title: Coordinating the initial steps of base excision repair. Apurinic/apyrimidinic endonuclease 1 actively stimulates thymine DNA glycosylase by disrupting the product complex.
abstract: DNA glycosylases initiate base excision repair by removing damaged or mismatched bases, producing apurinic/apyrimidinic (AP) DNA. For many glycosylases, the AP-DNA remains tightly bound, impeding enzymatic turnover. A prominent example is thymine DNA glycosylase (TDG), which removes T from G.T mispairs and recognizes other lesions, with specificity for damage at CpG dinucleotides. TDG turnover is very slow; its activity appears to reach a plateau as the [product]/[enzyme] ratio approaches unity. The follow-on base excision repair enzyme, AP endonuclease 1 (APE1), stimulates the turnover of TDG and other glycosylases, involving a mechanism that remains largely unknown. We examined the catalytic activity of human TDG (hTDG), alone and with human APE1 (hAPE1), using pre-steady-state kinetics and a coupled-enzyme (hTDG-hAPE1) fluorescence assay. hTDG turnover is exceedingly slow for G.T (k(cat)=0.00034 min(-1)) and G.U (k(cat)=0.005 min(-1)) substrates, much slower than k(max) from single turnover experiments, confirming that AP-DNA release is rate-limiting. We find unexpectedly large differences in k(cat) for G.T, G.U, and G.FU substrates, indicating the excised base remains trapped in the product complex by AP-DNA. hAPE1 increases hTDG turnover by 42- and 26-fold for G.T and G.U substrates, the first quantitative measure of the effect of hAPE1. hAPE1 stimulates hTDG by disrupting the product complex rather than merely depleting (endonucleolytically) the AP-DNA. The enhancement is greater for hTDG catalytic core (residues 111-308 of 410), indicating the N- and C-terminal domains are dispensable for stimulatory interactions with hAPE1. Potential mechanisms for hAPE1 disruption of the of hTDG product complex are discussed.
authors.affiliation: Department of Biochemistry and Molecular Biology, Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
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; D001483:Base Sequence; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D025842:SUMO-1 Protein / Q000378:metabolism; D044502:Thymine DNA Glycosylase / Q000378:metabolism*; D013997:Time Factors
chemical_list: D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D044502:Thymine DNA Glycosylase; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase