ENDONUCLEASE V EXPRESSION IN HUMAN CELLS

真核细胞DNA修复途径参与紫外线诱导的修复 损伤被认为至少与大肠杆菌UvrA、B、C系统一样复杂。 这种复杂性可以从10种不同的干皮病的鉴定中推断出来。 紫外线诱导下切口处缺失的色素(XP)补充组 损伤。相比之下，由猪瘟病毒DENV基因编码的内切酶V 噬菌体T4，一种嘧啶二聚体-DNA糖基化酶 脱嘧啶核酸内切酶活性，具有启动DNA修复的能力 作为一个单一的酶通过切割特定的嘧啶二聚体。DENV 基因已在大肠杆菌中被鉴定、测序和克隆， 便于构建适于导入的DENV质粒载体 转化为真核细胞，包括XP细胞。XP细胞一直是工具性的 在阐明紫外线诱导的光产物对生物的影响 进程，因为他们无法修复它们。在我们研究DENV的过程中- 编码内切酶V在真核细胞中催化的紫外线修复，三个新的 并进行了有趣的实验观察：(I)虽然紫外线 切除修复和报告基因的表达可以恢复到接近正常 DENV编码的内切酶V的水平，细胞存活不能恢复到野生状态 类型；(Ii)与内源性修复相反，内源性修复可以是非随机的和 限制在活性基因的限制下，核酸内切酶V催化的修复发生 在整个基因组中；以及(Iii)移除的核苷酸片段大小 在核酸内切酶V催化的嘧啶二聚体修复过程中， 在紫外线照射的正常细胞中。首先，有两个摄影产品， 丰富的嘧啶二聚体和不丰富的(6-4)TC嘧啶-嘧啶酮 照片产品[6-4]照片产品)，已经牵涉到不同的 紫外线的生物效应。因为无论是嘧啶二聚体还是(6- 4)照相产品被XP细胞去除，不可能 毫不含糊地区分他们对生物的个体贡献 体内紫外线损伤的后果。最近，一种XP回复细胞系已经 已经描述了修复(6-4)TC感光产物而不是嘧啶的方法 二聚体。将DENV基因导入XP和突变的XP细胞可提供 有机会分别分析嘧啶二聚体和 (6-4)TC光产物对DNA合成、转录、存活、修复和 人类细胞中的突变；并提供了研究 一种功能完善的DNA修复酶在正常和修复中的作用 缺乏人体细胞。

The eukaryotic DNA repair pathway involved in the repair of UV-induced lesions is thought to be at least as complex as the E. coli UvrA,B,C system. This complexity is inferred in the identification of ten different xeroderma pigmentosum (XP) complementation groups deficient in incision at UV-induced lesions. In contrast, endonuclease V encoded by the denV gene of bacteriophage T4, a pyrimidine dimer-DNA glycosylase with associated apyrimidinic endonuclease activity, has the capacity to initiate DNA repair as a single enzyme by incising specifically at pyrimidine dimers. The denV gene has been identified, sequenced and cloned in Escherichia coli, facilitating construction of denV plasmid vectors suitable for introduction into eukaryotic cells, including XP cells. XP cells have been instrumental in elucidating the effects of UV-induced photoproducts on biological processes due to their inability to repair them. During our study of denV- encoded endonuclease V-catalyzed UV repair in eukaryotic cells, three novel and interesting experimental observations have been made: (i) although UV excision repair and reporter gene expression can be restored to near normal levels by denV-encoded endonuclease V, cell survival is not restored to wild type; (ii) in contrast to the endogenous repair, which can be nonrandom and restricted to active genes, endonuclease V-catalyzed repair occurs throughout the entire genome; and (iii) the nucleotide patch size removed during endonuclease V-catalyzed pyrimidine dimer repair is much smaller than that in UV-irradiated normal cells. Primarily, two photoproducts, the abundant pyrimidine dimer and less abundant (6-4)TC pyrimidine-pyrimidone photoproducts [6-4]photoproducts), have been implicated in the diverse biological effects of UV light. Because neither pyrimidine dimers nor (6- 4)photoproducts are removed by XP cells, it has not been possible to unequivocally distinguish their individual contributions to the biological consequences of UV damage in vivo. Recently an XP revertant cell line has been described which repairs (6-4)TC photoproducts but not the pyrimidine dimers. Introducing the denV gene into XP and revertant XP cells provides an opportunity to separately analyze the effects of pyrimidine dimers and (6-4)TC photoproducts on DNA synthesis, transcription, survival, repair and mutations in human cells; and affords as the opportunity to examine the function of a well characterized DNA repair enzyme in normal and repair- deficient human cells.