Pol kappa: A DNA polymerase required for sister chromatid cohesion.

Establishment of cohesion between sister chromatids is coupled to replication fork passage through an unknown mechanism. Here we report that TRF4, an evolutionarily conserved gene necessary for chromosome segregation, encodes a DNA polymerase with beta-polymerase-like properties. A double mutant in the redundant homologs, TRF4 and TRF5, is unable to complete S phase, whereas a trf4 single mutant completes a presumably defective S phase that results in a failure of cohesion between the replicated sister chromatids. This suggests that TRFs are a key link in the coordination between DNA replication and sister chromatid cohesion. Trf4 and Trf5 represent the fourth class of essential nuclear DNA polymerases (designated DNA polymerase kappa) in Saccharomyces cerevisiae and probably in all eukaryotes.

The topoisomerase-related function gene TRF4 affects cellular sensitivity to the antitumor agent camptothecin.

Camptothecin is an antitumor agent that kills cells by converting DNA topoisomerase I into a DNA-damaging poison. Although camptothecin derivatives are now being used to treat tumors in a variety of clinical protocols, the cellular factors that influence sensitivity to the drug are only beginning to be understood. We report here that two genes required for sister chromatid cohesion, TRF4 and MCD1/SCC1, are also required to repair camptothecin-mediated damage to DNA. The hypersensitivity to camptothecin in the trf4 mutant does not result from elevated expression of DNA topoisomerase I. We show that Trf4 is a nuclear protein whose expression is cell cycle-regulated at a post-transcriptional level. Suppression of camptothecin hypersensitivity in the trf4 mutant by gene overexpression resulted in the isolation of three genes: another member of the TRF4 gene family, TRF5, and two genes that may influence higher order chromosome structure, ZDS1 and ZDS2. We have isolated and sequenced two human TRF4 family members, hTRF4-1 and hTRF4-2. The hTRF4-1 gene maps to chromosome 5p15, a region of frequent copy number alteration in several tumor types. The evolutionary conservation of TRF4 suggests that it may also influence mammalian cell sensitivity to camptothecin.

Mitotic chromosome condensation in the rDNA requires TRF4 and DNA topoisomerase I in Saccharomyces cerevisiae.

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 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 serve to define a previously unrecognized biological function of topo I.

A novel family of TRF (DNA topoisomerase I-related function) genes required for proper nuclear segregation.

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.

Isolation of mutants of Saccharomyces cerevisiae requiring DNA topoisomerase I.

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.