DNA REPAIR DEFECT IN FANCONI ANEMIA, GROUP A

范可尼贫血 A 组中的 DNA 修复缺陷

• 批准号: 2233342
• 负责人: Muriel W Lambert
• 金额: $ 25.03万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2233342
• 关键词: DNA; REPAIR; DEFECT; FANCONI; ANEMIA

DESCRIPTION: The applicant and her colleagues have isolated a DNA endonuclease complex from normal human chromatin that seems to be involved in the initial steps of the repair of DNA interstrand crosslinks. The complex contains the endonuclease and a damage recognition protein as well. In addition, this complex is abnormal in extracts from Fanconi Anemia (FA) cells of the A complementation group. She has found that these cells have absent or reduced activity of damage recognition and have a defect in the ability to endonucleolytically excise damaged DNA. In addition, using a strategy for site-specific trimethyl psoralen monoadducts, the FA complex is specifically defective in its capacity to make the an incision on the 3' side of the cross link while being competent to incise at the 5' side. Moreover, monoclonal antibodies have been raised by the applicant against specific proteins in the complex and have permitted the identification of a 41 Kda protein and a 225 Kda protein. The antibodies inhibit the capacity of normal complexes to create an incision on the 3' side of the crosslink. Finally, Western blot analysis shows that the FA cells are deficient in the 225 Kda protein. Based upon these observations the Applicant proposes to: (1) isolate DNA damage recognition protein and endonuclease involved in repair of interstrand crosslinks and to prepare tryptic peptide digests from each protein, (2) isolate and sequence CDNAS encoding these proteins and subsequently express these CDNAS in FA cells of the A complementation group to confirm their role in the repair process, (3) determine the chromosomal location of the gene and express recombinant proteins will be expressed in E.coli, yeast, and baculovirus to determine whether the recombinant molecules have functional activity in the cell-free system, (4) analyze the mutations in homologous CDNAS isolated from FA cells, (5) characterize the damage recognition protein and endonuclease in normal and FA cells and study their interactions on psoralen plus UVA light irradiated naked DNA.

描述：申请者和她的同事隔离了 一种来自正常人类染色质的DNA内切酶复合体 参与DNA链间修复的初始步骤 交联剂。该复合体含有内切酶和一种损伤 还有识别蛋白。另外，这个综合体是不正常的 在Fanconi贫血(FA)细胞的A互补提取物中 一群人。她发现这些细胞缺乏或减少了活动。 损害识别的能力有缺陷 核内溶解切除受损的DNA。此外，使用一种策略 对于位点特定的三甲基补骨脂素单加合物，FA络合物为 特别是有缺陷的，不能把伤口切开 交叉链的3‘边，同时能够在5’处切割 边上。此外，单克隆抗体已经由 针对复合体中的特定蛋白质提出申请，并允许 鉴定出一个41kDa的蛋白质和一个225kda的蛋白质。这个 抗体抑制正常的复合体产生 在交联剂的3‘侧的切口处。最后，西方印迹 分析表明，FA细胞缺乏225 KDA蛋白。 基于这些观察，申请人提议：(1)分离 DNA损伤识别蛋白和核酸内切酶参与的修复 链间交联剂和制备胰蛋白酶多肽 每种蛋白质，(2)分离并测序编码这些蛋白质的cDNA 并随后在A组的FA细胞中表达这些cDNA 补充小组确认其在修复过程中的作用，(3) 确定基因的染色体位置并表达重组 蛋白质将在大肠杆菌、酵母和杆状病毒中表达 确定重组分子是否具有功能 在无细胞系统中的活性，(4)分析 从FA细胞中分离的同源cDNA，(5)表征损伤 正常和FA细胞的识别蛋白和核酸内切酶 补骨脂素与UVA裸光照射的相互作用研究 DNA