title: Repair of thymine glycol by hNth1 and hNeil1 is modulated by base pairing and cis-trans epimerization.
abstract: Oxidation of thymine yields 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol. Tg) which, as cis 5S,6R and 5R,6S 2'-deoxyribonucleoside diastereoisomers (dTg1, dTg2), are in equilibrium with their trans 5S,6S and 5R,6R epimers. The stereoselective excision of Tg from DNA by the mammalian orthologs of E. coli DNA N-glycosylase/AP lyases Nth and Nei was reported using substrates in which Tg opposed adenine. Since we showed that Tg is the major product of oxidation of 5-methylcytosine, we asked if the opposing purine influenced stereospecific enzymatic excision. The human ortholog hNth1 released Tg2 much more rapidly than Tg1 regardless of the opposing purine. In contrast, hNeil1 released Tg non-stereoselectively, but the rate of excision was much greater when Tg opposed guanine. Remarkably, the kinetics of excision of Tg by hNth1 and hNeil1 were biphasic, describing a double exponential curve which yielded two rate constants. We suggest that the greater rate constant describes the rate of enzymatic excision of Tg. The smaller rate constant represents the equilibrium constant for the cis and trans epimerization of dTg1 and dTg2 in high molecular weight DNA. Thus, only one of the epimers of dTg1 and dTg2 are enzymatically processed but it is not yet known whether it is cis or trans. Thus, base excision repair of Tg in mammals is mediated by at least two DNA N-glycosylase/AP lyases which are affected by the nature of the diastereoisomer of dTg, the rate of cis-trans epimerization of each diastereoisomer, and the nature of the opposing purine.
authors.affiliation: Department of Pathology, New York University Medical Center, New York, NY 10016, USA.
authors.forename: Maria T
authors.initials: MT
authors.lastname: Ocampo-Hafalla
authors.forename: Alvin
authors.initials: A
authors.lastname: Altamirano
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Basu
authors.forename: Michael K
authors.initials: MK
authors.lastname: Chan
authors.forename: Jose Eliseo A
authors.initials: JE
authors.lastname: Ocampo
authors.forename: Archie
authors.initials: A
authors.lastname: Cummings
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Boorstein
authors.forename: Richard P
authors.initials: RP
authors.lastname: Cunningham
authors.forename: George W
authors.initials: GW
authors.lastname: Teebor
mesh_terms: D020029:Base Pairing; D002384:Catalysis; D004249:DNA Damage; D045647:DNA Glycosylases / Q000378:metabolism*; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism*; D003855:Deoxyribose / Q000138:chemical synthesis; D029968:Escherichia coli Proteins / Q000378:metabolism; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D009841:Oligonucleotides / Q000138:chemical synthesis; D011687:Purines / Q000378:metabolism; D013379:Substrate Specificity; D013941:Thymine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D011687:Purines; C029389:thymine glycol; D003855:Deoxyribose; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; C468347:NTHL1 protein, human; D045647:DNA Glycosylases; C473634:NEIL1 protein, human; D013941:Thymine