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: 2445310
• 负责人: Muriel W Lambert
• 金额: $ 25.99万
• 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/2445310
• 关键词: Baculoviridae; DNA damage; DNA repair; Escherichia coli; cell free system; complementary DNA; congenital aplastic anemia; crosslink; endonuclease; enzyme complex; gene complementation; gene mutation; genetic mapping; human subject; immunoaffinity chromatography; immunoprecipitation; monoclonal antibody; protein purification; protein sequence; psoralens; recombinant proteins; ultraviolet radiation; western blottings; xeroderma pigmentosum; yeasts

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：申请人及其同事分离出 一种来自正常人类染色质的DNA内切酶复合物， 参与DNA链间修复的初始步骤 交叉连接。 该复合物含有核酸内切酶和一个损伤 识别蛋白也是如此。 此外，这种情结是反常的 在A互补的范科尼贫血（FA）细胞提取物中 组她发现这些细胞缺乏或减少了活性， 损害识别的能力，并有缺陷， 内切核酸切除受损的DNA。 此外，使用一种策略 对于位点特异性的三甲基异戊烯单加合物，FA络合物是 特别是有缺陷的能力，使切口上的 3'侧的交叉连接，同时能够在5'侧切割 的方面想此外，单克隆抗体已经由 申请人针对复合物中的特定蛋白质，并且已经允许 41 Kda蛋白和225 Kda蛋白的鉴定。 的 抗体抑制正常复合物产生一种 在交叉连接的3'侧上的切口。 最后，Western blot 分析表明FA细胞缺乏225 Kda蛋白。 基于这些观察结果，申请人提出：（1）分离 DNA损伤识别蛋白和核酸内切酶参与修复 链间交联和由其制备胰蛋白酶肽酶 每种蛋白质，（2）分离编码这些蛋白质的cDNA并测序 并随后在A的FA细胞中表达这些CDNAS， 互补组，以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体 蛋白质将在大肠杆菌、酵母和杆状病毒中表达 以确定重组分子是否具有功能性 在无细胞系统中的活性，（4）分析 从FA细胞分离的同源CDNAS，（5）表征损伤 识别蛋白和核酸内切酶在正常和FA细胞中的表达， 用长波紫外线（UVA）照射裸体，研究了它们之间的相互作用 DNA.