A temperature-sensitive single-stranded DNA-binding protein from Escherichia coli.

Abstract

A temperature-sensitive single-stranded DNA-binding protein (SSB) has been purified from mutant Escherichia coli (ssb-1) cells by use of affinity chromatog- raphy on blue dextran-Sepharose.An altered amino acid sequence in the mutant protein is apparent in tryptic digests, confirming that the ssb mutation is in the structural gene.The mutant protein is less effective than the wild type in protecting single-stranded DNA from nuclease S1 digestion and in inhibiting DNA-dependent ATPases.The purified protein supports replication of phage G4 DNA in vitro at 30"C, although higher levels of mutant protein, 4-fold higher than wild type, are needed to do so.The mutant protein becomes less active in supporting replication above 30°C and becomes inactive at 42°C within 1 min.Activity is restored upon return to 20°C.Despite its temperature sensitivity in vivo and in vitro, the mutant binding protein can renature fully after exposure to 100°C.Thus, the mutant protein is both heat-stable and functionally temperature-sensitive.The single strand binding protein (SSB)' of Escherichia coli (previously designated DNA unwinding, DNA-binding, or helix-destabilizing protein (3-7)) is required for replication of single-stranded phage DNA in vitro (4, 6, 8, 9 ).However, the physiological significance of this requirement remained uncertain until a mutant carrying a temperature-sensitive lesion in SSB proved defective not only in replicating phage and E. coli DNA in vivo, but also in DNA repair and recombination (1, 2).The genetic locus for SSB is allelic with lenC,' a locus regarded as a control element in DNA metabolism (10).In this paper we report a simplified purification procedure for the mutant SSB and describe the properties of this temperature-sensitive protein.