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Structural basis for the excision repair of DNA glycosylse superfamily members

DNA糖基化超家族成员切除修复的结构基础

基本信息

批准号：
11672141
负责人：
YAMAGATA Yuriko
金额：
$ 2.3万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672141/
关键词：
DNA repair enzyme DNA glycosylase superfamily High resolution AlkA structure Enzymatic activity change Crystallizaion of protein-DNA complex DNA-蛋白質複合体の結晶化高分解能X線結晶構造解析 DNA修復 X線結晶構造解析 AIKA-DNA複合体

项目摘要

The integrity of the DNA is constantly challenged by a number of endogenous and environmental DNA-damaging agents. DNA glycosylases that recognize damaged bases and remove them by cleaving the N-glycosidic bond linking the base to deoxyribose are key enzymes in the base excision repair pathway.We previously determined the crystal structure of E.coli 3-methyladenine DNA glycosylase II (AlkA) which removes numerous alkylated bases from DNA at 2.3 Å resolution. The enzyme consists of three domains : one α+β fold domain with a similarity to one-half of the eukaryotic TATA box-binding protein, and two all helical domains similar to those of E.coli endonuclease III (EndoIII) with combined N-glycosylase/abasic (AP) lyase activity. The crystal structure and mutational studies revealed that the active site is located in the cleft between the two helical domains and that Asp288 is an essential catalytic residue in the N-glycosylase reaction. In this project we newly determined the structure at 1 … More .5 Å resolution. This structure reveals the accurate side chain conformations and hydration structures, of which the hydration detail in the active site is important for enzyme activity.The findings of the structural similarity between AlkA and EndoIII and many hypothetical proteins with similar sequences to them in genomes indicated the existence of a DNA glycosylase superfamily. The members possess a common fold, yet act upon remarkably diverse lesions. Some members are monofunctional and the others have DNA glycosylase/AP lyase activities. Our research purpose is to understand the structural basis for the recognition and removal of damaged bases from DNA by members of the DNA glycosylase superfamily. The three-dimensional structure-based sequence alignment among members of the superfamily shows the presence or absence of Lys in the active site (especially at the corresponding position to 218 of AlkA) is predictive of the presence or absence of an associated AP lyase activity in the members, respectively. Actually we have found that the replacement of Trp218 of AlkA by Lys results in enzymes that have AP lyase activity, in which Lys218 acts as a catalytic amine through the formation of an iminium ion intermediate (Schiff base). The appearance of date altered activity between the wild-type and mutant proteins suggests that the active-site structure of AlkA is much tolerant of different enzymatic activities.Now we are trying to crystallize the AlkA-DNA oligomer and AlkA-methylpurine nucleosidecomplexes. Less

DNA的完整性不断受到许多内源性和环境DNA损伤剂的挑战。DNA糖基化酶是碱基切除修复途径中的关键酶，它能识别受损碱基并通过切断连接碱基和脱氧核糖的N-糖苷键将其去除，我们以前测定了大肠杆菌3-甲基腺嘌呤DNA糖基化酶II（AlkA）的晶体结构，它能在2.3 nm分辨率下去除DNA中大量的烷基化碱基。该酶由三个结构域组成：一个α+β折叠结构域与真核生物TATA盒结合蛋白的一半相似，两个全螺旋结构域与大肠杆菌内切核酸酶III（EndoIII）的结构域相似，具有N-糖基化酶/脱碱基（AP）裂解酶活性。晶体结构和突变的研究表明，活性位点位于两个螺旋结构域之间的裂缝和Asp 288是在N-糖基化酶反应中的一个必不可少的催化残基。在这个项目中，我们重新确定了1 ...更多信息 0.5英寸分辨率。这种结构揭示了准确的侧链构象和水合结构，其中活性位点的水合细节对酶活性是重要的。AlkA和EndoIII与基因组中与它们序列相似的许多假设蛋白质之间的结构相似性的发现表明DNA糖基化酶超家族的存在。这些成员有一个共同的褶皱，但作用于明显不同的病变。一些成员是单功能的，而另一些则具有DNA糖基化酶/AP裂解酶活性。我们的研究目的是了解DNA糖基化酶超家族成员识别和去除DNA中受损碱基的结构基础。基于三维结构的序列比对表明，在活性位点（特别是在AlkA的218位对应位置）中存在或不存在Lys分别预测了成员中相关AP裂解酶活性的存在或不存在。实际上，我们已经发现，用Lys取代AlkA的Trp 218导致具有AP裂解酶活性的酶，其中Lys 218通过形成亚胺离子中间体（席夫碱）而充当催化胺。AlkA的活性中心结构对不同的酶活性有很大的耐受性，因此我们正在尝试将AlkA-DNA寡聚体和AlkA-甲基嘌呤核苷解复合物结晶化。少