| Budget Amount *help |
¥11,100,000 (Direct Cost: ¥11,100,000)
Fiscal Year 2000: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥7,900,000 (Direct Cost: ¥7,900,000)
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| Research Abstract |
DNA photolyase/Cryptochrom consists of fuctionally diversed proteins ; DNA repair and regulation of circadianrhythm. The aim of this project is to know the basic mechanism undelying this family. Here, we have analyzed the function of each type of protein.
1) 6-4 Photolyase ; The reaction mechanism of Xenopus (6-4) photolyase was investigatedusing several mutantenzymes. In the activesite, which is homologous between the CPD and (6-4) photolyases, four amino acid residues that are specific to (6-4) photolyase, Gln288, His354, Leu355, and His358, and two conserved tryptophans, Trp291 and Trp398, were substituted with alanine. Only the L355A mutant had a lower affinity for the substrate, which suggested a hydrophobic interaction with the (6-4) photoproduct. Both the H354A and H358A mutations resulted in an almost complete loss of the repair activity, although the Trp291 and Trp398 mutants retained some activity Taking the pH profile of the (6-4) photolyase reaction into consideration with t
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his observation, we propose a mechanism in which these histidines catalyze the formation of the four-memberedring intermediatein the repair process of this enzyme. When deuterium oxide was used as a solvent, the repair activity was decreased. The proton transfer shown by this isotope effect supports the proposed mechanism.
2) Cryptochrome ; Two types of animal CRYs are known, mammalian CRY and Drosophila CRY.Both CRYs participatein regulation of the circadianrhythm, but they are different in the light dependency for their reaction and have different effects on the negativefeedback loop which generates circadian oscillation of gene expression. Mammalian CRYs act as a potent inhibitor of transcriptional activator which reaction do not depend on light, but Drosophila CRY functions as a light-dependent suppressor of transcriptional inhibitor We cloned seven zebrafish genes that carry members of the DNA photolyase/cryptochromeprotein family ; one (6-4)photolyase and six cry genes. Sequence analysis and determination of their in vitro functions showed that these zebrafish cry genes constitute two groups. One has a high sequence similarity to mammaliancry genes and inhibits CLOCK : BMAL1 mediated transcription. The other, which has a higher sequence similarity to Drosophila cry gene rather than to mammaliancry genes, does not have transcription inhibitor activity. The expressions of these cry genes oscillate in a circadianmanner, but the pattern differs each other. These findings suggest that functionally diverse cry genes are present in zebrafish and each gene has differentrole on the molecular clock. Less
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