A dnaT mutant with phenotypes similar to those of a priA2::kan mutant in Escherichia coli K-12.
The ability to repair damaged replication forks and restart them is important for cell survival. DnaT is essential for replication restart in vitro and yet no definite genetic analysis has been done in Escherichia coli K-12. To begin, dnaT822, an in-frame six-codon (87-92) deletion was constructed. DnaT822 mutants show colony size, cell morphology, inability to properly partition nucleoids, UV sensitivity, and basal SOS expression similar to priA2::kan mutants. DnaT822 priA2::kan double mutants had phenotypes similar to those of the single mutants. DnaT822 and dnaT822 priA2::kan mutant phenotypes were fully suppressed by dnaC809. Previously, a dominant temperature-sensitive lethal mutation, dnaT1, had been isolated in E. coli 15T(-). DnaT1 was found to have a base-pair change relative to the E. coli 15T(-) and E. coli K-12 dnaT genes that led to a single amino acid change: R152C. A plasmid-encoded E. coli K-12 mutant dnaT gene with the R152C amino acid substitution did not display a dominant temperature-sensitive lethal phenotype in a dnaT(+) strain of E. coli K-12. Instead, this mutant dnaT gene was found to complement the E. coli K-12 dnaT822 mutant phenotypes. The significance of these results is discussed in terms of models for replication restart.
McCool
Jesse D
JD
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Ford
Christopher C
CC
Sandler
Steven J
SJ
D001483:Base Sequence; D003062:Codon / Q000235:genetics; D017931:DNA Primers; D004260:DNA Repair; D004261:DNA Replication / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D048168:Escherichia coli K12 / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion
D003062:Codon; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C104198:DnaT protein, E coli; D029968:Escherichia coli Proteins