title: Kinetics and binding of the thymine-DNA mismatch glycosylase, Mig-Mth, with mismatch-containing DNA substrates.
abstract: We have examined the removal of thymine residues from T-G mismatches in DNA by the thymine-DNA mismatch glycosylase from Methanobacterium thermoautrophicum (Mig-Mth), within the context of the base excision repair (BER) pathway, to investigate why this glycosylase has such low activity in vitro. Using single-turnover kinetics and steady-state kinetics, we calculated the catalytic and product dissociation rate constants for Mig-Mth, and determined that Mig-Mth is inhibited by product apyrimidinic (AP) sites in DNA. Electrophoretic mobility shift assays (EMSA) provide evidence that the specificity of product binding is dependent upon the base opposite the AP site. The binding of Mig-Mth to DNA containing the non-cleavable substrate analogue difluorotoluene (F) was also analyzed to determine the effect of the opposite base on Mig-Mth binding specificity for substrate-like duplex DNA. The results of these experiments support the idea that opposite strand interactions play roles in determining substrate specificity. Endonuclease IV, which cleaves AP sites in the next step of the BER pathway, was used to analyze the effect of product removal on the overall rate of thymine hydrolysis by Mig-Mth. Our results support the hypothesis that endonuclease IV increases the apparent activity of Mig-Mth significantly under steady-state conditions by preventing reassociation of enzyme to product.
authors.affiliation: Department of Biological Sciences, SUNY at Albany, Albany, NY 12222, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Begley
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Haas
authors.forename: Juan C
authors.initials: JC
authors.lastname: Morales
authors.forename: Eric T
authors.initials: ET
authors.lastname: Kool
authors.forename: Richard P
authors.initials: RP
authors.lastname: Cunningham
mesh_terms: D004247:DNA / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D007700:Kinetics; D017014:Methanobacterium / Q000201:enzymology / Q000235:genetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004247:DNA; D004720:Endonucleases; C445560:Mig.MthI DNA mismatch glycosylase; D009699:N-Glycosyl Hydrolases