Metal inhibition of human DNA glycosylases in the base excision repair pathway Free

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Occupational and environmental exposure to toxic metals and their compounds has been associated with increased human cancer risks. Cadmium (Cd(II)), nickel (Ni(II)) and lead (Pb(II)) are classified human and/or animal carcinogens, while zinc (Zn(II)) is an essential element to humans. Cobalt (Co(II)) is listed as a possible human carcinogen but is also a central component of the vitamin B-12. Metals were recently shown to inhibit several DNA repair proteins that utilize metal for their function and/or structure. However, we found that in vitro Cd(II), Ni(II), Pb(II) and Zn(II) can inhibit, albeit with different specificity and efficiency, activities of several human DMA glycosylases that do not require metal for their activity or structural integrity. These include N-methylpurine-DNA glycosylase (MPG), uracil-DNA glycosylase (UNG), thymine-DNA glycosylase (TDG) and single-strand selective monofunctional uracil-DNA glycosylase (SMUG1). Each of these enzymes recognizes and excises a subgroup of specific DNA base lesions and initiates the base excision repair pathway. An in vitro enzymatic assay was performed utilizing as substrate an oligonucleotide duplex that contains a single base modification. The MPG activity was measured against an ethenoadenine adduct and a uracil:guanine mismatch was tested for excision by the three enzymes that all act on uracil: UNG, TDG and SMUG1. Inhibition studies were carried out in the presence of metal ions at micromolar concentrations. Experiments using HeLa cell-free extracts were also performed to demonstrate potential metal inactivation of excision activity for ethenoadenine or uracil in these cell lysates. Metal ions were shown to be specific in inhibiting the activity of the DNA glycosylases studied. For example, both Cd(II) and Zn(II) showed dose-dependent and EDTA-reversible inhibition of the MPG catalytic activity. Ni(II) also inhibited MPG, but to a lesser extent. In contrast, divalent ions Co(II) and Mg(II) did not inhibit MPG activity. Metals were also shown to have different efficiencies in inhibition. For the three uracil-excising glycosylases, at a concentration range starting from 25 to 250 μM, Cd(II) is a potent inhibitor to all three enzymes. Zn(II) is also a potent inhibitor to TDG and SMUG1, but to a lesser extent to UDG. Both Ni(II) and Pb(II) showed moderate inhibition of UDG and TDG activity, but efficient inhibition of the SMUG1 activity. Mechanistic studies using molecular dynamics simulations with Zn(II) as a model ion showed that the MPG active site has a potential binding site for Zn(II), 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. Taken together, these data suggest that metal inhibition of non-metal dependent enzymes such as the four DNA glycosylases studied in this work can be an important mechanism contributing to metal genotoxicity.