Effect of enhanced synthesis of the epsilon subunit of DNA polymerase III on spontaneous and UV-induced mutagenesis of the Escherichia coli glyU gene.
We have studied spontaneous and UV mutagenesis of the glyU gene in Escherichia coli trpA461 (GAG) strains carrying the pIP11 plasmid, in which the dnaQ gene encoding the 3'-5' exonuclease subunit (epsilon) of DNA polymerase III is fused to the tac(trp-lac) promoter. We have used a pair of M13glyU phage in which the gene encoding the glycyl-tRNA is cloned in opposite orientations, consequently the phage present either GGG or CCC anticodon triplets for mutagenesis. The presence of IPTG, the inducer of the tac-dnaQ fusion, results in about 100-fold decrease in frequency of spontaneous Su+ (GAG) mutations arising in the CCC phage. The enhanced expression of tac-dnaQ reduces 10-fold the frequency of UV-induced Su+ (GAG) mutations in the CCC phage and nearly completely prevents generation by UV of Su+ (GAG) mutations in the GGG phage, in which UV-induced pyrimidine photo-products can be formed only in the vicinity of the target triplet. These results suggest that both locally and regionally targeted mutagenesis is affected by overproduction of the epsilon subunit. By delayed photoreversal mutagenesis we have shown that UV-induced chromosomal mutagenesis of the umuC36 trpA461 strain harboring pIP11 is completely abolished in the presence of IPTG. This result seems to indicate that the misinocorporation step of DNA translesion synthesis is affected by excess of the epsilon subunit. Finally, we have introduced the pIP13 plasmid carrying the dnaQ gene into the recA1207 strain, which is deficient in the recombinase activity of RecA but constitutive in the protease activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Ciesla
Z
Z
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
Jonczyk
P
P
Fijalkowska
I
I
D004258:DNA Polymerase III / Q000096:biosynthesis*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial / Q000528:radiation effects*; D009154:Mutation; D010957:Plasmids; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D012353:RNA, Transfer, Gly / Q000235:genetics*; D011938:Rec A Recombinases / Q000378:metabolism; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D012345:RNA, Transfer, Amino Acid-Specific; D012353:RNA, Transfer, Gly; D011938:Rec A Recombinases; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase