Mitotic chromosome condensation requires DNA topoisomerase 1 and TRF4. I. Castano, P. Brzoska, B. Sadoff, H. Chen, and M. Christman. Dept. of Radiation Oncology, Univ. of California San Francisco. 1855 Folsom St. San Francisco CA 94103, (415) 476-9085. DNA topoisomerase I (topo I) is not essential in S. cerevisiae, but it participates in DNA replication. The TRF4 gene is also nonessential and was identified in a synthetic lethal screen with a top1 null mutation. Here we report the surprising finding that a top1 trf4 (ts) double mutant is defective in several aspects of mitosis but not in DNA replication. Direct examination of rDNA-containing mitotic chromosomes shows that top1 trf4 (ts) mutants fail to establish and maintain chromosome condensation at mitosis. The condensation defect results in secondary failures of spindle elongation and nuclear segregation. We show that, the Trf4 protein physically associates with Smc2p, the S. cerevisiae homologue of a Xenopus protein that is required for mitotic chromosome condensation in vitro. The defect in the top1 trf4 (ts) mutant is sensed by the MAD1-dependent spindle assembly checkpoint but not by the RAD9-dependent DNA damage checkpoint, further supporting the notion that chromosome structure influences spindle assembly. These data indicate that TOP1 (encoding topo I) and TRF4 participate in overlapping or dependent steps in mitotic chromosome condensation and define a previously unrecognized bilogical function of topo I.