Structural basis for the excision repair of DNA glycosylse superfamily members

DNA的完整性不断受到一些内源性和环境中的DNA损伤剂的挑战。脱氧核糖基酶识别受损的碱基并通过裂解连接碱基和脱氧核糖的N-糖苷键来移除它们，是碱基切除修复途径中的关键酶。我们以前确定了大肠杆菌3-甲基腺嘌呤脱氧核糖基酶II(ALKA)的晶体结构，它以2.3Å的分辨率从DNA中去除大量的烷基化碱基。该酶由3个结构域组成：1个α+β折叠结构域，与真核细胞TATA盒结合蛋白的一半相似；2个全螺旋结构域，类似于大肠杆菌内切酶III(EndoIII)的结构域，具有N-糖基酶/碱性(AP)裂解酶的结合活性。晶体结构和突变研究表明，活性中心位于两个螺旋结构域之间的空隙中，Asp288是N-糖基酶反应中必不可少的催化残基。在这个项目中，我们新确定了1…的结构更高的0.5Å分辨率。这种结构揭示了准确的侧链构象和水化结构，其中活性部位的水化细节对酶的活性至关重要。Alka和EndoIII与基因组中许多序列相似的假想蛋白质的结构相似性表明，存在一个DNA糖基化酶超家族。这些成员拥有共同的褶皱，但作用于显著不同的病变。一些成员是单功能的，另一些成员具有DNA糖基酶/AP裂解酶活性。我们的研究目的是了解DNA糖基酶超家族成员识别和去除DNA中受损碱基的结构基础。超家族成员之间基于三维结构的序列比对表明，活性部位(特别是在与Alka的218的对应位置)存在或不存在Lys分别预测成员中相关AP裂解酶活性的存在或不存在。事实上，我们已经发现，Lys取代Alka的Trp218产生了具有AP裂解酶活性的酶，其中Lys218通过形成亚胺离子中间体(Schiff碱)作为催化胺。野生型和突变型蛋白之间活性日期变化的出现表明ALKA的活性中心结构对不同的酶活性具有很强的耐受性。现在，我们正在尝试结晶ALKA-DNA寡聚体和ALKA-甲基嘌呤核苷络合物。较少

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