Department of Biochemistry & Molecular Biology
نویسندگان
چکیده
Alkbh1 is a human homolog of the Escherichia coli AlkB protein that directs DNA repair by removing alkylation damage to DNA bases. While the in vivo role of Alkbh1 is still unknown, the protein was recently shown to possess abasic site lyase activity in vitro and to cleave abasic sites according to a betaelimination mechanism. Surprisingly, Alkbh1 forms a covalent adduct to the 5?-DNA product. In this project, different Alkbh1 variants were characterized, focusing on residues that may form the protein-DNA adduct as well as several amino acids which are predicted to bind metal ions. Site-directed mutagenesis was used to make variant proteins which differ in one or more amino acids from the wild type protein, with expression in E. coli and purification by affinity chromatography. Assays were carried out to investigate whether adduct formation and AP lyase activity differed from the wild type enzyme. This approach has begun to provide us with additional knowledge about key amino acids in Alkbh1, with the hope of identifying residues critical to adduct formation. Thus far, a series of variants of putative zinc-finger residues and selected other potential amino acids involved in adduct formation were shown to not affect adduct formation.
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تاریخ انتشار 2014