BIOLOGICAL FUNCTIONS OF TOPOISOMERASE I IN S CEREVISIAE

这项提案的长期目标是了解有丝分裂是如何 染色体凝聚（MCC）的发生以及它是如何在 细胞周期 虽然MCC的第一次观察早于 确定染色体作为遗传信息的载体， 染色体变化的分子机制 冷凝过程中结构仍然知之甚少， 但对它的细胞周期调控却一无所知。 为了更好地理解 MCC我们将使用S的组合。酿酒酵母分子遗传学 和非洲爪蟾无细胞染色体浓缩系统。 我们将 关注Top1 P（DNA拓扑异构酶I）和 拓扑异构酶相关功能，Tfr 4p和Trf 5 p。 参与 MCC代表了一种新的和以前未被认识的生物学 Top1 p的功能。 我们已经证明，top1 trf 4-ts和trf 4-tstrf 5突变体。Cerevisiae在MCC中存在缺陷 Trf 4p与Smc 2 p（一种蛋白质）相关， 在单拷贝区域中需要MCC。 SMC蛋白家族 与体内MCC所需的爪蟾蛋白有关， 提示MCC的发生机制具有进化保守性。 TRF 4基因家族在进化上也是高度保守的， 只代表了已知的第三类蛋白质， MCC。 我们的假设是Trf 4p/Smc 2 p复合物与 直接与所有浓缩的染色体区域，Top1 p是 需要释放与冷凝相关的扭转应力 过程 我们进一步假设， Trf 4p/Smc 2 p复合体是染色体凝聚所必需的， 复合物的功能在细胞周期中由 Cdc 28 p/Clb 2 p激酶。 在这个提议中，我们提出了实验， 设计来直接检验他的假设。

The long-term goal of this proposal is to understand how mitotic chromosome condensation (MCC) occurs and how it is regulated during the cell cycle. Whereas the first observations of MCC pre-date the identification of chromosomes as the carriers of genetic information, the molecular mechanisms underlying the changes of chromosome structure during condensation are still poorly understood and virtually nothing is known about its cell cycle regulation. To better unerstand MCC we will use a combination of S. Cerevisiae molecular-genetics and a Xenopus cell free chromosome condensation system. We will focus on the roles of Top1P (DNA topoisomerase I) and the Topoisomerase Related Functions, Tfr4p and Trf5p. Involvement in MCC represents a novel and previously unrecognized biological function of Top1p that we have discovered. We have shown that top1 trf4-ts and trf4-ts trf5 mutants of s. Cerevisiae are defective in MCC of the rDNA array, and that Trf4p associates with Smc2p, a protein required for MCC in single copy regions. The SMC family of proteins are related to Xenopus proteins that are required for MCC in viro, suggesting that the mechanism of MCC is evolutionarity conserved. The TRF4 gene family is also highly conserved evolutionarily and represents only the third class of proteins known to be required for MCC. It is our hypothesis that the Trf4p/Smc2p complex is associated directly with all condensed chromosomal regions and that Top1p is required to relieve the torsional stress associated with the condensation process. We further hypothesize that the other members of the Trf4p/Smc2p complex are required for chromosome condensation and that function of the complex is regulated during the cell cycle by the Cdc28p/Clb2p kinase. In this proposal, we present experiments designed to est htis hypothesis directly.