Reduced initiation frequency from oriC restores viability of a temperature-sensitive Escherichia coli replisome mutant

Abstract

ThednaXgene ofEscherichia coliencodesτandγclamp loader subunits of the replisome. Cells carrying the temperature-sensitivednaX2016mutation were induced for the SOS response at non-permissive temperature. The SOS induction most likely resulted from extensive replication fork collapse that exceeded the cells' capacity for restart. Seven mutations in thednaAgene that partly suppressed thednaX2016temperature sensitivity were isolated and characterized. Each of the mutations caused a single amino acid change in domains III and IV of the DnaA protein, where nucleotide binding and DNA binding, respectively, reside. The diversity ofdnaA(Sx) mutants obtained indicated that a direct interaction between the DnaA protein andτorγis unlikely and that the mechanism behind suppression is related to DnaA function. AlldnaA(Sx) mutant cells were compromised for initiation of DNA replication, and contained fewer active replication forks than their wild-type counterparts. Conceivably, this led to a reduced number of replication fork collapses within eachdnaX2016 dnaA(Sx) cell and prevented the SOS response. Lowered availability of wild-type DnaA protein also led to partial suppression of thednaX2016mutation, confirming that thednaA(Sx) mode of suppression is indirect and results from a reduced initiation frequency atoriC.