A brute force postgenome approach to identify temperature‐sensitive mutations that negatively interact with separase and securin plasmids

Abstract

Background: The fission yeast Schizosaccharomyces pombe separase/Cut1 and securin/Cut2 are required for anaphase‐specific activation of proteolysis that leads to proper sister chromatid separation. We intended to identify ts (temperature sensitive) strains whose growth was inhibited by multicopy plasmid pCUT1 or pCUT2 at the permissive temperature. Results: After a one‐by‐one transformation of 1015 randomly isolated ts strains, 18 transformants that retarded in colony formation at the permissive or semipermissive temperature were isolated. Six of them, in the absence of pCUT1 or pCUT2, produced mitotic phenotypes with condensed chromosomes at the restrictive temperature. Gene cloning established that these mutants were defective in either the subunits (Cut9, Cut23, Cut20 or Apc10) of APC (anaphase promoting complex)/cyclosome or Cut8, a regulator for 26S proteasome localization. The inhibitory effect of separase against APC/cyclosome mutations was abolished when the catalytic site mutation C1730A was introduced and overproduced, indicating that inhibition needs an active separase. Securin/Cut2 overproduction also caused a negative effect on these mutants. Surprisingly, the phenotypes of cut9 and cut23 in the presence of pCUT1 or pCUT2 were not the mitotic arrest, and they were strikingly different depending on pCUT1 or pCUT2. Conclusions: This study shows the functional link between separase/Cut1 and APC/cyclosome in a separase activity‐dependent manner. The negative effect of active separase overproduction on APC/cyclosome mutations is possibly due to the direct inhibition of APC/cyclosome. In addition, the manner of the inhibition by high copy securin and separase plasmids were quite different each other and did not result in the mitotic block.

Cdc48 is required for the stability of Cut1/separase in mitotic anaphase

“…1A. Plasmid pCUT1 carrying the separase gene was introduced into each of »1000 ts strains that were made by random mutagenesis, and a large number of resulting transformants were plated for colony formation at 36°C, the restrictive temperature (Matsumura et al, 2003;Hayashi et al, 2004;Yuasa et al, 2004). Two ts mutants (353 and 605) other than cut1 alleles were nearly fully rescued by plasmid pCUT1.…”

Cti1/C1D interacts with condensin SMC hinge and supports the DNA repair function of condensin

“…Schizosaccharomyces pombe haploid strains cut3-477, cut14-208, cnd2-1, ⌬cds1, rad1-1, ⌬rad3, and ⌬rad17 have been described (11,13,14). Cut14-H488 is another allele of cut14 ϩ that was isolated from a recent large-scale temperature-sensitive (ts) mutant screen (15). The complete yeast extract͞peptone͞ dextrose medium and Edinburgh minimal media 2 (EMM2) media were used (16).…”

Mapping epigenetic mutations in fission yeast using whole-genome next-generation sequencing

“…In a genetic screen for transcriptional silencing defects, we assayed a subset of a large collection of mutagenized S. pombe strains that were temperature sensitive (ts) for growth. In each case, the growth defect could be suppressed by one or more plasmids recovered from a genomic library that had been systematically transformed into each strain (Matsumura et al 2003;Hayashi et al 2004;Yuasa et al 2004). Twenty-seven ts strains were screened by reverse transcription polymerase chain reaction (RT-PCR) for repeat transcript accumulation at the nonpermissive temperature of 36°C.…”

“…Using genome sequencing, we were able to determine which of the 10 or more genes that rescued a ts lethal strain was responsible for the mutation. By sequencing each of the 1000 ts lethal strains in this collection (Matsumura et al 2003;Hayashi et al 2004;Yuasa et al 2004), the causative mutation underlying each strain will be revealed, identifying the other rescuing plasmids as allele-specific suppressors. This will rapidly lead to a genetic network of genes essential for growth, complementing more traditional and laborious approaches such as epistasis mapping (Roguev et al 2008).…”