Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4.
(41/2033)
The condensin complex in frog extracts, containing two SMC (structural maintenance of chromosomes) and three non-SMC subunits, promotes mitotic chromosome condensation, and its supercoiling activity increases during mitosis by Cdc2 phosphorylation. Here, we report that fission yeast has the same five-member condensin complex, each of which is essential for mitotic condensation. The condensin complex was purified and the subunits were identified by microsequencing. Cnd1, Cnd2, and Cnd3, three non-SMC subunits showing a high degree of sequence conservation to frog subunits, are essential for viability, and their gene disruption leads to a phenotype indistinguishable from that observed in cut3-477 and cut14-208, known mutations in SMC4 and SMC2-like subunits. Condensin subunits tagged with GFP were observed to alter dramatically their localization during the cell cycle, enriched in the nucleus during mitosis, but cytoplasmic during other stages. This stage-specific alteration in localization requires mitosis-specific phosphorylation of the T19 Cdc2 site in Cut3. The T19 site is phosphorylated in vitro by Cdc2 kinase and shows the maximal phosphorylation in metaphase in vivo. Its alanine substitution mutant fails to suppress the temperature-sensitive phenotype of cut3-477, and shows deficiency in condensation, probably because Cut3 T19A remains cytoplasmic. Therefore, direct Cdc2 phosphorylation of fission yeast condensin may facilitate its nuclear accumulation during mitosis. (+info)
Clustered adenine/thymine stretches are essential for function of a fission yeast replication origin.
(42/2033)
We have determined functional elements required for autonomous replication of the Schizosaccharomyces pombe ars2004 that acts as an intrinsic chromosomal replication origin. Internal deletion analysis of a 940-bp fragment (ars2004M) showed three regions, I to III, to be required for autonomously replicating sequence (ARS) activity. Eight-base-pair substitutions in the 40-bp region I, composed of arrays of adenines on a DNA strand, resulted in a great reduction of ARS activity. Substitutions of region I with synthetic sequences showed that no specific sequence but rather repeats of three or more consecutive adenines or thymines, without interruption by guanine or cytosine, are required for the ARS activity. The 65-bp region III contains 11 repeats of the AAAAT sequence, while the 165-bp region II has short adenine or thymine stretches and a guanine- and cytosine-rich region which enhances ARS activity. All three regions in ars2004M can be replaced with 40-bp poly(dA/dT) fragments without reduction of ARS activity. Although spacer regions in the ars2004M enhance ARS activity, all could be deleted when an 40-bp poly(dA/dT) fragment was added in place of region I. Our results suggest that the origin activity of fission yeast replicators depends on the number of adenine/thymine stretches, the extent of their clustering, and presence of certain replication-enhancing elements. (+info)
Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins.
(43/2033)
We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1(+)/cdc19(+) and mis5(+) genes, respectively, were associated with chromatin DNA only during the G(1) and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G(1) and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G(1) and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication. (+info)
Characterization and function in vivo of two novel phospholipases B/lysophospholipases from Saccharomyces cerevisiae.
(44/2033)
The yeast genome contains two genes, designated as PLB2 and PLB3, that are 67% and 62% identical, respectively, to PLB1, which codes for a phospholipase B/lysophospholipase in yeast (Lee, S. K., Patton, J. L., Fido, M., Hines, L. K., Kohlwein, S. D., Paltauf, F., Henry, S. A., and Levin, D. E. (1994) J. Biol. Chem. 269, 19725-19730). Deletion and overexpression studies and in vivo and in vitro activity measurements suggest that both genes indeed code for phospholipases B/lysophospholipases. In cell free extracts of a plb1 plb2 plb3 triple mutant, no phospholipase B activity was detectable. Upon overexpression of PLB2 in a plb1 plb3 mutant background, phospholipase B activity was detectable in the plasma membrane, periplasmic space extracts and the culture supernatant. Similar to Plb1p, Plb2p appears to accept all major phospholipid classes, with a preference for acidic phospholipids including phosphatidylinositol 3',4'-bisphosphate and phosphatidic acid. Consistent with a function as an extracellular lysophospholipase, PLB2 overexpression conferred resistance to lyso-phosphatidylcholine. Deletion of Plb2p function had no effect on glycerophosphoinositol or glycerophosphocholine release in vivo, in contrast to a deletion of Plb3p function, which resulted in a 50% reduction of phosphatidylinositol breakdown and glycerophosphoinositol release from the cells. In vitro, Plb3p hydrolyzes only phosphatidylinositol and phosphatidylserine and, to a lesser extent, their lyso-analogs. Plb3p activity in a plb1 plb2 mutant background was observed in periplasmic space extracts. Both Plb3p and Plb2p display transacylase activity in vitro, in the presence or absence, respectively, of detergent. (+info)
Identification of cohesin association sites at centromeres and along chromosome arms.
(45/2033)
A multisubunit cohesin complex holds sister chromatids together after DNA replication. Using chromatin immunoprecipitation, we detected cohesin association with centromeres and with discrete sites along chromosome arms from S phase until metaphase in S. cerevisiae. Short DNA sequences (130-280 bp) are sufficient to confer cohesin association. Cohesin association with a centromere depends on Mif2p, the centromere binding factor CBF3, and a centromere-specific histone variant, Cse4p. Because only active centromeres confer cohesin association with centromeric DNA, we suggest that cohesin is recruited by the same chromatin structure that confers the attachment of microtubules. Propagation of this structure might be partly epigenetic. Finally, cohesion associated with "minimal" centromeres is insufficient to resist the splitting force exerted by microtubules and appears to be reinforced by cohesion provided by their flanking DNA sequences. (+info)
DNA damage triggers disruption of telomeric silencing and Mec1p-dependent relocation of Sir3p.
(46/2033)
In eukaryotic cells, surveillance mechanisms detect and respond to DNA damage by triggering cell-cycle arrest and inducing the expression of DNA-repair genes [1]. In budding yeast, a single DNA double-strand break (DSB) is sufficient to trigger cell-cycle arrest [2]. One highly conserved pathway for repairing DNA DSBs is DNA non-homologous end-joining (NHEJ), which depends on the DNA end-binding protein Ku [3]. NHEJ also requires the SIR2, SIR3 and SIR4 gene products [4] [5], which are responsible for silencing at telomeres and the mating-type loci [6]. Because of the link between NHEJ and the Sir proteins, we investigated whether DNA damage influences telomeric silencing. We found that DNA damage triggers the reversible loss of telomeric silencing and relocation of Sir3p from telomeres. Complete Sir3p relocation was triggered by a single DNA DSB, suggesting that the singal is amplified. Consistent with this idea, Sir3p relocation depended on the DNA damage-signalling components Ddc1p and Mec1p. Thus, signalling of DNA damage may release Sir3p from telomeres and permit its subsequent association with other nuclear subdomains to regulate transcription, participate in DNA repair and/or enhance genomic stability by other mechanisms. (+info)
Saccharomyces cerevisiae checkpoint genes MEC1, RAD17 and RAD24 are required for normal meiotic recombination partner choice.
(47/2033)
Checkpoint gene function prevents meiotic progression when recombination is blocked by mutations in the recA homologue DMC1. Bypass of dmc1 arrest by mutation of the DNA damage checkpoint genes MEC1, RAD17, or RAD24 results in a dramatic loss of spore viability, suggesting that these genes play an important role in monitoring the progression of recombination. We show here that the role of mitotic checkpoint genes in meiosis is not limited to maintaining arrest in abnormal meioses; mec1-1, rad24, and rad17 single mutants have additional meiotic defects. All three mutants display Zip1 polycomplexes in two- to threefold more nuclei than observed in wild-type controls, suggesting that synapsis may be aberrant. Additionally, all three mutants exhibit elevated levels of ectopic recombination in a novel physical assay. rad17 mutants also alter the fraction of recombination events that are accompanied by an exchange of flanking markers. Crossovers are associated with up to 90% of recombination events for one pair of alleles in rad17, as compared with 65% in wild type. Meiotic progression is not required to allow ectopic recombination in rad17 mutants, as it still occurs at elevated levels in ndt80 mutants that arrest in prophase regardless of checkpoint signaling. These observations support the suggestion that MEC1, RAD17, and RAD24, in addition to their proposed monitoring function, act to promote normal meiotic recombination. (+info)
Genetic control of recombination partner preference in yeast meiosis. Isolation and characterization of mutants elevated for meiotic unequal sister-chromatid recombination.
(48/2033)
Meiotic exchange occurs preferentially between homologous chromatids, in contrast to mitotic recombination, which occurs primarily between sister chromatids. To identify functions that direct meiotic recombination events to homologues, we screened for mutants exhibiting an increase in meiotic unequal sister-chromatid recombination (SCR). The msc (meiotic sister-chromatid recombination) mutants were quantified in spo13 meiosis with respect to meiotic unequal SCR frequency, disome segregation pattern, sporulation frequency, and spore viability. Analysis of the msc mutants according to these criteria defines three classes. Mutants with a class I phenotype identified new alleles of the meiosis-specific genes RED1 and MEK1, the DNA damage checkpoint genes RAD24 and MEC3, and a previously unknown gene, MSC6. The genes RED1, MEK1, RAD24, RAD17, and MEC1 are required for meiotic prophase arrest induced by a dmc1 mutation, which defines a meiotic recombination checkpoint. Meiotic unequal SCR was also elevated in a rad17 mutant. Our observation that meiotic unequal SCR is elevated in meiotic recombination checkpoint mutants suggests that, in addition to their proposed monitoring function, these checkpoint genes function to direct meiotic recombination events to homologues. The mutants in class II, including a dmc1 mutant, confer a dominant meiotic lethal phenotype in diploid SPO13 meiosis in our strain background, and they identify alleles of UBR1, INP52, BUD3, PET122, ELA1, and MSC1-MSC3. These results suggest that DMC1 functions to bias the repair of meiosis-specific double-strand breaks to homologues. We hypothesize that the genes identified by the class II mutants function in or are regulators of the DMC1-promoted interhomologue recombination pathway. Class III mutants may be elevated for rates of both SCR and homologue exchange. (+info)