ENDONUCLEASE V EXPRESSION IN HUMAN CELLS

真核细胞DNA修复途径参与紫外线损伤修复 病变被认为至少和E. coliUvrA、B、C系统。 这种复杂性是推断在鉴定十种不同的干皮病 色素（XP）互补组在紫外线诱导的切口缺陷 病变 相比之下，由Diclavidae的denV基因编码的内切核酸酶V在大肠杆菌中表达。 噬菌体T4，嘧啶二聚体-DNA糖基化酶， 无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异性在嘧啶二聚体。 关于DenV 基因已经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 真核细胞，包括XP细胞。 XP细胞已经成为 在阐明紫外线诱导的光产物对生物的影响方面， 因为他们无法修复它们。 在我们对丹佛的研究中- 真核细胞中编码的核酸内切酶V催化的UV修复，三种新的 和有趣的实验观察已经作出：（一）虽然紫外线 切除修复和报告基因表达可以恢复到接近正常 在denV编码的核酸内切酶V的水平下，细胞存活没有恢复到野生型水平。 （ii）与内源性修复相反，内源性修复可以是非随机的， 仅限于活性基因，核酸内切酶V催化的修复发生在 在整个基因组中;和（iii）去除的核苷酸补丁大小 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。 Priestion，两个摄影产品， 丰富的嘧啶二聚体和较不丰富的（6-4）TC嘧啶-嘧啶酮 [6-4]photoproducts），已经牵连在各种各样的 紫外线的生物效应 因为无论是嘧啶二聚体还是（6- 4)光产物被XP细胞清除，这是不可能的， 明确区分它们各自对生物学的贡献 体内紫外线损伤的后果。 最近，XP回复突变细胞系 已经描述了修复（6-4）TC光产物，但不修复嘧啶 二聚体。 将denV基因引入XP和回复突变XP细胞提供了 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;并提供了检查 一个很好的特征DNA修复酶的功能，在正常和修复- 缺乏人类细胞。

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.