Structural basis for the excision repair of DNA glycosylse superfamily members

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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