ISOLATION AND ANALYSIS OF HUMAN DNA REPAIR GENES

该计划的目标是阐明两个新的哺乳动物基因的功能。 其中第一个是SNM 1，酵母DNA修复基因的哺乳动物同源物。 该基因是rad 3上位性组的成员，并且已知特异性地参与由链间交联剂产生的损伤的修复。 来自酵母研究的证据表明，该基因的表达产物在交联修复的后期发挥作用，以实现高分子量DNA的重建。 第二个基因与果蝇的北极星基因关系最密切。 在果蝇中对该基因的研究表明，它是一个有丝分裂因子，参与分裂后期染色体的分离。 北极星的确切功能尚不清楚，并已被假定参与许多过程，包括染色体凝聚，DNA修复或维持姐妹染色单体凝聚力的因素的溶解，如DNA缠绕或蛋白质-蛋白质相互作用。 我们计划通过多种方法分析这些基因在哺乳动物细胞中的功能。 这些包括作为细胞周期的函数的蛋白质表达和定位，与新的或已知的因子的蛋白质-蛋白质相互作用，以及通过包括反义表达、嵌合寡核苷酸转化和构建纯合-/-敲除小鼠的几种方法破坏功能。

The goals of this proposal are to elucidate the function of two novel mammalian genes. The first of these is SNM1, a mammalian homologue of the yeast DNA repair gene. This gene is a member of the rad3 epistasis group and is known to be specifically involved in the repair of lesions produced by interstrand crosslinking agents. Evidence from studies in yeast suggests that the expression product of this gene functions in the later stages of crosslink repair to effect reconstitution of high molecular weight DNA. The second gene is most closely related to the lodestar gene of Drosophila. Studies of this gene in Drosophila suggest that it is a mitotic factor involved in the segregation of chromosomes during anaphase. The exact function of lodestar is not clear, and has been postulated to be involved in a number of processes including chromosomal condensation, DNA repair, or dissolution of the factors that maintain sister chromatid cohesion, such as DNA intertwining or protein-protein interactions. We plan to analyze the function of these genes in mammalian cells by a variety of methods. These include protein expression and localization as a function of the cell cycle, protein-protein interactions with either novel or known factors, and disruption of function through several approaches including antisense expression, conversion by chimeric oligonucleotides, and construction of homozygous -/- knockout mice.