ENDONUCLEASE V EXPRESSION IN HUMAN CELLS

人类细胞中核酸内切酶 V 的表达

• 批准号: 3193803
• 负责人: EARL E HENDERSON
• 金额: $ 12.7万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3193803
• 关键词: DNA repair; DNA replication; Retroviridae; endonuclease; gene expression; genetic transcription; human tissue; photochemistry; pyrimidine dimers; radiation carcinogenesis; radiation genetics; tissue /cell culture; transposon /insertion element; ultraviolet radiation; xeroderma pigmentosum; DNA repair; DNA replication; Retroviridae; endonuclease; gene expression; genetic transcription; human tissue; photochemistry; pyrimidine dimers; radiation carcinogenesis; radiation genetics; tissue /cell culture; transposon /insertion element; ultraviolet radiation; xeroderma pigmentosum

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.

紫外线诱导的维修的真核DNA修复途径 病变被认为至少与大肠杆菌UVRA，B，C系统一样复杂。 在识别十个不同的xeroderma时，可以推断出这种复杂性 色素（XP）互补组缺乏紫外线诱导的切口 病变。 相比之下，核酸内切酶V v由DENV基因编码 噬菌体T4，一种与相关的嘧啶二聚体-DNA糖基酶 丙酰胺核酸内切酶活性具有启动DNA修复的能力 作为单个酶，通过在嘧啶二聚体上特异性切割。 DENV 基因已在大肠杆菌中鉴定，测序和克隆， 促进适合引入的DENV质粒载体的构造 进入包括XP细胞在内的真核细胞。 XP细胞已经有用 阐明紫外线诱导的光产物对生物学的影响 过程由于无法修复它们。 在我们研究denv- 真核细胞中编码的内切酶V催化的紫外线修复，三个新型 并进行了有趣的实验观察：（i）尽管紫外线 切除修复和报告基因表达可以恢复到接近正常 DENV编码的核酸内切酶V的水平，细胞存活未恢复为野生 类型; （ii）与内源性修复相反，这可以是非随机的 核酸内切酶V催化修复限制为活性基因 在整个基因组中； （iii）除去核苷酸斑块大小 在核酸内切酶中V催化的嘧啶二聚体修复远小于 在紫外线辐射的正常细胞中。 主要是两个光产物， 丰富的嘧啶二聚体和较少丰富的（6-4）TC嘧啶 - 吡啶酮 光产物[6-4]光产物）已与多样化有关 紫外光的生物学作用。 因为嘧啶二聚体也没有（6- 4）XP细胞去除光吸收，不可能 明确区分他们对生物学的个人贡献 体内紫外线损伤的后果。 最近，XP恢复细胞系具有 被描述了哪些修复（6-4）TC光产物，而不是嘧啶 二聚体。 将DENV基因引入XP和Reververtant XP细胞提供 有机会分别分析嘧啶二聚体和 （6-4）DNA合成，转录，生存，修复和修复的TC光产物 人类细胞中的突变；并提供了检查的机会 在正常和修复中表征良好的DNA修复酶的功能 - 不足的人类细胞。