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Translesion synthesis across bulky N2-alkyl guanine DNA adducts by human DNA polymerase κ
被引:117
作者:
Choi, Jeong-Yun
Angel, Karen C.
Guengerich, F. Peter
机构:
[1] Vanderbilt Univ, Sch Med, Dept Biochem, Nashville, TN 37232 USA
[2] Vanderbilt Univ, Sch Med, Ctr Mol Toxicol, Nashville, TN 37232 USA
[3] Ewha Womans Univ, Coll Med, Dept Pharmacol, Seoul 158710, South Korea
关键词:
D O I:
10.1074/jbc.M602246200
中图分类号:
Q5 [生物化学];
Q7 [分子生物学];
学科分类号:
071010 ;
081704 ;
摘要:
DNA polymerase (pol) kappa 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 kappa, 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 kappa was studied with a series of eight N2-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH2(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 N-2-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with k(cat)/K-m values similar to 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 (k(p) 5.9-10.3 s(-1)). The rapid bursts were abolished opposite BPG when alpha-thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols eta and iota; pol kappa is the most resistant to N-2-bulk and the most quantitatively efficient of these in catalyzing dCTP incorporation opposite bulky guanine N-2-adducts, particularly the largest (N-2-BPG).
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页码:21062 / 21072
页数:11
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