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Title: Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair

Abstract

Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations thatmore » show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.« less

Authors:
; ;
Publication Date:
Research Org.:
Ernest Orlando Lawrence Berkeley NationalLaboratory, Berkeley, CA (US)
Sponsoring Org.:
USDOE Director, Office of Science; National Institutes ofHealth
OSTI Identifier:
919754
Report Number(s):
LBNL-59790
Journal ID: ISSN 0378-4274; TOLED5; R&D Project: 865B1C; BnR: 400412000; TRN: US200822%%518
DOE Contract Number:
DE-AC02-05CH11231; NIHCA72079
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology Letters; Journal Volume: 166; Journal Issue: 3; Related Information: Journal Publication Date: 10/25/2006
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; DNA REPAIR; EXCISION REPAIR; IN VIVO; REPAIR; INHIBITION; CARCINOGENS; ALKYLATION; DAMAGE

Citation Formats

Wang, Ping, Guliaev, Anton B., and Hang, Bo. Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair. United States: N. p., 2006. Web. doi:10.1016/j.toxlet.2006.06.647.
Wang, Ping, Guliaev, Anton B., & Hang, Bo. Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair. United States. doi:10.1016/j.toxlet.2006.06.647.
Wang, Ping, Guliaev, Anton B., and Hang, Bo. Tue . "Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair". United States. doi:10.1016/j.toxlet.2006.06.647. https://www.osti.gov/servlets/purl/919754.
@article{osti_919754,
title = {Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair},
author = {Wang, Ping and Guliaev, Anton B. and Hang, Bo},
abstractNote = {Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.},
doi = {10.1016/j.toxlet.2006.06.647},
journal = {Toxicology Letters},
number = 3,
volume = 166,
place = {United States},
year = {Tue Feb 28 00:00:00 EST 2006},
month = {Tue Feb 28 00:00:00 EST 2006}
}
  • DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct frommore » that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.« less
  • Oxidative damage is emerging as one of the most important mechanisms responsible for mutagenesis, carcinogenesis, aging, and various diseases (Farr and Kogma, 1991). One of the potential targets for oxidation is cellular DNA. While exposure to exogenous agents, such as ionizing radiation and chemicals, contributes to damaging DNA, the most important oxidative agents are endogenous, such as the reactive free radicals produced during normal oxidative metabolism (Adelman et., 1988). To mitigate the potentially deleterious effects of oxidative DNA damage virtually all aerobic organisms have developed complex repair mechanisms (Petit and Sancar, 1999). One repair mechanism, base excision repair (BER), appearsmore » to be responsible for replacing most oxidative DNA damage (David and Williams, 1998). Formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa, is a key BER enzyme in prokaryotes (Boiteaux et al., 1987). Substrates recognized and released by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6 diamino-4-hydroxy-5-formamido pyrimidine (Fapy-G), the adenine equivalents 8-oxoA and Fapy-A, 5-hydroxycytosine, 5-hydroxyuracil, B ureidoisobutiric acid, and a-R-hydroxy-B-ureidoisobutiric acid (Freidberg et al., 1995). In vitro Fpg bind double-stranded DNA and performs three catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase.« less
  • We have tested the hypothesis that the inhibition by hydroxyurea of repair patch ligation and chromatin rearrangement during u.v.-induced DNA excision repair results from a reduction in cellular deoxyribonucleotide concentrations and not from a direct effect of hydroxyurea on the repair process. Using permeable human fibroblasts, we have shown that hydroxyurea has no direct effect on either repair synthesis or repair patch ligation. We also have shown that by reducing the deoxyribonucleoside triphosphate concentrations in the permeable cell reaction mixture, we can mimic the inhibition of repair patch ligation and chromatin rearrangement seen when u.v.-damaged intact confluent fibroblasts are treatedmore » with hydroxyurea. Our results are consistent with the concept that hydroxyurea inhibits DNA repair in intact cells by inhibiting deoxyribonucleotide synthesis through its effect on ribonucleotide reductase and, conversely, that continued deoxyribonucleotide synthesis is required for the excision repair of u.v.-induced DNA damage even in resting cells.« less
  • The antineoplastic agent arabinofuranosyl cytosine (ara-C) produces an inhibition of the pyrimidine dimer excision system of human DNA repair. Alkaline sucrose gradient analysis of DNA from normal human skin fibroblasts exposed to 20 J/m/sup 2/ of ultraviolet radiation (254 nm) shows an accumulation of DNA single-strand breaks when DNA repair is attempted in the presence of 10 ..mu..M ara-C. Cells from complementaion groups of xeroderma pigmentosum that are defective in early steps of excision repair show reduced numbers of DNA single strand breaks/10/sup 8/ daltons when compared with normal cells. Cesium chloride gradient analysis of radioactive precurser uptake during repairmore » replication indicates that ara-C causes a 6 to 56% reduction in the number of nucleotide bases inserted into the DNA at concentrations of 1 and 10 ..mu..M, respectively. These concentrations result in the substitution for deoxycytidine (dCyd) by ara-C of 40 and 100%, respectively, in repaired regions. Repair inhibition is reversed by 50% upon removal of ara-C and by > 95% with the addition of 100 ..mu..M dCyd. Chromatography of digested DNA shows that incorporated ara-C is not removed during dCyd reversal, suggesting that ara-C incorporation per se does not play a significant role in the inhibition of repair synthesis. The repair inhibition observed here is dependent on 2 mM hydroxyurea, presumably due to reduction in the intracellular pool of dCyd. The overall results suggest the possibility that ara-C is a weak competitive inhibitor of DNA polymerases associated with ultraviolet-induced excision repair.« less