Specificity of Human Thymine DNA Glycosylase Depends on N-Glycosidic Bond Stability

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• 作者：Matthew T. Bennett ; M. T. Rodgers ; Alexander S. Hebert ; Lindsay E. Ruslander ; Leslie Eisele ; Alexander C. Drohat
• 刊名：Journal of the American Chemical Society
• 出版年：2006
• 出版时间：September 27, 2006
• 年：2006
• 卷：128
• 期：38
• 页码：12510 - 12519
• 全文大小：217K
• 年卷期：v.128,no.38(September 27, 2006)
• ISSN：1520-5126

文摘

Initiating the DNA base excision repair pathway, DNA glycosylases find and hydrolytically excisedamaged bases from DNA. While some DNA glycosylases exhibit narrow specificity, others remove multipleforms of damage. Human thymine DNA glycosylase (hTDG) cleaves thymine from mutagenic G·T mispairs,recognizes many additional lesions, and has a strong preference for nucleobases paired with guaninerather than adenine. Yet, hTDG avoids cytosine, despite the million-fold excess of normal G·C pairs overG·T mispairs. The mechanism of this remarkable and essential specificity has remained obscure. Here,we examine the possibility that hTDG specificity depends on the stability of the scissile base-sugar bondby determining the maximal activity (k_max) against a series of nucleobases with varying leaving-group ability.We find that hTDG removes 5-fluorouracil 78-fold faster than uracil, and 5-chlorouracil, 572-fold fasterthan thymine, differences that can be attributed predominantly to leaving-group ability. Moreover, hTDGreadily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine,and 5-hydroxycytosine, indicating that cytosine has access to the active site. A plot of log(k_max) versusleaving-group pK_a reveals a Brimages/entities/oslash.gif">nsted-type linear free energy relationship with a large negative slope of images/gifchars/beta2.gif" BORDER=0 ALIGN="middle">_lg= -1.6 ± 0.2, consistent with a highly dissociative reaction mechanism. Further, we find that the hydrophobicactive site of hTDG contributes to its specificity by enhancing the inherent differences in substrate reactivity.Thus, hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine isdue largely to its very poor leaving ability rather than its exclusion from the active site.