Quantitative analysis of translesion DNA synthesis across a benzo[a]pyrene-guanine adduct in mammalian cells: The role of DNA polymerase

Sharon Avkin, Moshe Goldsmith, Susana Velasco-Miguel, Nicholas Geacintov, Errol C. Friedberg, Zvi Livneh

Research output: Contribution to journalArticle

147 Citations (Scopus)

Abstract

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 benze[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 κ (polκ). In contrast, polη 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 polκ. Expression of polκ from a plasmid introduced into PelK-/- MEFs restored both the extent and fidelity of bypass across BP-G. Polκ was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both polκ and polη, suggesting that the biological role of polκ 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 polκ bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.

Original languageEnglish (US)
Pages (from-to)53298-53305
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number51
DOIs
StatePublished - Dec 17 2004

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Benzo(a)pyrene
Guanine
DNA-Directed DNA Polymerase
Cells
Fibroblasts
DNA
Chemical analysis
Embryonic Structures
Plasmids
pyrene
Tobacco
Smoke
Escherichia coli
Cultured Cells
Assays
Recovery
Cell Line

ASJC Scopus subject areas

  • Biochemistry

Cite this

Quantitative analysis of translesion DNA synthesis across a benzo[a]pyrene-guanine adduct in mammalian cells : The role of DNA polymerase. / Avkin, Sharon; Goldsmith, Moshe; Velasco-Miguel, Susana; Geacintov, Nicholas; Friedberg, Errol C.; Livneh, Zvi.

In: Journal of Biological Chemistry, Vol. 279, No. 51, 17.12.2004, p. 53298-53305.

Research output: Contribution to journalArticle

Avkin, Sharon ; Goldsmith, Moshe ; Velasco-Miguel, Susana ; Geacintov, Nicholas ; Friedberg, Errol C. ; Livneh, Zvi. / Quantitative analysis of translesion DNA synthesis across a benzo[a]pyrene-guanine adduct in mammalian cells : The role of DNA polymerase. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 51. pp. 53298-53305.
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