An Escherichia coli mutant defective in single-strand binding protein is defective in DNA replication.

Abstract

An Escherichia coli mutant, temperaturesensitive for DNA synthesis in vivo and in vitro, is defective in single-strand binding protein (SSB; DNA-binding protein). Conversion of phage G4 single strands to the duplex form is defective in crude enzyme fractions of the mutant and is complemented by pure wild-type SSB. Radioimmunoassays of mutant extracts show normal levels of material crossreacting with anti-SSB antibody. SSB purified to homogeneity from the mutant is active, with lower specific activity, in the reconstituted G4 replication assay at 30'C, but virtually inactive at 420C. Surprisingly, the mutant protein, like the wild-type protein, survives heating at 1000C. Thus, mutant SSB is structurally heat-resistant but is functionally thermosensitive in vitro and in vivo. Both the in vivo and in vitro defects are tightly linked in transductions by phage P1. SSB is required for the conversion, in vitro, of single-stranded phage DNA to the duplex replicative form (3) and in the conversion of the latter to single-stranded viral circles (5). Despite this dependence on SSB for phage DNA replication in vitro, it was not clear whether replication in vivo had a similar requirement. For lack of a mutant defective in SSB, or a specific inhibitor of it, the question of an essential or auxiliary role in replication, repair, and recombination remained in doubt.We report here that SSB is an essential component in phage and cellular DNA metabolism in vivo. A mutant has been identified with a temperature-sensitive defect in SSB. DNA synthesis stops immediately when the temperature is raised from 30'C to 420C. Single-stranded phage (ST-1) production is also blocked at 420C. The isolated mutant SSB shows a similar temperature sensitivity in an in vitro phage DNA replication system. Additional descriptions of the biochemical and genetic features of SSB will be reported elsewhere. MATERIALS AND METHODSE. coli strains were obtained as follows: strain SG1635 (thy A3, dnaM710, ssb-1) and parent DG17 (thyA3) (7) from D. Glaser (University of California, Berkeley, CA), strain KY2750 (dnaP18) and its parent KY2053 (8) from T. Yura (Kyoto University, Kyoto, Japan), and strains AB1157 (9) and AN385 (ubiA -, gal-, strR, X +) (10) from G. Weinstock of this department. SG1635 contains two mutations resulting in temperature-sensitive cell growth. One mutation, originally designated dnaM710, maps at about 75 min on the E. coil genetic map (ref. 7; unpublished results). This mutation is not responsible for temperature-sensitive DNA replication, has no effect on SSB (unpublished results), and thus is irrelevant to the results reported here. The effects of the other mutation (ssb-1) are the subject of this paper. The origins of strains JGC155 and JGC158 are described in this paper.A set of Hfr strains was obtained from B. Bachmann (E. col' Genetic Stock Center, Yale University, New Haven, CT). Hfr mapping was performed as described (11).Complementation assays were carried out in 25-Al reaction mixtures containing 20 mM Tris-HCl (pH 7.5)...

Indirect stimulation of genetic recombination

“…In a recB21 sbcA8 double mutant, the basal level of ,B-galactosidase was 10 The only mutation that we found to have an effect on indirect stimulation was ssb-1 (20). This mutation decreased drastically (to 1/8th of) the magnitude of indirect stimulation [by A (UV)] in crosses of AlacZ118 and AlacZ813 in an ssb-1 host (KL922) compared to an ssb+ host (KL921).…”

“…In crosses of AlacZ118 with F-lacZ813, the ssb-1 mutation (strain EG339) eliminated completely the indirect stimulation. Recombination in these experiments was carried out at 430C to inactivate ssb function (20).…”

Interaction of the heat shock protein GroEL of Escherichia coli with single-stranded DNA-binding protein: suppression of ssb-113 by groEL46

“…While the most dramatic effects of the ssb-113 and ssb-1 mutations are on DNA replication and repair (10, 21,34,42,50,51,53,55,70), these DNA-binding protein mutations also exhibit a less dramatic but significant decrease in recombination (21,23). We have examined the recombination frequency of two different genes in the ssb-113 and suppressor strains, as assayed by phage P1 transduction.…”

The ssb‐113 allele suppresses the dnaQ49 mutator and alters DNA supercoiling in Escherichia coli

“…Mutants of the ssb gene exhibit conditional lethality above 42ЊC owing to defective DNA replication Meyer et al, 1979). At permissive temperatures, they are defective in DNA repair Glasberg et al, 1979), including deficiencies in the inducibility of the SOS response (Baluch et al, 1980;Vales et al, 1980;Meyer et al, 1982) and in DNA recombination (Glasberg et al, 1979;Golub and Low, 1983;Quiñ ones and Piechocki, 1987).…”