Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes)
Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes)
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DOI：
10.1111/php.12658
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发表时间：
2017
影响因子：
3.3
通讯作者：
Tomoko Ishikawa-Fujiwara;Eri Shiraishi;Y. Fujikawa;Toshio Mori;T. Tsujimura;T. Todo
中科院分区：
文献类型：
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作者：
Tomoko Ishikawa-Fujiwara;Eri Shiraishi;Y. Fujikawa;Toshio Mori;T. Tsujimura;T. Todo
隐花色素/光解酶家族（CPF）的蛋白质具有序列和结构保守性，但它们的功能是不同的。光裂合酶是一种DNA修复酶，催化紫外线（UV）诱导的光产物的光依赖性修复，而隐花色素则充当光感受器或生物钟蛋白。存在两种类型的DNA光解酶：CPD光解酶，修复环丁烷嘧啶二聚体（CPD），和6 - 4光解酶，修复6 - 4嘧啶-嘧啶酮光产物（6 - 4PP）。虽然Cry‐DASH蛋白被归类为隐花色素，但它也具有光依赖性DNA修复活性。为了确定三种光依赖性修复酶在生物体水平上从太阳紫外线诱导的DNA损伤中恢复的重要性，我们使用CRISPR基因组编辑技术在青鳉的每个基因中产生突变体。在体外培养细胞和体内皮肤组织中检查突变体的光依赖性修复活性。CPD光裂合酶缺陷突变体中CPD的光依赖性修复丧失，而6 - 4光裂合酶缺陷突变体中对6 - 4PP的弱修复活性持续存在。这些结果表明存在一种迄今未知的6 - 4PP修复途径，从而提高了我们对脊椎动物防御太阳紫外线机制的理解。
Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light‐dependent repair of ultraviolet (UV)‐induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6‐4 photolyase, which repairs 6‐4 pyrimidine–pyrimidone photoproducts (6‐4PPs). Although the Cry‐DASH protein is classified as a cryptochrome, it also has light‐dependent DNA repair activity. To determine the significance of the three light‐dependent repair enzymes in recovering from solar UV‐induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light‐dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light‐dependent repair of CPD was lost in the CPD photolyase‐deficient mutant, whereas weak repair activity against 6‐4PPs persisted in the 6‐4 photolyase‐deficient mutant. These results suggest the existence of a heretofore unknown 6‐4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates.