Genetic analysis of the requirements for SOS induction by nalidixic acid in Escherichia coli

Abstract

Nalidixic acid, the prototype antibacterial quinolone, induces the SOS response by a mechanism that requires the RecBCD nuclease/helicase. A key step inferred for this induction pathway is the conversion of a drug-induced gyrase cleavage complex into a DNA break that can be processed by RecBC. We tried to clarify the nature of this step by searching for additional gene products that are specifically necessary for SOS induction following nalidixic acid treatment. A transposon library of approximately 19,000 insertion mutants yielded 18 mutants that were substantially reduced for SOS induction following nalidixic acid but not UV treatment, and which were also hypersensitive to nalidixic acid. All 18 mutants turned out to have insertions in recB or recC. As expected, recA insertion mutants were uncovered as being uninducible by either nalidixic acid or UV treatment. Insertions in 11 other genes were found to cause partial defects in SOS induction by one or both pathways, providing possible leads in understanding the detailed mechanisms of SOS induction. Overall, these results suggest that nalidixic acid-induced DNA breaks are generated either by RecBC itself, by redundant activities, and/or by an essential protein that could not be uncovered with transposon mutagenesis.

Introduction

Type II DNA topoisomerases are essential enzymes involved in numerous cellular processes, including DNA replication, DNA segregation and transcription. These enzymes catalyze changes in DNA topology by creating a transient double strand break in one segment of duplex DNA and passing a second duplex segment through that break. Many clinically important antibacterial and antitumor agents target type II topoisomerases by altering the equilibrium of the reaction cycle to stabilize the normally transient intermediate called the cleavage complex (Drlica and Zhao, 1997, Burden and Osheroff, 1998). These include the antibacterial quinolones (e.g., nalidixic acid) that target DNA gyrase (and topoisomerase IV in certain bacteria), and antitumor drugs such as mitoxantrone, adriamycin and etoposide, which target mammalian type II topoisomerase. In each case, the drug-stabilized cleavage complex consists of the topoisomerase linked via phosphotyrosine bonds to both 5′ ends of the staggered double-strand break. Results from numerous systems demonstrate that the cytotoxicity of these inhibitors is dependent on formation of cleavage complexes rather than inhibition of topoisomerase catalytic activity (for reviews, see Chen and Liu, 1994, Drlica and Zhao, 1997).

Although much research has been devoted to understanding how stabilized cleavage complexes lead to cell death, the detailed mechanism(s) remains unclear. Formation of cleavage complexes is necessary but not sufficient for cytotoxicity. The 5′ ends of the broken DNA within the cleavage complex are safely linked to protein, and cleavage complexes are readily reversible upon drug removal both in vivo and in vitro (Hsiang and Liu, 1989; also see Drlica and Zhao, 1997). The energy poison dinitrophenol blocks cytotoxicity of mammalian type II topoisomerase inhibitors but does not prevent cleavage complex formation (Kupfer et al., 1987). Likewise, dinitrophenol (as well as chloramphenicol) protects Escherichia coli from the cytotoxic action of nalidixic acid (Cook et al., 1966). Evidently, a cellular processing event must occur to convert a subset of the cleavage complexes into cytotoxic lesions. What is this event, and what is the exact nature of the cytotoxic lesion?

A number of observations strongly suggest that the cytotoxic lesion is some type of double-strand break (for review, see Chen and Liu, 1994, Drlica and Zhao, 1997). For example, mutational inactivation of functionally conserved recombination proteins in phage, bacterial and eukaryotic systems leads to drug hypersensitivity. The gene products required for the repair of topoisomerase-mediated DNA damage are similar or identical to those required for the repair of endonuclease-generated double-strand breaks. Furthermore, there is evidence for overt chromosomal breaks after nalidixic acid treatment, but the location of the breaks and the possibility of covalently attached protein were not determined (Chen et al., 1996).

A variety of models could in principle explain the relationship between drug-stabilized cleavage complexes and cytotoxicity. First, a nuclease such as SbcCD might directly recognize the covalent protein–DNA complex and cleave the DNA nearby (Connelly et al., 2003). Second, the replication complex or associated helicase may be able to extract the lagging strand template from the cleavage complex upon collision (Howard et al., 1994). Third, DNA breaks might result as “collateral damage” from recombination nucleases that act after replication fork blockage by the cleavage complex, as in the phage T4 system (Hong and Kreuzer, 2003). We have recently found that quinolone-stabilized gyrase cleavage complexes block E. coli replication forks on plasmid pBR322 in vivo (Pohlhaus and Kreuzer, 2005). In that study, some of the blocked forks were broken, consistent with the collateral damage model.

To approach the mechanism of cytotoxicity, we have taken advantage of an SOS reporter system and screened for E. coli mutants specifically deficient in SOS induction upon nalidixic acid treatment. The primary target of nalidixic acid in E. coli is DNA gyrase (see Maxwell and Critchlow, 1998), and this drug was one of the first inducers of the SOS regulon studied in detail. The SOS regulon consists of about 30 different genes, many of which are involved in damage repair or bypass reactions (Friedberg et al., 1995). The LexA protein normally represses SOS genes, but is cleaved to trigger SOS as a result of DNA damage. Cleavage of LexA depends on the activated form of the RecA protein, bound to single-stranded DNA.

DNA gyrase cleavage complexes stabilized by nalidixic acid are necessary but not sufficient for induction of the SOS response. There is conflicting evidence as to whether DNA replication is required for induction of SOS by nalidixic acid (Gudas and Pardee, 1976, Sassanfar and Roberts, 1990). Aside from RecA, the only known protein that is required for SOS induction by nalidixic acid is the multifunctional RecBCD enzyme (for review, see Myers and Stahl, 1994). Since RecBCD generally requires a DNA end to gain entry to DNA, its involvement in SOS induction suggests that a free DNA break is somehow generated from the nalidixic acid-stabilized cleavage complex.

The above results provide strong parallels between the mechanism of cytotoxicity and the mechanism of SOS induction by nalidixic acid, particularly since recA, recB and recC mutants are all hypersensitive to the drug (McDaniel et al., 1978). It seems highly likely that whatever mechanism creates DNA breaks to induce the SOS pathway is also a mechanism that leads to cytotoxicity. In this report, we identify and analyze E. coli mutants with deficiencies in the SOS response to nalidixic acid.