MECHANISMS OF HOMOLOGOUS RECOMBINATION IN HUMAN CELLS

我们研究的长期目标是理解 致癌物致癌机制的研究进展 人类的呼唤。现在有了强有力的证据。摘自几个 不同的领域，认为致癌是一个多步骤的过程。 一个或多个步骤可能涉及体细胞突变。然而， 从分子生物学研究中获得的最新遗传证据 来自遗传性视网膜母细胞瘤患者的细胞， 表明等位基因之间的有丝分裂重组可以导致 通过使细胞纯合来促进肿瘤的发展 突变的等位基因，允许表达隐性性状。 基因重组的机制目前还知之甚少。 哺乳动物细胞。我们最近证明了化学物质和 物理致癌物可诱导同源重组 哺乳动物细胞中的染色体基因(Wang等人，Mol.牢房。 Biol，8,196-202,1988)。我们打算在 建议的研究是：这些致癌物是如何诱发 重组？其中涉及哪些机制？是重组 由致癌物诱导的损伤干扰的存在刺激 在S期有DNA复制？或者，是 DNA断裂的引入刺激重组 由切除修复过程或单链区域引起 在修复过程中产生的父母DNA？DNA连接酶有什么作用 玩重组吗？细胞来自癌症高发者吗？ 比正常人的细胞更敏感？我们建议 整合到人类细胞的染色体中的单个拷贝 含有编码可选基因的两个拷贝的质粒 遗传标记。每个基因在一个独特的位置被一个 限制性内切酶的8个碱基连接位点 需要生产性重组事件才能获得功能性 (可选)基因产物。质粒将被稳定整合 进入不同DNA修复的人类细胞的基因组 能力，包括来自干皮病患者的细胞 色素性共济失调毛细血管扩张症，以及布鲁姆综合征 作为正常人的细胞。

The long range objective of our studies is to understand the mechanism by which carcinogens cause malignant transformation of human calls. There is now strong evidence. drawn from several different fields, that carcinogenesis is a multistepped process. One or more steps may involve somatic cell mutations. However, recent genetic evidence, obtained from molecular biology studies of cells derived from persons with hereditary retinoblastoma, indicates that mitotic recombination between allelic genes can lead to the development of tumors by making the cell homozygous for the mutated allele, which permits expression of a recessive trait. Little is known about the mechanism of genetic recombination in mammalian cells. We recently demonstrated that chemical and physical carcinogens can induce homonomous recombination between chromosomal genes in mammalian cells (Wang et al., Mol. Cell. Biol, 8, 196-202, 1988). The question we intend to address in the proposed research are: How do these carcinogens induce recombination? What mechanisms are involved? Is recombination stimulated by the presence of carcinogen-induced damage interfering with DNA replication during S-phase? Alternatively, is recombination stimulated by the introduction of breaks in DNA caused by excision repair processes, or by single stranded regions of parental DNA generated during repair? What role do DNA ligases play in recombination? Are cells from cancer prone individuals more sensitive than cells from normal persons? We propose to integrate into the chromosome of human cells a single copy of a plasmid containing two copies of a gene coding for a selectable genetic marker. Each gene is interrupted at a unique site by an 8 bp linker site for a restriction endonuclease so that a productive recombinational event is required to get a functional (selectable) gene product. The plasmids will be stably integrated into the genome of human cells that differ in their DNA repair capacity, including cells derived from patients with xeroderma pigmentosum ataxia telangiectasia, and Bloom's syndrome, as well as cells from normal persons.