DNA Photolyase and Blue Light Receptor

DNA光解酶/隐色素由功能多样化的蛋白质组成；DNA修复和昼夜节律的调节。这个项目的目的是了解这个家族的基本机制。在这里，我们分析了每种蛋白质的功能。1) 6-4光解酶；利用几种突变酶对爪蟾（6-4）光解酶的反应机理进行了研究。在CPD与（6-4）光解酶同源的活性位点中，（6-4）光解酶特异性的4个氨基酸残基Gln288、His354、Leu355和His358以及两个保守的色氨酸Trp291和Trp398被丙氨酸取代。只有L355A突变体对底物的亲和力较低，这表明它与（6-4）光产物存在疏水相互作用。H354A和H358A突变导致修复活性几乎完全丧失，尽管Trp291和Trp398突变体保留了一些活性。考虑到（6-4）光解酶反应的pH谱，我们提出了一种机制，这些组氨酸在该酶的修复过程中催化了四元中间体的形成。当氧化氘作为溶剂时，修复活性降低。这种同位素效应所显示的质子转移支持了所提出的机制。2)隐花色素；已知有两种动物的哭泣，哺乳动物的哭泣和果蝇的哭泣。这两种CRYs都参与昼夜节律的调节，但它们的反应对光的依赖性不同，对产生基因表达昼夜振荡的负反馈回路的影响也不同。哺乳动物的CRYs是一种不依赖于光的转录激活因子的有效抑制剂，但果蝇的CRYs是一种依赖于光的转录抑制因子。一个（6-4）光解酶和六个哭泣基因。序列分析和体外功能测定表明，这些斑马鱼哭泣基因可分为两类。一个与哺乳动物基因序列高度相似，抑制CLOCK: BMAL1介导的转录。另一个与果蝇哭泣基因的序列相似性高于与哺乳动物哭泣基因的序列相似性，不具有转录抑制剂活性。这些哭泣基因的表达以昼夜节律的方式振荡，但模式彼此不同。这些发现表明，斑马鱼中存在功能多样的哭泣基因，每个基因在分子钟中都有不同的作用。少

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 … More 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