BIOLOGICAL FUNCTIONS OF TOPOISOMERASE I IN S CEREVISIAE

这项提议的长期目标是了解有丝分裂如何 染色体凝聚(MCC)的发生及其调控 细胞周期。鉴于MCC的第一次观测早于 将染色体识别为遗传信息的载体， 染色体改变的分子机制 冷凝过程中的结构仍然知之甚少，而且几乎 关于它的细胞周期调控，我们一无所知。为了更好地理解 我们将使用酿酒酵母分子遗传学的组合 和非洲爪哇细胞自由染色体凝聚系统。我们会 关注Top1P(DNA拓扑异构酶I)的作用和 拓扑异构酶相关功能，Tfr4p和Trf5p。参与 MCC代表了一种以前未被认识到的新生物 我们已经发现了Top1p的功能。我们已经证明了Top1 酿酒酵母S的trf4-ts和trf4-ts trf5突变体在MCC中存在缺陷 并且Trf4p与Smc2p，一种蛋白质 单副本区域中的MCC需要。SMC蛋白家族 与病毒中MCC所需的非洲爪哇蛋白有关， 提示MCC的发病机制是进化保守的。 TRF4基因家族在进化上也高度保守， 仅代表已知的第三类蛋白质 MCC.我们的假设是Trf4p/Smc2p复合体与 直接与所有浓缩的染色体区域结合，而Top1p是 用来缓解与冷凝有关的扭转应力 进程。我们进一步假设，委员会的其他成员 Trf4p/Smc2p复合体是染色体凝聚和 该复合体的功能在细胞周期中由 CDC28p/Clb2p激酶。在这个提案中，我们介绍了实验 旨在直接检验这一假说。

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.