Sequence analysis and phenotypes of five temperature sensitive mutator alleles of dnaE, encoding modified alpha-catalytic subunits of Escherichia coli DNA polymerase III holoenzyme.
In the 1970s, several thermosensitive alleles of dnaE (encoding the alpha-catalytic subunit of pol III) were isolated. Genetic characterization of these dnaE mutants revealed that some are mutator alleles at permissive temperature. We have determined the nucleotide changes of five such temperature sensitive mutator alleles (dnaE9, dnaE74, dnaE486, dnaE511, and dnaE1026) and find that most are single missense mutations. The exception is dnaE1026 which is a compound allele consisting of multiple missense mutations. When the previously characterized mutator alleles were moved into a lexA51(Def) recA730 strain, dnaE486, dnaE1026 and dnaE74 conferred a modest approximately two-six-fold increase in spontaneous mutagenesis when grown at the permissive temperature of 28 degrees C, while dnaE9 and dnaE511 actually resulted in a slight decrease in spontaneous mutagenesis. In isogenic DeltaumuDC derivatives, the level of spontaneous mutagenesis dropped significantly, although in each case, the overall mutator effect conferred by the dnaE allele was relatively larger, with all five dnaE alleles conferring an increased spontaneous mutation rate approximately 5-22-fold over the isogenic dnaE+ DeltaumuDC strain. Interestingly, the temperature sensitivity conferred by each allele varied considerably in the lexA51(Def) recA730 background and in many cases, this phenotype was dependent upon the presence of functional pol V (UmuD'2C). Our data suggest that pol V can compete effectively with the impaired alpha-subunit for a 3' primer terminus and as a result, a large proportion of the phenotypic effects observed with strains carrying missense temperature sensitive mutations in dnaE can, in fact, be attributed to the actions of pol V rather than pol III.
Vandewiele
Dominique
D
Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2725, USA.
Fernández de Henestrosa
Antonio R
AR
Timms
Andrew R
AR
Bridges
Bryn A
BA
Woodgate
Roger
R
D001426:Bacterial Proteins / Q000235:genetics; D020134:Catalytic Domain; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D004258:DNA Polymerase III / Q000235:genetics* / Q000378:metabolism; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000502:physiology*; D009154:Mutation; D010641:Phenotype; D011938:Rec A Recombinases / Q000235:genetics; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000235:genetics; D013696:Temperature; D014161:Transduction, Genetic; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004258:DNA Polymerase III; D004251:DNA Transposable Elements; D011938:Rec A Recombinases; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein; C074904:DNA polymerase III, alpha subunit