The Reaction Mechanism of DNA Glycosylase/AP Lyases at Abasic Sites

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• 作者：Amanda K. McCullough ; Ana Sanchez ; M. L. Dodson ; Praveen Marapaka ; John-Stephen Taylor ; and R. Stephen Lloyd
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：January 16, 2001
• 年：2001
• 卷：40
• 期：2
• 页码：561 - 568
• 全文大小：170K
• 年卷期：v.40,no.2(January 16, 2001)
• ISSN：1520-4995

文摘

DNA glycosylase and glycosylase/abasic (AP) lyases are the enzymes responsible for initiatingthe base excision repair pathway by recognizing the damaged target base and catalyzing the breakage ofthe base-sugar glycosyl bond. The subset of glycosylases that have an associated AP lyase activity alsocatalyze DNA strand breakage at the resulting or preexisting AP site via a mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-elimination reaction, proceedingfrom an enzyme-DNA imino intermediate. Two distinct mechanisms have been proposed for the formationof this intermediate. These mechanisms essentially differ in the nature of the first bond broken and thetiming of the opening of the deoxyribose ring. The data presented here demonstrate that the combinedrate of sugar ring opening and reduction of the sugar is significantly slower than the rate of formation ofa 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-pdgreaction can initiate at the ring-closed form, albeit at a drastically reduced rate. T4-pdg preferentiallycleaved the mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-anomer of the methyl-deoxyribofuranose AP site analogue. This is consistent with amechanism in which the methoxy group is backside-displaced by the amino group from the mages/gifchars/alpha.gif" BORDER=0>-face of thedeoxyribofuranose ring. In addition, studies examining rates of sugar-aldehyde reduction and the sodiumborohydride concentration dependence of the rate of formation of the covalent imine intermediate suggestthat the reduction of the intermediate is rate-limiting in the reaction.