Substrate specificity of human methylpurine DNA N-glycosylase

The activity of human methylpurine DNA N-glycosylase (hMPG) for major subst rates was directly compared using two types of substrates, i.e., natural DM A and synthetic oligonucleotides. By the use of ARP assay detecting abasic sites in DNA, we first investigated the activity on the natural DNA substra tes containing methylpurines, ethenopurines, or hypoxanthine (Hx) prepared by the conventional methods. After the treatment with hMPG, the amount of A P sites in methylated DNA was much higher than that in DNA containing ethen opurines or Hx. The oligodeoxynucleotide having a single 7-methylguanine (7 -mG) was newly synthesized in addition to 1,N-6-ethenoadenine (epsilon A)-, Hx-, and 8-oxoguanine-containing oligonucleotides. 7-mG was effectively ex cised by hMPG, though it might be less toxic than the other methylated base s with respect to mutagenesis and cell killing. The kinetic study demonstra ted that k(cat)/K-m ratios of the enzyme for epsilon A, Hx, and 7-mG were 2 .5 x 10(-3), 1.4 x 10(-3), and 4 x 10(-4) min(-1) nM(-1), respectively. The oligonucleotides containing epsilon A effectively competed against 7-mG, w hile Hx substrates showed unexpectedly low competition. Concerning the effe ct of the base opposite damage, hMPG much preferred Hx . T to other Hx pair s, and epsilon A . C and epsilon A . A pairs were better substrates than ep silon A . T.