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J. Biol. Chem., Vol. 279, Issue 51, 53298-53305, December 17, 2004

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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, and Zvi Livneh||

From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel, the Laboratory of Molecular Pathology, the Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9072, and the ^¶Chemistry Department, New York University, New York, New York 10003-5180

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

Received for publication, August 10, 2004 , and in revised form, September 27, 2004.

^* This work was supported in part by Israel Science Foundation Grant 78/00 (to Z. L.) and by NIH/NCI Grant CA099194 (to N. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Incumbent of the Maxwell Ellis Professorial Chair in Biomedical Research. To whom all correspondence should be addressed: Dept. of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: 972-8-934-3203; Fax: 972-8-934-4169; E-mail: zvi.livneh{at}weizmann.ac.il.

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