Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases

Jeong Yun Choi, Goutam Chowdhury, Hong Zang, Karen C. Angel, Choua C. Vu, Lisa A Peterson, F. Peter Guengerich

Research output: Contribution to journalArticle

100 Scopus citations

Abstract

Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O6-methyl and -benzyl (Bz) G. Recombinant human polymerase (pol) δ also copied past these two lesions but was totally blocked by O6-[4-oxo-4-(3-pyridyl) butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone. The human translesion pols ι and κ produced mainly only 1-base incorporation opposite O6-MeG and O 6-BzG and had very low activity in copying O6-PobG. Human pol η copied past all three adducts. Steady-state kinetic analysis showed similar efficiencies of insertion opposite the O6-alkylG adducts for dCTP and dTTP with pol η and κ; pol ι showed a strong preference for dTTP. pol η, ι, and κ showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O6-MeG but little, if any, for O6-BzG or O6-PobG. Analysis of the pol η O 6-PobG products indicated that the insertion of G was opposite the base (C) 5′ of the adduct, but this product was not extended. Mass spectrometry analysis of all of the pol η primer extension products indicated multiple components, mainly with C or T inserted opposite O 6-alkylG but with no deletions in the cases of O6-MeG and O6-PobG. With pol η and O6-BzG, products were also obtained with -1 and -2 deletions and also with A inserted (opposite O 6-BzG). The results with pol η may be relevant to some mutations previously reported with O6-alkylG adducts in mammalian cells.

Original languageEnglish (US)
Pages (from-to)38244-38256
Number of pages13
JournalJournal of Biological Chemistry
Volume281
Issue number50
DOIs
StatePublished - Dec 15 2006

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