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).
Highlights
Repair of damage, and the processing of DNA to generate permanent changes in the genetic information
A group of DNA polymerases first discovered in the late 1990s participates in “translesion” synthesis beyond small and large DNA adducts, the process is, in general, less efficient and more error-prone than the reactions that replicative polymerases catalyzed with unmodified DNA [1, 5]
The ability of pol to bypass a number of types of DNA damage has been reported, including guanine modified with N-acetylaminofluorene or BPDE (9 –14), xanthine [15], 8-nitroguanine [16], and guanine modified with oxidized estrogens [17]
Summary
Methyl; Et, ethyl; Ib, isobutyl; Bz, benzyl; N2-Naph, N2-CH2(2-naphthyl); N2-Anth, N2-CH2(9-anthracenyl); N2-BP, N2-CH2(6-benzo[a]pyrenyl); BPDE, benzo[a]pyrene diol epoxide; dCTP␣S, 2Ј-deoxycytidine 5Ј-O-(1-thiotriphosphate); PCNA, proliferating cell nuclear antigen; pol, DNA polymerase; RT, reverse transcriptase; T7Ϫ, bacteriophage DNA polymerase T7, exonuclease-deficient; HIV-1, human immunodeficiency virus, type 1. Some steady-state kinetic studies have been done, but many only report steady-state rates as percentages and cannot be converted to kcat values. To obtain a better perspective on the role of human pol , we prepared a purified full-length enzyme and utilized this in kinetic and other studies with a defined oligonucleotide containing a G or each of eight N2-substituted Gs at the same site. The system varies only in the size of the lesion (with associated changes in hydrophobicity and electronic properties), and the results obtained with pol can be compared directly with corresponding studies done with HIV-1 RT and bacteriophage T7Ϫ [28] and two other human Y-family polymerases, pol [29] and pol [30]. Our results indicate that pol is resilient to blockage with the larger adducts, as judged by several criteria, and that incorporation opposite the adducts is as efficient as extension
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