No abstract

We have identified an abundant ribonucleoprotein particle from Schizosaccharomyces pombe with properties related to those of the vertebrate signal recognition particle (SRP), including cytoplasmic localization, association with microsomes and ribosomes at low, but not high, salt concentrations and high resistance to micrococcal nuclease. The 256‐nucleotide RNA component carries a 5′‐triphosphate group and shows close secondary structure, and limited primary sequence homology to vertebrate 7SL RNA. 7SL‐like RNAs were also detected in a number of other fungi. The single copy gene (SRP7) encoding S.pombe 7SL was disrupted by insertion of a transposon carrying the selective marker LEU2, and the disrupted gene was used to replace one chromosomal SRP7 gene in a diploid strain. Haploid srp7[unk] strains fail to germinate.

Sap1 is a dimeric sequence-specific DNA binding-protein, initially identified for its role in mating-type switching of the fission yeast Schizosaccharomyces pombe. The protein is relatively abundant, around 10,000 dimers/cell, and is localized in the nucleus. sap1؉ is essential for viability, and transient overexpression is accompanied by rapid cell death, without an apparent checkpoint response and independently of mating-type switching. Time lapse video microscopy of living cells revealed that the loss of viability is accompanied by abnormal mitosis and chromosome fragmentation. Overexpression of the C terminus of Sap1 induces minichromosome loss associated with the "cut" phenotype (uncoupling mitosis and cytokinesis). These phenotypes are favored when the C terminus of Sap1 is overexpressed during DNA replication. Fluorescence in situ hybridization experiments demonstrated that the cut phenotype is related to precocious centromere separation, a typical marker for loss of cohesion. We propose that Sap1 is an architectural chromatin-associated protein, required for chromosome organization.Eukaryotic chromosomes are organized into discrete domains or loops defining independent and functional units that are attached to a proteinaceous nuclear scaffold (27). This higher-order structural organization is not static but rather is dramatically remodeled during the cell cycle such as the DNA replication and mitotic phases. In addition, transcriptional regulation and site-specific recombination are also linked to modulation of the chromatin architecture during interphase (for reviews, see references 16, 24, and 38).It is generally accepted that during DNA replication, sister chromatids are aligned and held together by a tight chromosomal connection that is conserved throughout the G 2 phase, until the metaphase/anaphase transition. The beginning of the mitotic phase is accompanied by chromosome condensation, which is essential for topoisomerase II-dependent DNA decatenation of intertwined sister chromatids. It has been proposed that the disruption of both linkages between sister chromatids allows them to segregate to opposite poles of the cell (34).The main candidate for organizing sister chromatids together is the cohesin-condensing complexes, which have also been implicated in recombination and DNA repair (36, 37) and more recently in postreplicative double-strand-break repair (48). Interestingly, Scc1p, known as Rad21 in Schizosaccharomyces pombe, was first identified as a DNA repair protein (13). Other players, not part of these complexes, interact with the replication machinery and have been proposed to participate in the establishment of cohesion between sister chromatids (for a review, see reference 14).These recent findings indicate a direct connection between cohesion and the replication-repair machinery, supporting the notion that cohesion participates in maintaining genomic integrity and ensuring segregation of two identical copies of the genetic material into daughter cells. The result is the production of t...

The sap1 gene from Schizosaccharomyces pombe, which is essential for mating-type switching and for growth, encodes a sequence-specific DNA-binding protein with no homology to other known proteins. We have used a reiterative selection procedure to isolate binding sites for sap1, using a bacterially expressed protein and randomized double-strand oligonucleotides. The sap1 homodimer preferentially selects a pentameric motif, TA(A/G)CG, organized as a direct repeat and spaced by 5 nucleotides. Removal of a C-terminal dimerization domain abolishes recognition of the direct repeat and creates a new specificity for a DNA sequence containing the same pentameric motif but organized as an inverted repeat. We present evidence that the orientation of the DNA-binding domain is controlled by two independent oligomerization interfaces. The C-terminal dimerization domain allows a head-to-tail organization of the DNA-binding domains in solution, while an N-terminal domain is involved in a cooperative interaction on the DNA target between pairs of dimers.

Mating type switching in fission yeast, Schizosaccharomyces pombe, is initiated by a site-specific double-strand break (DSB) at the mat1 locus. The DSB is controlled from a distance by cis- and trans-acting elements. The switch-activating protein, Sap1 binds to the SAS1 cis-acting element which controls the frequency of the DSB at the mat1 locus and, consequently the efficiency of mating type switching. We developed a general method for screening randomly mutagenized expression libraries of DNA-binding protein in E.coli. Sap1 gene was mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA was expressed in E.coli and screened for SAS1-recognition. This method was used to isolated 16 point mutations that abolished SAS1 interaction together with 18 mutations that did not affect binding. The position of these point mutations allowed the identification of three protein domains located in the N-terminal part of Sap1 that are essential for DNA-binding. Deletions and biochemical analysis showed that Sap1 is a dimer both in solution and when bound to SAS1 sequence. The dimerization domain was localized C-terminally to the three domains described above and when used in exess it inhibited DNA binding.