Quantitative Analysis of Translesion DNA Synthesis across a Benzoapyrene-Guanine Adduct in Mammalian Cells: The Role of DNA Polymerase Kappa

• Published in: The Journal of biological chemistry 2004-12, Vol.279 (51), p.53298-53305
• Main Authors: Avkin, Sharon, Goldsmith, Moshe, Velasco-Miguel, Susana, Geacintov, Nicholas, Friedberg, Errol C, Livneh, Zvi
• Format: Article
• Language: English
• Subjects: Escherichia coli
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M409155200

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 benzoapyrene-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 super(+/+) 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 super(-/-) MEFs, bypass was 16 +/-1%, suggesting that at least two-thirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase Kappa (pol Kappa ). In contrast, pol eta 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 pol Kappa . Expression of pol Kappa from a plasmid introduced into PolK super(-/-) MEFs restored both the extent and fidelity of bypass across BP- G. Pol Kappa was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both pol Kappa and pol eta , suggesting that the biological role of pol Kappa 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 pol Kappa bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.