title: Translesion synthesis across bulky N2-alkyl guanine DNA adducts by human DNA polymerase kappa.
abstract: 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 N(2)-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH(2)(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, k(cat)/K(m)) 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 approximately 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).
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146; Department of Pharmacology, College of Medicine, Ewha Womans University, 911-1 Mok-6-Dong, Yangcheon-Gu, Seoul 158-710, Republic of Korea.
authors.forename: Jeong-Yun
authors.initials: JY
authors.lastname: Choi
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
authors.forename: Karen C
authors.initials: KC
authors.lastname: Angel
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146. Electronic address: f.guengerich@vanderbilt.edu.
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D018736:DNA Adducts / Q000737:chemistry*; D004249:DNA Damage; D018076:DNA, Complementary / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D003845:Deoxycytosine Nucleotides / Q000737:chemistry; D006147:Guanine / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009841:Oligonucleotides / Q000737:chemistry; D013997:Time Factors; D000099270:Y-Family DNA Polymerases
chemical_list: D018736:DNA Adducts; D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase; D003845:Deoxycytosine Nucleotides; D006147:Guanine; D009841:Oligonucleotides; C024107:2'-deoxycytidine 5'-triphosphate; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases