Efficiency, specificity and DNA polymerase-dependence of translesion replication across the oxidative DNA lesion 8-oxoguanine in human cells.
The oxidation product of guanine, 8-oxoguanine, is a major lesion formed in DNA by intracellular metabolism, ionizing radiation, and tobacco smoke. Using a recently developed method for the quantitative analysis of translesion replication, we have studied the bypass of 8-oxoguanine in vivo by transfecting human cells with a gapped plasmid carrying a site-specific 8-oxoguanine in the ssDNA region. The efficiency of bypass in the human large-cell lung carcinoma cell line H1299 was 80%, and it was similar when assayed in the presence of aphidicolin, an inhibitor of DNA polymerases alpha, delta and epsilon. A similar extent of bypass was observed also in XP-V cells, defective in pol eta, both in the absence and presence of aphidicolin. DNA sequence analysis indicated that the major nucleotide inserted opposite the 8-oxoguanine was the correct nucleotide C, both in H1299 cells (81%) and in XP-V cells (77%). The major mutagenic event was the insertion of an A, both in H1299 and XP-V cells, and it occurred at a frequency of 16-17%, significantly higher than previously reported. Interestingly, the misinsertion frequency of A opposite 8-oxoguanine was decreased in XP-V cells in the presence of aphidicolin, and misinsertion of G was observed. This modulation of the mutagenic specificity at 8-oxoguanine is consistent with the notion that while not essential for the bypass reaction, pol eta and pol delta, when present, are involved in bypass of 8-oxoguanine in vivo.
Avkin
Sharon
S
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Livneh
Zvi
Z
D001483:Base Sequence; D002478:Cells, Cultured; D004249:DNA Damage; D004258:DNA Polymerase III / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D000099270:Y-Family DNA Polymerases
D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D006147:Guanine; C024829:8-hydroxyguanine; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases