Interaction of the recombinant human methylpurine-DNA glycosylase (MPG protein) with oligodeoxyribonucleotides containing either hypoxanthine or abasic sites.
Methylpurine-DNA glycosylases (MPG proteins, 3-methyladenine-DNA glycosylases) excise numerous damaged bases from DNA during the first step of base excision repair. The damaged bases removed by these proteins include those induced by both alkylating agents and/or oxidizing agents. The intrinsic kinetic parameters (k(cat) and K(m)) for the excision of hypoxanthine by the recombinant human MPG protein from a 39 bp oligodeoxyribonucleotide harboring a unique hypoxanthine were determined. Comparison with other reactions catalyzed by the human MPG protein suggests that the differences in specificity are primarily in product release and not binding. Analysis of MPG protein binding to the 39 bp oligodeoxyribonucleotide revealed that the apparent dissociation constant is of the same order of magnitude as the K(m) and that a 1:1 complex is formed. The MPG protein also forms a strong complex with the product of excision, an abasic site, as well as with a reduced abasic site. DNase I footprinting experiments with the MPG protein on an oligodeoxyribonucleotide with a unique hypoxanthine at a defined position indicate that the protein protects 11 bases on the strand with the hypoxanthine and 12 bases on the complementary strand. Competition experiments with different length, double-stranded, hypoxanthine-containing oligodeoxyribonucleotides show that the footprinted region is relatively small. Despite the small footprint, however, oligodeoxyribonucleotides comprising <15 bp with a hypoxanthine have a 10-fold reduced binding capacity compared with hypoxanthine-containing oligodeoxyribonucleotides >20 bp in length. These results provide a basis for other structural studies of the MPG protein with its targets.
Miao
F
F
Beckman Research Institute, City of Hope National Medical Center, 1450 East Duarte Road, Duarte, CA 91010, USA.
Bouziane
M
M
O'Connor
T R
TR
D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018983:DNA Footprinting; D045647:DNA Glycosylases; D004260:DNA Repair; D006801:Humans; D019271:Hypoxanthine; D007288:Inosine; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019271:Hypoxanthine; D007288:Inosine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases