Translesion synthesis across bulky N2-alkyl guanine DNA adducts by human DNA polymerase κ

Abstract

DNA polymerase (pol) κ is one of the so-called translesion polymerases involved in replication past DNA lesions. Bypass events have been studied with a number of chemical modifications with human pol κ, and the conclusion has been presented, based on limited quantitative data, that the enzyme is ineffective at incorporating opposite DNA damage but proficient at extending beyond bases paired with the damage. Purified recombinant full-length human pol κ was studied with a series of eight N2-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH 2(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, kcat/Km) were similar for insertion of dCTP opposite the lesions and for extension beyond the N2-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with k cat/Km values ∼10-3 less than for dCTP with all adducts; a similar differential was found for extension beyond a paired adduct. Pre-steady-state kinetic analysis showed moderately rapid burst kinetics for dCTP incorporations, even opposite the bulky methyl(9-anthracenyl)- and BPG adducts (kp 5.9-10.3 s-1). The rapid bursts were abolished opposite BPG when α-thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols η and ι; pol κ is the most resistant to N2-bulk and the most quantitatively efficient of these in catalyzing dCTP incorporation opposite bulky guanine N2-adducts, particularly the largest (N2-BPG). © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.