title: Active-site determination of a pyrimidine dimer glycosylase.
abstract: One mechanism for the repair of UV-induced DNA damage is the base excision repair pathway. The initial step in this pathway and the specificity for the type of damage that is to be repaired reside in DNA glycosylase/abasic (AP) lyases. Cleavage of the glycosyl bond of the 5' pyrimidine of a cyclobutane pyrimidine dimer is hypothesized to occur through the destabilization of the glycosyl bond by protonation of the base or sugar with a concomitant nucleophilic attack on C1' of the deoxyribose moiety. Based on mechanistic biochemical information from several glycosylase/AP lyases and the structural information on the bacteriophage T4 pyrimidine dimer glycosylase (T4-pdg), the catalytic mechanism has been investigated for the Chlorella virus pyrimidine dimer glycosylase (cv-pdg). As predicted from modeling studies and reaction mechanisms, the primary amine that initiates the nucleophilic displacement reaction could be trapped as a covalent imine intermediate and its identity determined by sequential Edman degradation. The primary amine was identified as the alpha-amino group on the N-terminal Thr2. Site-directed mutagenesis was subsequently used to confirm the conclusions that the alpha-amino group of cv-pdg is the active-site nucleophile.
authors.affiliation: Department of Microbiology, University of Texas Medical Branch, Galveston, TX 77555-1071, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Garvish
authors.forename: R S
authors.initials: RS
authors.lastname: Lloyd
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002708:Chlorella / Q000821:virology; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011740:Pyrimidine Dimers
chemical_list: D017931:DNA Primers; D011740:Pyrimidine Dimers; D004247:DNA; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)