The domains of mammalian base excision repair enzyme w-methylpurine-dna glycosylase: Interaction, conformational change and role in dna-binding and damage recognition

The mouse N-methylpurine-DNA glycosylase (MPG) removes N-alkylpurines, ethenoadenine, and 8-hydroxyguanine from DNA. Analysis of polypeptide fragments generated by controlled proteolysis with various proteases shows that the protein is organized as three non-overlapping domains of molecular masses 8 kDa (at amino terminus), 6 kDa, and 13 kDa (at carboxyl terminus). Based on DNA-cellulose chromatography and the protease protection assay, it appears that the 6 kDa and 13 kDa domains are necessary for nontarget DNA binding, and that the 8 kDa domain, in cooperation with the other two domains, participates in recognition of damaged bases. Another approach, using PCR mutagenesis, also shows that the minimal sequence needed for substrate recognition includes the 6 kDa domain plus most of the 8 kDa and nearly half of the 13 kDa domains. Furthermore, chemical crosslinking and protease protection studies indicate that in the presence of substrate DNA, the 8 kDa and 6 kDa domains undergo con-formational changes as is also evident from the reduced availability of cysteine residues for thiol exchange reaction.