Isolation of mutants of Saccharomyces cerevisiae requiring DNA topoisomerase I.

Abstract

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.

The nuclear RNA polymerase II surveillance system targets polymerase III transcripts

“…All three strains showed wild‐type (WT) growth rates, indicating that the fusion proteins were functional. The trf4 Δ strain is cs‐lethal at 18°C (Sadoff et al , 1995), and impairs growth at all temperatures. The cs‐lethal phenotype was fully complemented by the tagged construct (Supplementary Figure S1A).…”

Chromosome cohesion: A polymerase for chromosome bridges

“…Polymerase κ was previously known as Trf4 in budding yeast. Trf4 was initially identified as a topoisomerase I-related activity in a screen for mutations that are lethal in the absence of the non-essential topoisomerase I [11]. Trf4 itself is not, however, a topoisomerase, and unexpectedly trf4 mutant cells turned out to be defective in sister chromatid cohesion [1].…”