Substrate specificity of human methylpurine DNA N-glycosylase.
The activity of human methylpurine DNA N-glycosylase (hMPG) for major substrates was directly compared using two types of substrates, i.e., natural DNA and synthetic oligonucleotides. By the use of ARP assay detecting abasic sites in DNA, we first investigated the activity on the natural DNA substrates containing methylpurines, ethenopurines, or hypoxanthine (Hx) prepared by the conventional methods. After the treatment with hMPG, the amount of AP sites in methylated DNA was much higher than that in DNA containing ethenopurines or Hx. The oligodeoxynucleotide having a single 7-methylguanine (7-mG) was newly synthesized in addition to 1, N(6)-ethenoadenine (epsilonA)-, Hx-, and 8-oxoguanine-containing oligonucleotides. 7-mG was effectively excised by hMPG, though it might be less toxic than the other methylated bases with respect to mutagenesis and cell killing. The kinetic study demonstrated that k(cat)/K(m) ratios of the enzyme for epsilonA, 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 epsilonA effectively competed against 7-mG, while Hx substrates showed unexpectedly low competition. Concerning the effect of the base opposite damage, hMPG much preferred Hx.T to other Hx pairs, and epsilonA.C and epsilonA.A pairs were better substrates than epsilonA.T.
Asaeda
A
A
Department of Veterinary Radiology, School of Veterinary Medicine, College of Agriculture, Osaka Prefecture University, Gakuen-cho, Sakai, Osaka 599-8531, Japan.
Ide
H
H
Asagoshi
K
K
Matsuyama
S
S
Tano
K
K
Murakami
A
A
Takamori
Y
Y
Kubo
K
K
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D045647:DNA Glycosylases; D004260:DNA Repair; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D009841:Oligonucleotides; D011994:Recombinant Proteins; D006151:Guanosine; C004981:1,N(6)-ethenoadenine; D019271:Hypoxanthine; C046215:8-hydroxyguanosine; D006147:Guanine; C008450:7-methylguanine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D000225:Adenine