Escherichia coli DNA polymerase II catalyzes chromosomal and episomal DNA synthesis in vivo.
We have investigated a role for Escherichia coli DNA polymerase II (Pol II) in copying chromosomal and episomal DNA in dividing cells in vivo. Forward mutation frequencies and rates were measured at two chromosomal loci, rpoB and gyrA, and base substitution and frameshift mutation frequencies were measured on an F'(lacZ) episome. To amplify any differences in polymerase error rates, methyl-directed mismatch repair was inactivated. When wild-type Pol II (polB+) was replaced on the chromosome by a proofreading-defective Pol II exo- (polBex1), there was a significant increase in mutation frequencies to rifampicin resistance (RifR) (rpoB) and nalidixic acid resistance (NalR) (gyrA). This increased mutagenesis occurred in the presence of an antimutator allele of E. coli DNA polymerase III (Pol III) (dnaE915), but not in the presence of wild-type Pol III (dnaE+), suggesting that Pol II can compete effectively with DnaE915 but not with DnaE+. Sequencing the RifR mutants revealed a G --> A hot spot highly specific to Pol II exo-. Pol II exo- caused a significant increase in the frequency of base substitution and frameshift mutations on F' episomes, even in dnaE+ cells, suggesting that Pol II is able to compete with Pol III for DNA synthesis on F episomes.
Rangarajan
S
S
Department of Biological Sciences, University of Southern California, Los Angeles 90089-1340, USA.
Gudmundsson
G
G
Qiu
Z
Z
Foster
P L
PL
Goodman
M F
MF
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000378:metabolism; D027081:DNA Gyrase; D004252:DNA Mutational Analysis; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D004258:DNA Polymerase III / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases / Q000235:genetics; D016368:Frameshift Mutation / Q000235:genetics; D007763:Lac Operon; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009268:Nalidixic Acid / Q000494:pharmacology; D010957:Plasmids / Q000096:biosynthesis*; D012319:RNA Polymerase II / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C074431:polB protein, E coli; D009268:Nalidixic Acid; C074904:DNA polymerase III, alpha subunit; D012319:RNA Polymerase II; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D012293:Rifampin