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 glyco sylases) excise numerous damaged bases from DNA during the first step of base excision repair. The damaged bases removed by these proteins i nclude those induced by both alkylating agents and/or oxidizing agents . The intrinsic kinetic parameters (k(cat) and K-m) for the excision o f hypoxanthine by the recombinant human MPG protein from a 39 bp oligo deoxyribonucleotide harboring a unique hypoxanthine were determined. C omparison with other reactions catalyzed by the human MPG protein sugg ests that the differences in specificity are primarily in product rele ase and not binding. Analysis of MPG protein binding to the 39 bp olig odeoxyribonucleotide revealed that the apparent dissociation constant is of the same order of magnitude as the K-m and that 1:1 complex is f ormed. The MPG protein also forms a strong complex with the product of excision, an abasic site, as well as with a reduced abasic site. DNas e I footprinting experiments with the MPG protein on an oligodeoxyribo nucleotide with a unique hypoxanthine at a defined position indicate t hat 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 oligodeoxy ribonucleotides show that the footprinted region is relatively small. Despite the small footprint, however, oligodeoxyribonucleotides compri sing <15 bp with a hypoxanthine have a 10-fold reduced binding capacit y compared with hypoxanthine-containing oligodeoxyribonucleotides >20 bp in length. These results provide a basis for other structural studi es of the MPG protein with its targets.