MECHANISMS OF DNA CROSSLINK REPAIR IN HIGHER EUKARYOTES

高等真核生物 DNA 交联修复机制

• 批准号: 2462943
• 负责人: KENNETH Charles BURTIS
• 金额: $ 14.03万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2462943
• 关键词: DNA binding protein; DNA repair; Drosophilidae; congenital aplastic anemia; crosslink; gene mutation; molecular cloning; protein purification; yeast two hybrid system

The primary goal of this proposal is to clone the Fanconi anemia A (FA-A) gene of man through an analysis of the functionally homologous mus3O8 gene of Drosophila. This hematological disorder is a candidate for human gene therapy. Current support for gene homology between these two organisms is provided by the observation that among several mutagen sensitive disorders in man only Fanconi anemia is characterized by hypersensitivity to DNA cross-linking agents and concurrent insensitivity to monofunctional alkylating agents. An extensive search for analogous mutants in Drosophila has revealed only one autosomal gene with this property; the mus3O8 gene. The presence of this unique spectrum of mutagen sensitivity in mutants of only one disorder in man and one in Drosophila strongly suggests that the mus3O8 gene encodes a function analogous to one of the Fanconi genes. That suggestion is further supported by the demonstration that both Fanconi anemia cells and mus308 cells exhibit an elevated frequency of spontaneous chromosomal aberrations. Two additional rare phenotypes are common to both the mus3O8 mutants and cells deficient in the FA-A gene. These are a failure to recover DNA synthesis following mutagen treatment and the unique alteration of a deoxyribonuclease. Because the mus3O8 mutants and FA-A cells share a total of four rare properties, there is therefore a strong probability that they are functionally related. This potential functional homology has stimulated efforts to clone the Drosophila mus3O8 gene in preparation for cloning the human analogue. The feasibility of that approach is supported by numerous examples in which cloned genes from one of these organisms has been employed to recover the homologous gene from the alternate organism. Recovery of the FA-A gene can be definitively verified by testing the capacity of the cloned gene to complement the hypersensitivity of FA-A cells to DNA crosslinking agents following transfection. This approach has been chosen because all of the direct mammalian efforts, including an approach recently employed to clone the FA C gene, have thus far failed to recover the FA-A gene. Drosophila was chosen for this study because gene cloning in this organism is facilitated by genetic analysis, the presence of giant polytene chromosomes and a small genome size. It is anticipated that recovery of the human gene will place our collaborators in a position to determine if it can be employed to reverse the lethal effects of Fanconi anemia through gene therapy. This approach is particularly pertinent to Fanconi anemia because the most frequent cause of lethality in this disease is a failure of bone marrow cells to proliferate in late adolescence. This disorder can be reversed in Fanconi children, however, by bone marrow transplantation when a compatible donor is available. If, however, the patients own stem cells could be provided with the normal gene, then each individual could serve as his or her own donor.

该提案的主要目标是克隆范可尼贫血 A (FA-A) 通过功能同源 mus3O8 分析人类基因 果蝇的基因。 这种血液疾病是人类的候选疾病 基因疗法。 目前对这两者之间基因同源性的支持 生物体是通过观察提供的，在几种诱变剂中 仅限男性的敏感疾病 范可尼贫血的特点是 对 DNA 交联剂过敏并并发不敏感 至单官能烷基化剂。 广泛寻找类似的 果蝇突变体仅揭示了一种常染色体基因 财产; mus3O8 基因。 这种独特光谱的存在 仅一种人类疾病和一种疾病的突变体对诱变剂的敏感性 果蝇强烈表明 mus3O8 基因编码一种功能 类似于范可尼基因之一。 这个建议更进一步 范可尼贫血细胞和 mus308 的证明支持 细胞表现出自发染色体频率升高 像差。 另外两种罕见的表型是两者共有的 mus3O8 突变体和 FA-A 基因缺陷的细胞。 这些都是失败的 诱变剂处理后恢复 DNA 合成以及独特的 脱氧核糖核酸酶的改变。 因为 mus3O8 突变体和 FA-A 细胞共有四种罕见的特性，因此具有很强的 它们在功能上相关的概率。 这种潜在的功能同源性刺激了克隆的努力 果蝇 mus3O8 基因为克隆人类类似物做准备。 该方法的可行性得到了许多例子的支持 从这些生物体之一克隆基因已被用于 从替代生物体中恢复同源基因。 恢复的 FA-A基因可以通过测试FA-A基因的能力来明确验证 克隆基因以补充 FA-A 细胞对 DNA 的超敏反应 转染后的交联剂。 这种方法已被 选择是因为所有直接的哺乳动物努力，包括一种方法 最近被用来克隆FA C基因，迄今为止未能恢复 FA-A基因。 选择果蝇进行这项研究是因为基因克隆 在这种生物体中，遗传分析促进了 巨大的多线染色体和较小的基因组大小。 预计人类基因的恢复将使我们 合作者能够确定是否可以使用它来逆转 通过基因治疗范可尼贫血的致命影响。 这种做法 与范可尼贫血特别相关，因为最常见的 这种疾病的致死原因是骨髓细胞无法 在青春期后期增殖。 这种疾病可以逆转 然而，范科尼儿童在接受骨髓移植时 可以使用兼容的供体。 然而，如果患者拥有干细胞 可以提供正常的基因，然后每个人都可以服务 作为他或她自己的捐赠者。