involvement in cleavage complex processing. However, two of the gene products have known or suspected DNA cleavage activities: McrA, which is a restriction endonuclease, and IntQ, which is encoded by a cryptic prophage and is related to the integrase family of proteins.
An inherent limitation of our genetic screen raises a third possible explanation for finding only recBC
mutants with a strong SOS defect that is specific for nalidixic acid treatment. Perhaps the mutants of interest were missed in our screen because they were partially constitutive for the SOS response. That is, even in the absence of nalidixic acid, the desired mutants might have levels of β-galactosidase similar to or greater than the levels in wild-type cells with nalidixic acid. Insertions in a variety of important DNA metabolism genes cause an SOS constitutive phenotype (O’Reilly and Kreuzer, 2004
), and we are currently analyzing these to see if any are defective for SOS induction after nalidixic acid treatment.
Our collection also included insertions in four genes that caused SOS defects after either nalidixic acid or UV treatment. Mutations in each of these genes are known to cause pleiotropic effects by altering nucleiod structure and gene expression (hns
), protease function (clpP
) or the cyclic AMP regulatory system (cyaA
) (Dorman, 2004
; Saier et al., 1996
; Gross, 1996
). Although the SOS defects in each of these four mutants is likely to be caused by some indirect effect of their pleiotropy, these results may provide clues about subtleties of the SOS regulatory system. The cAMP system, involving the products of the crp
genes, has previously been linked to the SOS system in the context of cAMP-dependent SOS induction in resting cells (Taddei et al., 1995