Quantitative analysis of translesion DNA synthesis across a benzo[a]pyrene-guanine adduct in mammalian cells: the role of DNA polymerase kappa.
Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK(+/+) mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 +/- 4%, which is an order of magnitude higher than in Escherichia coli. In PolK(-/-) MEFs, bypass was 16 +/- 1%, suggesting that at least two-thirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase kappa (polkappa). In contrast, poleta was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking polkappa. Expression of polkappa from a plasmid introduced into PolK(-/-) MEFs restored both the extent and fidelity of bypass across BP-G. Polkappa was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both polkappa and poleta, suggesting that the biological role of polkappa in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that polkappa bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.
Avkin
Sharon
S
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Goldsmith
Moshe
M
Velasco-Miguel
Susana
S
Geacintov
Nicholas
N
Friedberg
Errol C
EC
Livneh
Zvi
Z
D000818:Animals; D001483:Base Sequence; D001564:Benzo(a)pyrene / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis* / Q000378:metabolism; D018736:DNA Adducts; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D001564:Benzo(a)pyrene; D004247:DNA; D018736:DNA Adducts; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases