Slupphaug G, Mol C D, Kavli B, Arvai A S, Krokan H E, Tainer J A
UNIGEN Center for Molecular Biology, The Norwegian University of Science and Technology, Trondheim, Norway.
Nature. 1996 Nov 7;384(6604):87-92. doi: 10.1038/384087a0.
Any uracil bases in DNA, a result of either misincorporation or deamination of cytosine, are removed by uracil-DNA glycosylase (UDG), one of the most efficient and specific of the base-excision DNA-repair enzymes. Crystal structures of human and viral UDGs complexed with free uracil have indicated that the enzyme binds an extrahelical uracil. Such binding of undamaged extrahelical bases has been seen in the structures of two bacterial methyltransferases and bacteriophage T4 endonuclease V. Here we characterize the DNA binding and kinetics of several engineered human UDG mutants and present the crystal structure of one of these, which to our knowledge represents the first structure of any eukaryotic DNA repair enzyme in complex with its damaged, target DNA. Electrostatic orientation along the UDG active site, insertion of an amino acid (residue 272) into the DNA through the minor groove, and compression of the DNA backbone flanking the uracil all result in the flipping-out of the damaged base from the DNA major groove, allowing specific recognition of its phosphate, deoxyribose and uracil moieties. Our structure thus provides a view of a productive complex specific for cleavage of uracil from DNA and also reveals the basis for the enzyme-assisted nucleotide flipping by this critical DNA-repair enzyme.
DNA中任何尿嘧啶碱基，无论是胞嘧啶错掺入还是脱氨的结果，都会被尿嘧啶-DNA糖基化酶（UDG）去除，UDG是碱基切除DNA修复酶中效率最高、特异性最强的酶之一。与游离尿嘧啶复合的人类和病毒UDG的晶体结构表明，该酶结合一个螺旋外尿嘧啶。在两种细菌甲基转移酶和噬菌体T4内切核酸酶V的结构中也观察到了未受损螺旋外碱基的这种结合。在此，我们对几种工程化人类UDG突变体的DNA结合和动力学进行了表征，并给出了其中一种突变体的晶体结构，据我们所知，这代表了任何真核DNA修复酶与其受损靶DNA复合的首个结构。沿着UDG活性位点的静电取向、通过小沟将一个氨基酸（第272位残基）插入DNA以及尿嘧啶侧翼DNA主链的压缩，都导致受损碱基从DNA大沟中翻转出来，从而使其磷酸、脱氧核糖和尿嘧啶部分得到特异性识别。因此，我们的结构展示了一种特异性切割DNA中尿嘧啶的有效复合物，并揭示了这种关键DNA修复酶协助核苷酸翻转的基础。
