The domains of mammalian base excision repair enzyme N-methylpurine-DNA glycosylase. Interaction, conformational change, and role in DNA binding and damage recognition.
Repair of a variety of alkylated base adducts in DNA is initiated by their removal by N-methylpurine-DNA glycosylase. The 31-kDa mouse N-methylpurine-DNA glycosylase, derived by deletion of 48 amino acid residues from the 333-residue wild type protein without loss of activity, was analyzed for the presence of protease-resistant domains with specific roles in substrate binding and catalysis. Increasing proteolysis with trypsin generated first a 29-kDa polypeptide by removal of 42 amino-terminal residues, followed by production of 8-, 6-, and 13-kDa fragments with defined, nonoverlapping boundaries. The 8- and 13-kDa domains include the amino and carboxyl termini, respectively. Based on DNA-affinity chromatography and the protease protection assay, it appears that the 6- 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. Furthermore, chemical cross-linking studies indicated that, in the presence of substrate DNA, the 8- and 6-kDa domains undergo conformational changes reflected by both protection from proteolysis and reduced availability of cysteine residues for the thiol-exchange reaction.
Roy
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Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Kumar
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A
Lee
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JC
Mitra
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D000595:Amino Acid Sequence; D000818:Animals; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D005091:Exons; D005796:Genes; D051379:Mice; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D010446:Peptide Fragments; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases