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Volume 271, Number 45, Issue of November 8, 1996 pp. 28391-28398
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Replication across O⁶-Methylguanine by Human DNA Polymerase in Vitro
INSIGHTS INTO THE FUTILE CYTOTOXIC REPAIR AND MUTAGENESIS OF O⁶-METHYLGUANINE

(Received for publication, February 6, 1996, and in revised form, August 3, 1996)

From the Nelson Institute of Environmental Medicine, New York University Medical Center, Tuxedo, New York 10987

Replication in vivo across unrepaired O⁶-methylguanine (m⁶dG) lesions by mammalian DNA polymerase (pol ) during short patch repair may contribute to the cytotoxicity and mutagenesis of m⁶dG. We have employed in vitro steady state kinetic analysis to investigate the replication of oligonucleotide templates containing site-specific m⁶dG by human pol . Our results show that m⁶dG is a strong but not absolute block to replication by pol . pol exhibits mixed kinetic discrimination during overall replication across dG and m⁶dG. pol preferentially inserts dTMP rather than dCMP opposite m⁶dG. However, pol extends from the dC-m⁶dG base pair more efficiently than from the dT-m⁶dG base pair. This is in strong contrast to other polymerases such as the exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (exoKF) that preferentially extends dT-m⁶dG by a factor of 10 over dC-m⁶dG. When both insertion and extension are considered, pol has a 15-fold overall preference for incorporation of the mutagenic substrate dTTP rather than the nonmutagenic substrate dCTP during replication across m⁶dG. This suggests that pol , in concert with the T:G-specific thymine DNA glycosylase, may be intricately involved in the futile cytotoxic repair induced by m⁶dG. Our results also suggest that replication across m⁶dG by pol may contribute to m⁶dG-induced G A transition mutations.

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