The reaction mechanism of DNA glycosylase/AP lyases at abasic sites

DNA glycosylase and glycosylase/abasic (AP) lyases are the enzymes responsi ble for initiating the base excision repair pathway by recognizing the dama ged 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 intermedia te. Two distinct mechanisms have been proposed for the formation of this in termediate. 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 an d reduction of the sugar is significantly slower than the rate of formation of a T4-pyrimidine dimer glycosylase (T4-pdg)-DNA intermediate. Using a me thyl-deoxyribofuranose AP-site analogue that is incapable of undergoing sug ar 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 prefe rentially 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 deoxyrib ofuranose ring. In addition, studies examining rates of sugar-aldehyde redu ction and the sodium borohydride concentration dependence of the rate of fo rmation of the covalent imine intermediate suggest that the reduction of th e intermediate is rate-limiting in the reaction.