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 One mutation, originally designated dnaM710, maps at about 75 min on the E. coil genetic map (ref. 7; unpublished results).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)...