title: Stoichiometry and affinity for thymine DNA glycosylase binding to specific and nonspecific DNA.
abstract: Deamination of 5-methylcytosine to thymine creates mutagenic G · T mispairs, contributing to cancer and genetic disease. Thymine DNA glycosylase (TDG) removes thymine from these G · T lesions, and follow-on base excision repair yields a G · C pair. A previous crystal structure revealed TDG (catalytic domain) bound to abasic DNA product in a 2:1 complex, one subunit at the abasic site and the other bound to undamaged DNA. Biochemical studies showed TDG can bind abasic DNA with 1:1 or 2:1 stoichiometry, but the dissociation constants were unknown, as was the stoichiometry and affinity for binding substrates and undamaged DNA. We showed that 2:1 binding is dispensable for G · U activity, but its role in G · T repair was unknown. Using equilibrium binding anisotropy experiments, we show that a single TDG subunit binds very tightly to G · U mispairs and abasic (G · AP) sites, and somewhat less tightly G · T mispairs. Kinetics experiments show 1:1 binding provides full G · T activity. TDG binds undamaged CpG sites with remarkable affinity, modestly weaker than G · T mispairs, and exhibits substantial affinity for nonspecific DNA. While 2:1 binding is observed for large excess TDG concentrations, our findings indicate that a single TDG subunit is fully capable of locating and processing G · U or G · T lesions.
authors.affiliation: Department of Biochemistry and Molecular Biology and Greenebaum Cancer Center, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Morgan
authors.forename: Atanu
authors.initials: A
authors.lastname: Maiti
authors.forename: Megan E
authors.initials: ME
authors.lastname: Fitzgerald
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Drohat
mesh_terms: D020137:Base Pair Mismatch; D018899:CpG Islands; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D007700:Kinetics; D011485:Protein Binding; D044502:Thymine DNA Glycosylase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004247:DNA; D044502:Thymine DNA Glycosylase