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Photobiological significance of DNA photolgase/Blue-light photoreceptor family

DNA光酶/蓝光光感受器家族的光生物学意义

基本信息

批准号：
09833003
负责人：
TODO Takeshi
金额：
$ 1.47万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09833003/
关键词：
DNA Repair UV damage (6-4)Photolyase Cryptochrome CRY Blue light photoreceptor Circadian Rhythm 光回復酵素

项目摘要

We report isolation of a Xenopus laevis (6-4)photolyase gene, and show that the (6-4)photolyase binds noncovalently to stoichiometric amounts of FAD.Xenopus (6-4) photolyase binds with high affinity to DNA bearing a (6-4) photoproduct and repairs it in a light-dependent reaction. To clarify its repair mechanism of (6-4) photolyase, we determined its binding and catalytic properties using synthetic DNA substrate which carries a photoproduct at a single location. The (6-4)photolyase binds to T[6-4]T in double stranded DNA with high affinity (kD = 10_<-9>) and to T[6-4]T in single stranded DNA and T[Dewar]T in double- and single-stranded DNA although with slightly lower affinity (k_D = -2 x 10_<-8>). Majority of the T[6-4]T (6-4) photolyase-complex dissociates very slowly (koff = 2.9 x 10_<-5 S-1>). Its absolute action spectrum without a second chromophore in the 350-600 nm region closely matches the absorption spectrum of the enzyme. The quantum yield (f) of repair is approximately 0.11. … More The fully reduced form (E-FADH-) of (6-4)photolyase is catalytically active. Direct analysis of the photoreactivated product showed that (6-4) photolyase restores the original pyrimidines. These findings demonstrate that CPD photolyase and (6-4)photolyase are quite similar, but they are different with regard to the binding properties.Light is the major environmental signal for the entrainment of circadian rhythms. Light signals can entrain these rhythms by shifting their phases. However, little is known about the molecular mechanism for the perception and transduction of the light signal. The members of the photolyase/cryptochrome family contain flavin adenine dinucleotide (FAD) as chromophore and are involved in two diverse functions, DNA repair and photoreception of environmental light signals. We report the cloning of a new member of this family, dcry, from Drosophila. Northern blot analysis shows that this gene is expressed in various tissues. . The dcry mRNA is expressed in a circadian manner in adult heads, while such rhythmic fluctuation is abolished in the clock-defective per0 and tim0 mutants. The circadian expression damps in constant darkness. Over-expression of the dcry gene alters the light-induced phase delay in the locomotor activity rhythms of flies. These results suggest that DCRY is a circadian photoreceptor and its expression is regulated by circadian clock genes.. Less

我们报道了非洲爪蟾（6-4）光解酶基因的分离，并表明（6-4）光解酶与化学计量量的FAD非共价结合。爪蟾（6-4）光解酶与携带（6-4）光产物的DNA具有高亲和力，并在依赖光的反应中对其进行修复。为了阐明其对（6-4）光解酶的修复机制，我们利用在单个位置携带光产物的合成DNA底物测定了其结合和催化性能。（6-4）光解酶与双链DNA中的T[6-4]T具有高亲和力（kD = 10_<-9>），与单链DNA中的T[6-4]T和双链和单链DNA中的T[杜瓦]T具有较低亲和力（k_D = -2 × 10_<-8>）。大部分T[6-4]T（6-4）光解酶复合物解离非常缓慢（koff = 2.9 × 10_<-5 S-1>）。在350- 600nm范围内，不含第二个发色团的绝对作用谱与酶的吸收谱非常接近。修复的量子产率(f)约为0.11。（6-4）光解酶的完全还原形式（E-FADH-）具有催化活性。对光再激活产物的直接分析表明，（6-4）光解酶恢复了原有的嘧啶。这些结果表明CPD光解酶和（6-4）光解酶非常相似，但它们的结合性质不同。光是昼夜节律的主要环境信号。光信号可以通过改变它们的相位来控制这些节律。然而，人们对光信号感知和转导的分子机制知之甚少。光解酶/隐色素家族成员以黄素腺嘌呤二核苷酸（FAD）为发色团，参与DNA修复和环境光信号的光接受两种不同的功能。我们报道克隆了这个家族的一个新成员，dcry，来自果蝇。Northern blot分析表明，该基因在多种组织中均有表达。dcry mRNA在成人头部以昼夜节律方式表达，而这种节律波动在per0和tim0突变体中被消除。昼夜节律表达在持续的黑暗中受到抑制。dcry基因的过度表达改变了果蝇运动活动节律中光诱导的相位延迟。这些结果表明DCRY是一种昼夜光感受器，其表达受生物钟基因的调控。少