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.
