The reaction mechanism of DNA glycosylase/AP lyases at abasic sites.
DNA glycosylase and glycosylase/abasic (AP) lyases are the enzymes responsible for initiating the base excision repair pathway by recognizing the damaged target base and catalyzing the breakage of the base-sugar glycosyl bond. The subset of glycosylases that have an associated AP lyase activity also catalyze DNA strand breakage at the resulting or preexisting AP site via a beta-elimination reaction, proceeding from an enzyme-DNA imino intermediate. Two distinct mechanisms have been proposed for the formation of this intermediate. These mechanisms essentially differ in the nature of the first bond broken and the timing of the opening of the deoxyribose ring. The data presented here demonstrate that the combined rate of sugar ring opening and reduction of the sugar is significantly slower than the rate of formation of a T4-pyrimidine dimer glycosylase (T4-pdg)-DNA intermediate. Using a methyl-deoxyribofuranose AP-site analogue that is incapable of undergoing sugar ring opening, it was demonstrated that the T4-pdg reaction can initiate at the ring-closed form, albeit at a drastically reduced rate. T4-pdg preferentially cleaved the beta-anomer of the methyl-deoxyribofuranose AP site analogue. This is consistent with a mechanism in which the methoxy group is backside-displaced by the amino group from the alpha-face of the deoxyribofuranose ring. In addition, studies examining rates of sugar-aldehyde reduction and the sodium borohydride concentration dependence of the rate of formation of the covalent imine intermediate suggest that the reduction of the intermediate is rate-limiting in the reaction.
McCullough
A K
AK
Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1071, USA.
Sanchez
A
A
Dodson
M L
ML
Marapaka
P
P
Taylor
J S
JS
Lloyd
R S
RS
D017122:Bacteriophage T4 / Q000201:enzymology; D001894:Borohydrides / Q000737:chemistry; D019757:Carbon-Oxygen Lyases / Q000737:chemistry*; D045647:DNA Glycosylases; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D003853:Deoxyribonucleosides / Q000138:chemical synthesis; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D046911:Macromolecular Substances; D009699:N-Glycosyl Hydrolases / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012545:Schiff Bases / Q000737:chemistry; D013873:Thionucleotides / Q000138:chemical synthesis
D001894:Borohydrides; D003853:Deoxyribonucleosides; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D012545:Schiff Bases; D013873:Thionucleotides; C025364:sodium borohydride; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C036401:deoxyribopyrimidine endonucleosidase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase