The inhibitory effect of novel triterpenoid compounds, fomitellic acids, on DNA polymerase beta.
We previously found new triterpenoid compounds, designated fomitellic acid A and B, which selectively inhibit the activities of mammalian DNA polymerase alpha and beta in vitro. On DNA polymerase beta, the fomitellic acids acted by competing with both the substrate and the template primer, but on DNA polymerase alpha, they acted non-competitively. At least on DNA polymerase beta, the evidence suggests that each of the fomitellic acids bind to the active region competing with the substrate and/or template primer, and subsequently inhibits the catalytic activity. We therefore further investigated the enzyme-binding properties by using DNA polymerase beta and its proteolytic fragments. The 39 kDa enzyme was proteolytically separated into two fragments of the template-primer-binding domain (8 kDa) and the catalytic domain (31 kDa). The fomitellic acids bound tightly to the 8 kDa fragment, but not to the 31 kDa fragment. The immuno-precipitation by antibodies against the enzyme or each of the fragments also proved the binding. These results suggest that the fomitellic acid molecule competes with the template-primer molecule on its 8 kDa binding site, binds to the site, and the fomitellic acid molecule simultaneously disturbs the substrate incorporation into the template primer.
Mizushina
Y
Y
Department of Applied Biological Science, Science University of Tokyo, Noda, Chiba 278-8510, Japan.
Tanaka
N
N
Kitamura
A
A
Tamai
K
K
Ikeda
M
M
Takemura
M
M
Sugawara
F
F
Arai
T
T
Matsukage
A
A
Yoshida
S
S
Sakaguchi
K
K
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D004247:DNA / Q000096:biosynthesis; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors*; D017931:DNA Primers; D004261:DNA Replication / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D011089:Polydeoxyribonucleotides; D013698:Templates, Genetic; D013729:Terpenes / Q000494:pharmacology*; D013950:Thymus Gland / Q000201:enzymology
D017931:DNA Primers; D011089:Polydeoxyribonucleotides; D013729:Terpenes; C111927:fomitellic acid A; C111928:fomitellic acid B; C108692:poly(dA)-oligo(dT); D004247:DNA; D004256:DNA Polymerase I; D019951:DNA Polymerase beta