DNA topoisomerase I (topo I) is known to participate in the process of DNA replication, but is not essential in Saccharomyces cerevisiae. The TRF4 gene is also nonessential and was identified in a screen for mutations that are inviable in combination with a top1 null mutation. Here we report the surprising finding that a top1 trf4-ts double mutant is defective in the mitotic events of chromosome condensation, spindle elongation, and nuclear segregation, but not in DNA replication. Direct examination of rDNA-containing mitotic chromosomes demonstrates that a top1 trf4-ts mutant fails both to establish and to maintain chromosome condensation in the rDNA at mitosis. We show that the Trf4p associates physically with both Smclp and Smc2p, the S. cerevisiae homologs of Xenopus proteins that are required for mitotic chromosome condensation in vitro. The defect in the top1 trf4-ts mutant is sensed by the MADl-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 serve to define a previously unrecognized biological function of topo I.[Key Words: DNA topoisomerase; chromosome condensation; mitosis; nuclear division; checkpoint] Received July 29, 1996; revised version accepted August 26, 1996.Chromosomes are involved in dynamic cellular processes such as DNA replication, transcription, chromatin assembly, mitotic condensation, and genetic recombination that can lead to the formation of local domains of torsional stress (for review, see Wang and Lynch 1993).In Saccharomyces cerevisiae, DNA topoisomerases I and II (topo I and II) appear to act together during DNA replication as a swivel to relieve torsional stress at the replication fork. When both topo I and II are inactivated, DNA replication stops rapidly (Brill et al. 1987), with elongation of new DNA chains continuing for only a few thousand nucleotides (Kim and Wang 1989). Transcription can also lead to the formation of locally supercoiled domains in DNA. In the twin domain model (Liu and Wang 1987), movement of a transcription complex along the helical backbone generates positive supercoils ahead of the complex and negative supercoils behind the complex. In S. cerevisiae, topo I and II function together to facilitate rRNA transcription and, to a lesser extent, mRNA transcription (Brill et al. 1987).Despite the considerable evidence for the involvement ~Present address:

We recently reported the identification of a gene, TRF4 (for DNA topoisomerase related function), in a screen for mutations that are synthetically lethal with mutations in DNA topoisomerase I (top1). Here we describe the isolation of a second member of the TRF4 gene family, TRF5. Overexpression of TRF5 complements the inviability of top1 trf4 double mutants. The predicted Trf5 protein is 55% identical and 72% similar to Trf4p. As with Trf4p, a region of Trf5p is homologous to the catalytically dispensable N-terminus of Top1p. The TRF4/5 function is essential as trf4 trf5 double mutants are inviable. A trf4 (ts) trf5 double mutant is hypersensitive to the anti-microtubule agent thiabendazole at a semi-permissive temperature, suggesting that TRF4/5 function is required at the time of mitosis. Examination of nuclear morphology in a trf4 (ts) trf5 mutant at a restrictive temperature reveals the presence of many cells undergoing aberrant nuclear division, as well as many anucleate cells, demonstrating that the TRF4/5 function is required for proper mitosis. Database searches reveal the existence of probable Schizosaccharomyces pombe and human homologs of Trf4p, indicating that TRF4 is the canonical member of a gene family that is highly conserved evolutionarily.

AU the chromosomes from isogenic TOP) and top) strains have similar mobility on pulsed-field gels except for chromosome XII, which fails to migrate into the gels in top) mutants. Chromosome XII contains the tandem repeats of rRNA-encoding DNA (rDNA). When a segment ofchromosome XII containing only rDNA is transferred to chromosome m by a recombination event, chromosome m fails to enter a pulsedfield gel in extracts from top) strains, indicating that the aberrant migration of chromosome XII in topl mutants is caused by the presence of rDNA. Failure of chromosome XII to migrate into a pulsed-field gel occurs only in preparations from exponentially growing topl cultures and not in preparations from stationary-phase topl cultures. rDNA from a top) strain does enter the gel if it is cut with an enzyme (Pst I) that cuts the tandem rDNA array into single 9-kb repeat units, indicating that more than a single repeat unit is required to maintain the aberrant structure.

Despite evidence that DNA topoisomerase I is required to relieve torsional stress during DNA replication and transcription, yeast strains with a top1 null mutation are viable and display no gross defects in DNA or RNA synthesis, possibly because other proteins provide overlapping functions. We isolated mutants whose inviablility or growth defect is relieved when TOP1 is expressed [trf mutants (topoisomerase one-requiring function)]. The TRF genes define at least four complementation groups. TRF3 is allelic to TOP2. TRF1 is allelic to HPR1, previously shown to be homologous to TOP1 over two short regions. TRF4 encodes a novel 584-amino acid protein with homology to the N-terminus of Saccharomyces cerevisiae topo I. Like top1 mutants, trf4 mutants have elevated rDNA recombination and fail to shut off RNA polymerase II transcription in stationary phase. trf4 null mutants are cs for viability, display reduced expression of GAL1 and Cell Cycle Box UAS::LacZ fusions, and are inviable in combination with trfI null mutants, indicating that both proteins may share a common function with DNA topoisomerase I. The existence of multiple TRF complementation groups suggests that not all biological functions of topo I can be carried out by topo II.