A Covalent Protein-DNA 5'-Product Adduct is Generated Following AP Lyase Activity of Human AlkB Homolog 1 (ALKBH1)

ALKBH1 is a mammalian AlkB homolog that possesses abasic site (AP) lyase activity. The AP lyase reaction is catalyzed by imine formation with an active site Lys, and a covalent intermediate can be trapped in the presence of NaBH4. Surprisingly, ALKBH1 also forms a stable protein-DNA adduct in the absence of a reducing agent. Experiments with different substrates demonstrated that the protein covalently binds to the 5' DNA product; i.e., the fragment containing an α,β-unsaturated aldehyde. The amino terminal domain of ALKBH1 was identified as the main site of linkage with DNA. By contrast, mutagenesis studies suggest that the primary catalytic residue forming the imine linkage is Lys133, with Lys154 and other Lys residues in this region serving in opportunistic roles. These findings confirm the classification of ALKBH1 as an AP lyase, identify the primary and a secondary Lys involved in the lyase reaction, and demonstrate the protein forms a covalent adduct with the 5' DNA product. We propose two plausible chemical mechanisms to account for the covalent attachment.
Source: BJ Gene - Category: Biochemistry Authors: Tags: BJ Gene Source Type: research