| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2000: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus, electively inhibits the activities of mammalian DNA polymerase beta and lambda (pol beta and lambda) in vitro. The IC50 values of the compound were 30 microm for pol beta and 37 microm for poi lambda. Because poi beta and lambda are in a family and their three-dimensional structures are thought to be highly similar to each other, we used pol beta to. analyze the biochemical relationship with solanapyrone A. On poi beta, solanapyrone A antagonistically competed with both the DNA template and the nucleotide substrate. BlAcore analysis demonstrated that solanapyrone A bound selectively to the N-terminal 8-kDa domain of poi beta. This domain is known to bind single-stranded DNA, provide 5 -phosphate recognition of gapped DNA, and cleave the sugar-phosphate bond 3 to an intact apurinic/apyrimidinic (AP) site (i. e. AP lyase activity) including 5 -deoxyribose phosphate lyase activity. Solanapyrone A inhibited the singl
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e-stranded DNA-binding activity but did not influence the activities of the 5 -phosphate recognition in gapped DNA structures and the AP lyase. Dehydroaltenusin was found to be an inhibitor of mammalian DNA polymerase alpha (pol. alpha) in vitro, but did not influence the activities of the other replicative DNA polymerases including even other vertebrate pol. alpha. In this study, we purified or synthesized various slightly modified derivatives of dehydroaltenusin, and using them, investigated the relationship between the chemical structure and the inhibitory effects, and the in vitro and in vivo effects of dehydroaltenusin to determine to what extent the pol. alpha activity inhibition influences cell proliferation. Dehydroaltenusin inhibited the cell proliferation of the human gastric cancer cell line NTJGC-3 by arresting the cells at G1/S-phase, and prevented the incorporation of thymidine into the cells, indicating that it blocks the primary step of in vivo DNA replication by inhibiting pol. alpha. Less
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