or sulA, which encodes an SOS-inducible inhibitor of septation. ... alkylation damage and hyperinduced for the SOS response, but had unpredictable ...
JOURNAL
OF
BACTERIOLOGY, Aug. 1990, p. 4719-4720
Vol. 172, No. 8
0021-9193/90/084719-02$02.00/0 Copyright © 1990, American Society for Microbiology
Escherichia coli Strains with Multiple DNA Repair Defects Are Hyperinduced for the SOS Response PATRICIA L. FOSTER Division of Environmental Health, Boston University School of Public Health, and Department of Pathology, Boston University School of Medicine, Boston, Massachusetts 02118 Received 22 February 1990/Accepted 24 May 1990
Escherichia coli strains defective for the repair of apurinic/apyrimidinic sites and for the UvrABC excision repair pathway could be constructed if they also carried a mutation in ung, which encodes uracil glycosylase, or sulA, which encodes an SOS-inducible inhibitor of septation. The resultant strains were sensitive to alkylation damage and hyperinduced for the SOS response, but had unpredictable spontaneous mutation rates. Bacterial strains deficient in DNA repair activities are useful to identify mutagenic and lethal DNA lesions. However, the pathway for the repair of apurinic/apyrimidinic (AP) sites has proved difficult to manipulate genetically because several different enzymes act upon these lesions. Escherichia coli possesses two major AP endonucleases that have overlapping substrate specificities: exonuclease III, encoded by the xthA gene; and endonuclease IV, encoded by the nfo gene (1). Although these enzymes primarily function in different repair pathways, both can initiate the repair of simple AP sites arising from DNA alkylation and glycosylase activity (1). Two recent papers have suggested that the UvrABC excision repair pathway of E. coli can act upon some substrates for AP endonucleases. Saporito et al. (12) reported that a uvrA6 AxthA nfo::kan triple mutant could not be constructed, and Goerlich et al. (6) reported a similar failure to combine uvrA6 with a null mutation in nth, which codes for a third AP endonuclease, endonuclease III. However, the inviability of bacteria with multiple defects in DNA repair could be due simply to the accumulated burden of unrepaired DNA lesions. One endogenous source of AP sites is the activity of uracil glycosylase, the product of the ung gene (3). Thus, I used an ung mutation to test whether relieving the burden of AP sites would allow a null allele of nfo, nfo::kan (1), to be transduced into repair-defective cells. The parent strains were PF260 [F- ara A(gpt-lac-pro) thi his-5 supD60] and PF456, its A(xth-pncA) derivative (4). Genetic construction was by P1 vir transduction as described previously (10). uvrA6 was transduced from AB1886 (8) via linkage to mal::TnlO; ung-J was transduced from BW367 (obtained from B. Weiss) via linkage to tyrA::TnlO. Both TnlO transposons were removed by subsequent transduction to Mal' and Tyr'. nfo: :kan could indeed be transduced into the uvrA6 A(xthA) ung-J triple mutant, but not into the uvrA6 A(xthA) ung+ parent (Table 1). The resulting uvrA6 A(xthA) ung-J nfo::kan strain was poorly viable and extremely sensitive to methyl methanesulfonate, hydrogen peroxide, and tert butyl hydroperoxide (data not shown). In addition, it filamented extensively, suggesting that chronic induction of the SOS response was contributing to its poor viability. To test this hypothesis, I transduced into the various backgrounds a sulA::Mu d(Ap lac)XCam (chloramphenicol resistant) fusion (11). Mutations in sulA prevent lethal filamentation during SOS induction (5). All strains carrying the sulA fusion could be readily transduced with nfo::kan, although the uvrA6 allele decreased and the ung-l allele
increased the transduction frequencies into the A(xthA) background (Table 1). The sensitivities of the resulting strains to DNA alkylation are shown in Fig. 1. uvrA6 A(xthA) nfo::kan strains were much more sensitive to methyl methanesulfonate than uvrA+ A(xthA) nfo::kan strains, but the uvrA6 allele had little effect on methyl methanesulfonate sensitivity in any other background. Since sulA is an SOS controlled gene (9), the sulA::Mu d(Ap lac)XCam fusion allowed the basal level of SOS induction to be measured (Table 2). SOS induction in uvrA6 A&(xthA) nfo::kan cells was increased over 25-fold relative to wild type. In nfo+ cells, the uvrA6 and A(xthA) alleles each independently conferred a twofold increase in induction. The nfo::kan allele had no effect alone but when combined with A(xthA) resulted in a further threefold increase in induction in uvrA+ cells and a sixfold increase in induction in uvrA6 cells. In contrast, the ung-J allele had no effect in any background. To determine the spontaneous mutation rates of these strains, I introduced an episome carrying the his operon with the hisG46 allele from Salmonella typhimurium (4). The ung-J allele confers a high mutation rate because of the persistence in the DNA of uracil residues created by the spontaneous deamination of cytosine (2). I expected that, among strains with the same ung allele, the spontaneous mutation frequency would reflect the degree of SOS induction, but this proved not to be the case (Fig. 2). Among the ung+ strains, only the uvrA6 allele significantly increased mutation frequencies, and this increase was not seen in the uvrA6 A(xthA) nfo::kan strain, which was strongly induced for SOS (Table 2). In the ung-J background, the mutation frequencies correlated with the level of SOS induction among A(xthA) strains, but not among xthA+ strains. TABLE 1. Transduction efficiencies of nfo::kan into strains with various repair defects' Kanr transductants/109 cells
Strain
uvrA+ ung+ uvrA ung+ uvrA+ ung uvrA ung
xthA+ suIA+
xthA suLA+
xthA + sulA
xthA sulA
2.1 1.3 1.5 1.7
4.6
