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Genetics, Vol. 167, 569-578, June 2004, Copyright © 2004
doi:10.1534/genetics.103.025296

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A dnaT Mutant With Phenotypes Similar to Those of a priA2::kan Mutant in Escherichia coli K-12

Jesse D. McCool1, Christopher C. Ford and Steven J. Sandler2

Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003

2 Corresponding author: Department of Microbiology, Morrill Science Center IV N203, University of Massachusetts, Amherst, MA 01003.
E-mail: sandler{at}microbio.umass.edu

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.




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