Abstract
Replication in vivo across unrepaired O6-methylguanine (m6dG) lesions by mammalian DNA polymerase beta (pol beta) 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 beta. Our results show that m6dG is a strong but not absolute block to replication by pol beta. pol beta exhibits mixed kinetic discrimination during overall replication across dG and m6dG. pol beta preferentially inserts dTMP rather than dCMP opposite m6dG. However, pol beta 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 (exo-KF) that preferentially extends dT-m6dG by a factor of 10 over dC-m6dG. When both insertion and extension are considered, pol beta 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 beta, 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 beta may contribute to m6dG-induced G --> A transition mutations.
Highlights
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
In this paper we report the kinetic parameters of steady state replication across dG and m6dG by pol 
Summary—This is the first report to dissect the kinetic parameters of replication across m6dG by pol  in vitro
Summary
Materials—Purified human pol  was very generously supplied by Dr Samuel Wilson (Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, TX). Primer Extension Assays—10 –100 pmol of 32P-labeled primer-template were replicated with different DNA polymerases for different times at 37 °C in 10-l reactions containing variable concentrations of dNTPs. Primer extension with pol  was carried out in the presence of 50 mM Tris-HCl (pH 7.5), 2 mM DTT, and 10 mM MgCl2. 3 units of Sequenase 2.0, 40 M of all four dNTP’s, 40 M each dideoxynucleotide triphosphate, 50 mM NaCl, 3.6 mM MnCl2, and 16.7 mM DTT were mixed and primer extension was carried out for 5 min before termination of the reaction by the addition of stop solution. The sequencing lanes were run side-by-side with the primer extension reactions on denaturing polyacrylamide gels, as described above
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
