DNA Photolyase and Blue Light Receptor

DNA光裂合酶/隐色素是一种功能多样的蛋白质，参与DNA修复和昼夜节律的调节。本研究的目的是了解这个家族的基本机制。1）6-4光裂解酶;用几种突变酶对爪蟾（6-4）光裂解酶的反应机理进行了研究。在CPD和（6-4）光解酶之间同源的活性位点中，对（6-4）光解酶特异的四个氨基酸残基Gln 288、His 354、Leu 355和His 358以及两个保守的色氨酸Trp 291和Trp 398被丙氨酸取代。只有L355 A突变体对底物的亲和力较低，这表明与（6-4）光产物的疏水相互作用。H354 A和H358 A突变导致几乎完全丧失修复活性，尽管Trp 291和Trp 398突变体保留了一些活性。 关于我们 通过观察，我们提出了一种机制，在这种机制中，这些组氨酸催化了这种酶修复过程中四元环中间体的形成。当使用氧化氘作为溶剂时，修复活性降低。2）隐花色素：哺乳动物的CRY和果蝇的CRY都参与了昼夜节律的调节，但它们的反应对光的依赖性不同，对基因表达的昼夜振荡负反馈环的作用也不同。哺乳动物的CRY是一种不依赖于光的转录激活因子的有效抑制因子，而果蝇的CRY是一种依赖于光的转录抑制因子。我们克隆了7个斑马鱼基因，它们携带DNA光裂合酶/隐色蛋白家族的成员：1个（6-4）光裂合酶和6个cry基因。序列分析和体外功能测定表明，这些斑马鱼cry基因分为两大类。其中一个与CLOCK：BMAL 1基因具有高度的序列相似性，并抑制CLOCK：BMAL 1介导的转录。另一个基因与果蝇cry基因的序列相似性较高，但不具有转录抑制活性。这些cry基因的表达呈昼夜振荡，但模式各不相同。这些发现表明，在斑马鱼中存在功能多样的cry基因，并且每个基因对分子钟具有不同的作用。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