Translesion Synthesis across O6-Alkylguanine DNA Adducts by Recombinant Human DNA Polymerases

Abstract

Chemical and physical damage to DNA can cause mutations and ultimately cancer, cardiovascular disease, aging, and other diseases (1-4). This damage can result from endogenous agents or exogenous chemicals. Many types of damage are known, and the biological effects can vary considerably. One of the prominent types of damage is alkylation at the O-6 atom of guanine (5, 6). O 6 -AlkylG 4 lesions are some of the more mutagenic lesions formed from DNA-alkylating agents (5, 7). The ability of O 6 -alkylG adducts to cause mutations has been demonstrated directly in site-specific mutagenesis experiments with defined adducts (8 -12). Further support for the view that these are deleterious species derives from the existence of specific DNA repair systems for this type of damage in almost all species, ranging from most bacteria to humans (13,14).Different alkylating agents form O 6 -alkylG adducts, and structure-activity relationships are important for understanding the basic mechanisms of how DNA polymerases function as well as issues such as carcinogenesis. Ϫ and HIV-1 RT (16) and by others with several DNA polymerases, mostly bacterial (7,17,18). Some of the more significant conclusions with pol T7Ϫ and HIV-1 RT were that the bulk of the adduct at the O-6 atom had an inhibitory effect and that an inactive polymerase-oligonucleotide complex is in equilibrium with the functional form (16,19).Studies with pol T7 Ϫ and HIV-1 RT indicated that the effect of size at the N-2 atom is more severe than at the O-6 atom (16, * This work was supported in part by United States Public Health Service Grants R01 CA059887, R01 CA115309 (to L. A. P.), R01 ES010375, and P30 ES000267 (to F. P. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains a MALDI-TOF mass spectrum and capillary gel electrophoresis analysis of the O 6 -PobG 36-mer, an electrophoretic gel showing the lack of extension of an O 6 -PobG:G mispair by pol , and mass spectral analyses used to obtain the results shown in Fig. 7 4 The generic term "alkyl" is used to include both alkyl and aralkyl (Bz) groups for convenience. 5 The abbreviations used are: Bz, benzyl; CID, collision-induced dissociation; dCTP␣S, 2Ј-deoxycytidine 5Ј-O-(1-thiotriphosphate); DTT, dithiothreitol; ESI, electrospray ionization; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; MS, mass spectrometry; Pob, 4-oxo-4-(3-pyridyl)butyl; PCNA, proliferating cell nuclear antigen; pol, (DNA) polymerase; pol T7 Ϫ , bacteriophage pol T7 exonuclease-deficient; RT, reverse transcriptase; UDG, uracil DNA glycosylase; HIV-1, human immunodeficiency virus, type 1.

Bypass of Mutagenic O6-Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases

“…This low primer extension capacity is consistent with previous observations for Pol ι bypassing adducts such as O 6 -MeG, O 6 -BnG, 3-methylcytosine, T-T dimers or an abasic site. 24,46,47 Despite the limited activity of Pol ι, it could be shown that the enzyme displayed a preference for incorporating dTMP over dCMP opposite O 6 -CMG, which corresponds as well with previous observations for the bypass of O 6 -alkylG adducts by Pol ι. 24,48 30,31 However, the latter is derived from a structure of the free duplex and does not account for the important influences of the enzyme.…”

“…The extension efficiency from the C:O 6 -CMG pair was around 4.5-fold higher compared to incorporation of dCMP opposite O 6 -CMG, but was in the same range as previously reported for the extension from O 6 -MeG. 24,45 These data indicate a potentially important miscoding difference between carboxymethyl and other O 6 -alkyl-G adducts, whereby the carboxyl group avoids interfering with the correct insertion of dCMP by Pol κ.…”

“…A potential explanation for this observation might be the known property of Pol κ to create -1 frameshift mutations. 24,39 The visualized elongation band might therefore still resemble the correct full-length extension but after slippage of Pol κ during the synthesis.…”

“…In this process, TLS Pols are deployed to overcome blockages, protecting cells from acute toxicity however at the possible cost of base misincorporation and genomic instability. [40][41][42][43] The potential for base misincorporation during DNA replication by a variety of human Pols in bypassing O 6 -alkylG adducts has been described; 24 however the presence of O 6 -CMG in the template has never been examined for human Y-and B-family polymerases. Herein, we examined the chemical basis of this process by characterizing the propensity of four human TLS Pols, Pol η, κ, ι and ζ vs the replicative Pol δ, to catalyze the bypass and extension past an O 6 -CMG adduct with primer extension and steady-state kinetics studies.…”

“…[24][25][26] These O 6 -alkylguanine adducts are relevant in terms of endogenous methylation, tobacco nitrosamine exposure, and the model hepatocarcinogen methylbenzylnitrosamine. In comparing these previously characterized O 6 -alkylG TLS substrates, the larger the alkyl group on the O 6 -position of guanine, the greater the reduction in bypass capacity of the human enzymes.…”