Mutator activity and specificity of Escherichia coli dnaQ49 allele--effect of umuDC products.
The high fidelity of DNA replication in Escherichia coli is ensured by the alpha (DnaE) and epsilon (DnaQ) subunits of DNA polymerase providing insertion fidelity, 3'-->5' exonuclease proofreading activity, and by the dam-directed mismatch repair system. dnaQ49 is a recessive allele that confers a temperature-sensitive proofreading phenotype resulting in a high rate of spontaneous mutations and chronic induction of the SOS response. The aim of this study was to analyse the mutational specificity of dnaQ49 in umuDC and DeltaumuDC backgrounds at 28 and 37 degrees C in a system developed by J.H. Miller. We confirmed that the mutator activity of dnaQ49 was negligible at 28 degrees C and fully expressed at 37 degrees C. Of the six possible base pair substitutions, only GC-->AT transitions and GC-->TA and AT-->TA transversions were appreciably increased. However, the most numerous mutations were frameshifts, -1G deletions and +1A insertions. All mutations which increased in response to dnaQ49 damage were to a various extent umuDC-dependent, especially -1G deletions. This type of mutations decreased in CC108dnaQ49DeltaumuDC to 10% of the value found in CC108dnaQ49umuDC+ and increased in the presence of plasmids producing UmuD'C or UmuDC proteins. In the recovery of dnaQ49 mutator activity the plasmid harbouring umuD'C genes was more effective than the one harbouring umuDC. Analysis of mutational specificity of pol III with defective epsilon subunit indicates that continuation of DNA replication is allowed past G:T, C:T, T:T (or C:A, G:A, A:A) mismatches but does not allow for acceptance of T:C, C:C, A:C (or A:G, G:G, T:G) (the underlined base is in the template strand).
Nowosielska
Anetta
A
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warszawa, Poland.
Wrzesiński
Michał
M
Nieminuszczy
Jadwiga
J
Janion
Celina
C
Grzesiuk
Elzbieta
E
D000483:Alleles; D004258:DNA Polymerase III / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics*; D016296:Mutagenesis; D010957:Plasmids
D029968:Escherichia coli Proteins; D004258:DNA Polymerase III; C120926:DNA polymerase V, E coli; D004259:DNA-Directed DNA Polymerase; C468316:dnaQ protein, E coli