Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: isolation of a new ssb allele, ssb-3

Abstract

Mutations in several dna genes of Escherchia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-1-Dgalactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, muking the cel X-Gal extrasensitive (phenotypically Xgx), possibly becaue of enhed permeability to X-Gal or leakage of J-galactosWdae. ( well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found.Although many genes involved in DNA replication and modulation of DNA structure in Escherichia coli have been identified, several replication proteins have not yet been matched with any gene (21), suggesting that other DNA replication genes, designated dna genes, remain to be identified. Furthermore, several suppressors of mutations in dna genes are known, and these suppressors may represent new dna genes (1, 39).Bacterial genes whose products are involved in DNA replication have been identified by several strategies that entail detection of mutants under nonpermissive growth conditions. Temperature-sensitive dna mutants have been selected by their resistance to UV irradiation resulting from their failure to incorporate 5-bromouracil into DNA at high temperatures (7,8). Such dna mutants have also been selected by their resistance to thymine starvation, which does not affect nonreplicating DNA (44). A variation on the same theme is to take advantage of the inability of mutants to incorporate labeled thymine at high temperatures while remaining able to synthesize protein and RNA (30, 32, 37, 38).Our isolation procedure attempts to identify bacterial genes whose products are involved in DNA replication. The method was designed to detect dna mutants by virtue of defects in the structure of their DNA, but it apparently succeeded for other reasons. number of unlinked genes collectively called the SOS regulon (28). The promoters of a number of SOS genes have been fused to the lac operon (17); these lac fusion genes produce 0-galactosidase in response to DNA damage. The presence of DNA defects in a dna mutant, particularly an excess of single-stranded DNA regions, would be expected to result in induction of an SOS fusion gene, causing a blue colony on a plate containing 5-bromo-4-chloro-3-indolyl-,-D-galactoside (X-Gal), a chromogenic substrate for P-galactosidase.Evidence that some dna mutations can indeed induce the SOS systen) comes from the work of Schuster et al. (31), although induction was obtained only at temperatures nonpermissive for growth of the dna mutants. They found that strains mutated in dnaB, dnaC, dnaE, or dnaG, when incubated for 3 h at a nonpermissive temperature and then shifted to 30°C, produced an SOS response, namely induction of prophage lambda. Mutations in dnaB produced particularly strong induction of...

A common core for binding single‐stranded DNA: structural comparison of the single‐stranded DNA‐binding proteins (SSB) from E. coli and human mitochondria

“…Although it has been previously believed that no arginine residues were involved in ssDNA binding [37], six of them in this region of the protein (arginines 21, 41, 56, 72, 86 and 96) are also oriented into the DNA‐binding cleft in a manner that may suggest a possible role in ssDNA binding. Interestingly, the Eco SSB ssb3 mutation, in which Gly‐15 is substituted for aspartic acid, giving rise to an extremely UV‐sensitive phenotype in affected cells [38], can also now be explained by the Eco SSB structure: the observed mutation would produce a striking clash with the essential Trp‐54 residue that is one of the key determinants of DNA binding (Fig. 3 ).…”

SSB binds to the RecG and PriA helicases in vivo in the absence of DNA

“…2015). Support for the implication of membranes in SSB function comes from a previous study identifying the ssb-3 allele (Schmellik-Sandage & Tessman 1990). This mutation (G15D) renders the cell extremely sensitive to UV irradiation, with a survival curve similar to that for a Δ recA mutation.…”

A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB.

“…At least one region important for protein-protein interactions seems to be conserved in the amino-terminus of all four SSBs. The existence of this region was suggested from the phenotype associated with E. coli ssb-3 mutant (Schmellik-Sandage and Tessman, 1990;Meyer and Laine, 1990 The EcoRI restriction site used to clone the partial cDNA fragment is underlined. Bold underlined nucleotides represent the oligonucleotide used as the 3' primer for the isolation and amplification of RlMI partial cDNA.…”