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

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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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.",
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