MECHANISMS OF HOMOLOGOUS RECOMBINATION IN HUMAN CELLS

我们研究的长远目标是了解 致癌物质引起恶性转化的机制 人类的呼唤 现在有强有力的证据。 从几个 在不同领域，癌症的发生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，在这方面， 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， 突变的等位基因，允许隐性性状的表达。 对基因重组的机制知之甚少， 哺乳动物细胞 我们最近证明了化学和 物理致癌物可以诱导同源重组之间 哺乳动物细胞中的染色体基因（Wang等，摩尔Cell. ，8，196-202，1988）。 我们打算在本报告中讨论的问题是， 建议的研究是：这些致癌物质如何诱导 重组？ 涉及哪些机制？ 是重组 受致癌物诱导的损伤干扰的刺激， 与S期的DNA复制有关吗 替代地 通过在DNA中引入断裂而刺激的重组 由切除修复过程或单链区域引起 在修复过程中产生的亲本DNA DNA连接酶的作用 在重组中发挥作用？ 是来自癌症易感个体的细胞 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， 含有两个拷贝的编码可选择的 遗传标记 每个基因在一个独特的位点被一个 限制性内切核酸酶的8bp接头位点，使得 需要生产性重组事件来获得函数 （可选择的）基因产物。 质粒将稳定整合 人类细胞的基因组中， 能力，包括干皮病患者的细胞 色素性共济失调毛细血管扩张症和布卢姆综合征 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.