TY - JOUR
T1 - Reduced initiation frequency from oriC restores viability of a temperature-sensitive Escherichia coli replisome mutant
AU - Skovgaard, Ole
AU - Løbner-Olesen, Anders
PY - 2005/3
Y1 - 2005/3
N2 - The dnaX gene of Escherichia coli encodes tau and gamma clamp loader subunits of the replisome. Cells carrying the temperature-sensitive dnaX2016 mutation 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 the dnaA gene that partly suppressed the dnaX2016 temperature 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 of dnaA(Sx) mutants obtained indicated that a direct interaction between the DnaA protein and tau or gamma is unlikely and that the mechanism behind suppression is related to DnaA function. All dnaA(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 each dnaX2016 dnaA(Sx) cell and prevented the SOS response. Lowered availability of wild-type DnaA protein also led to partial suppression of the dnaX2016 mutation, confirming that the dnaA(Sx) mode of suppression is indirect and results from a reduced initiation frequency at oriC.
AB - The dnaX gene of Escherichia coli encodes tau and gamma clamp loader subunits of the replisome. Cells carrying the temperature-sensitive dnaX2016 mutation 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 the dnaA gene that partly suppressed the dnaX2016 temperature 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 of dnaA(Sx) mutants obtained indicated that a direct interaction between the DnaA protein and tau or gamma is unlikely and that the mechanism behind suppression is related to DnaA function. All dnaA(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 each dnaX2016 dnaA(Sx) cell and prevented the SOS response. Lowered availability of wild-type DnaA protein also led to partial suppression of the dnaX2016 mutation, confirming that the dnaA(Sx) mode of suppression is indirect and results from a reduced initiation frequency at oriC.
KW - Bacterial Proteins/genetics
KW - DNA Polymerase III/genetics
KW - DNA Replication
KW - DNA-Binding Proteins/genetics
KW - Escherichia coli/genetics
KW - Escherichia coli Proteins/genetics
KW - Gene Expression Regulation, Bacterial
KW - Genes, Suppressor
KW - Mutation
KW - Replication Origin/genetics
KW - SOS Response (Genetics)
KW - Temperature
U2 - 10.1099/mic.0.27630-0
DO - 10.1099/mic.0.27630-0
M3 - Journal article
C2 - 15758241
SN - 1350-0872
VL - 151
SP - 963
EP - 973
JO - Microbiology
JF - Microbiology
IS - Pt 3
ER -