Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability ofSaccharomyces cerevisiae. CBF5 specifies a 55-kDa highly charged protein that contains a repeating KKD/E sequence domain near the C terminus, similar to known microtubule-binding domains in microtubule-associated proteins 1A and 1B. CBF5p A 240-kDa multisubunit protein complex (CBF3) that binds specifically to the CDEIII region of the yeast centromere has been purified and characterized (20). This protein complex is thought to be absolutely essential for centromere and kinetochore function, since it binds in vitro to the wild-type CEN sequence but not to a functionally inactive mutated CEN DNA that contains a single-base alteration in CDEIII (20,24). Affinity-purified CBF3 consists of at least three tightly associated subunits: 110 kDa (CBF3A), 64 kDa (CBF3B), and 58 kDa (CBF3C). Significantly, affinity-purified preparations of CBF3 contain an ATP-dependent motor activity that mediates movement of CEN DNA-coated microbeads along microtubules in a plus-to-minus direction (15). Thus, it is likely that CBF3 contains a kinetochore component that brings about attachment and movement of the chromosomes on the spindle microtubules. The gene (CBF2) encoding the 110-kDa subunit of this complex was cloned by using tryptic peptide sequences obtained from gel-purified CBF3A as a basis to prepare synthetic oligodeoxyribonucleotide hybridization probes, which were subsequently used to screen a yeast genomic library (17). At the same time, Goh and Kilmartin (12) cloned the identical gene by complementation of a conditional mutation (ndclO) leading to a defect in chromosome segregation. The CBF2/ NDC10 gene product is essential for viability of S. cerevisiae and for proper chromosome segregation. Although this protein contains a consensus nucleotide-binding site, no homologies to known molecular motors are apparent.In this paper, we describe the isolation of two yeast low-affinity centromere-binding proteins, topoisomerase II (Topo II) and CBF5p, and the cloning and sequencing of the CBF5 gene. The CBF5 protein contains a (KKD/E)n sequence domain highly homologous to known microtubulebinding domains in microtubule-associated protein 1A (MAP1A) and MAPlB (19,26). Evidence indicating that 4884 on May 12, 2018 by guest

Yeast two-hybrid and genetic interaction screens indicate that Bir1p, a yeast protein containing phylogenetically conserved antiapoptotic repeat domains called baculovirus inhibitor of apoptosis repeats (BIRs), is involved in chromosome segregation events. In the two-hybrid screen, Bir1p specifically interacts with Ndc10p, an essential component of the yeast kinetochore. Although Bir1p carries two BIR motifs in the N-terminal region, the C-terminal third of the protein is sufficient to provide strong interaction with Ndc10p and moderate interaction with Skp1p, another essential component of the yeast kinetochore. In addition, deletion of BIR1 is synthetically lethal with deletion of CBF1 or CTF19, genes specifying two other components of the yeast kinetochore. Yeast cells deleted of BIR1 have a chromosome-loss phenotype, which can be completely rescued by elevating NDC10 dosage. Furthermore, overexpression of either full-length or the C-terminal region of Bir1p can efficiently suppress the chromosome-loss phenotype of both bir1⌬ null and skp1-4 mutants. Our data suggest that Bir1p participates in chromosome segregation events, either directly or via interaction with kinetochore proteins, and these effects are apparently not mediated by the BIR domains of Bir1p.

Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolarcytoplasmic shuttling (U. T. Meier, and G. Blobel, J. Cell Biol. 127:1505-1514, 1994). The temperaturesensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal. The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures. A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype. SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor. A cbf5⌬ null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription.Cbf5p of the yeast Saccharomyces cerevisiae was originally isolated as one of the major low-affinity centromeric DNA (CEN) binding proteins (23). CBF5 is an essential gene encoding a highly charged protein with a domain containing ten tandem KKE/D repeats. These repeats are homologous to portions of microtubule-associated proteins 1A and 1B in the domain responsible for microtubule binding (40), and Cbf5p has been shown to bind microtubules in vitro (23). Yeast cells containing C-terminal truncated CBF5 genes delay, with replicated genomes, at the G 2 /M phase of the cell cycle, with the replicated DNA being located at the bud junction (23). Overexpression of Cbf5p suppresses the ndc10-1 temperature-sensitive (ts) mutation in the gene specifying the 110-kDa subunit (Cbf2p/Ndc10p) of the multisubunit yeast centromere DNA binding complex, CBF3, and overexpression of meiosis and centromere regulatory kinase Mck1p suppresses a ts mutation in the gene for either Cbf2p or Cbf5p (22). These genetic interactions support a direct or indirect link between Cbf5p and centromeres. However, both Cbf5p and a homologous protein (NAP57) from rats were found to be nucleolar proteins (21,33). While a functional relationship between a nucleolar protein and centromeres is not obvious, some proteins are associated with both the nucleolus and the centromere in higher eukaryotes. It has been known for some time that centromere autoantigens associate with the nucleolus (43), and experiments with autoimmune sera have also shown that a set of nucleolar proteins and ribonuclear proteins relocate around chromosomes during mitosis (16,17).The nucleolar location of Cbf5p may be indicative of a function unrelated to centromeres and chromos...

The CDC7 protein of Saccharomyces cerevisiae may be involved in the G1/S-phase transition and/or in the initiation of mitotic DNA synthesis. The CDC7 gene has two in-frame AUG codons as possible translation start sites, which would produce 58-and 56-kDa proteins, respectively. Both p58 and p56 derived from recombinant plasmids complement the temperature-sensitive growth defect of the cdc7-1 allele. To determine the biochemical function of the CDC7 protein, the CDC7 gene was cloned and polyclonal antibodies were produced against the CDC7 protein. CDC7 immune complexes prepared from yeast with these antibodies phosphorylate histone H1. Kinase activity is thermolabile in strains carrying the cdc7-1 temperature-sensitive mutant allele and is elevated >10-fold in strains carrying plasmids overexpressing either p56 or p58, confirming that the kinase in the immunoprecipitates is the CDC7 gene product. In addition, we show that CDC7 is a phosphoprotein itself. Indirect immunofluorescence and biochemical fractionation show that the CDC7 protein is present at relatively high concentrations in the nucleus compared with the cytoplasm, suggesting that nuclear proteins may be substrates for the CDC7 protein.Two major events define the eukaryotic cell cycle: replication of the chromosomes during S phase and segregation of the chromosomes during mitosis. Replication and segregation are separated by two gap periods, G1 and G2, during which the cells prepare for these events. There are also two major control points for the cell cycle in G1 and G2, the point of commitment to DNA synthesis in G1 and the regulation of progression into mitosis in G2. Although we have made great strides in understanding mitotic control in the past three years, relatively little is known about the events in G1 that lead to S phase. In yeast, the point of commitment to DNA synthesis is defined as "Start", early in G1. DNA synthesis does not ensue immediately after Start, however, and it is not clear whether the lag is due to assembly of the replication apparatus or to a cascade of controls initiated at Start. CDC28, CDC4, and CDC7 define a dependent series of events set in motion at Start (1-3). CDC28 encodes a protein kinase subunit, the analog of the highly conserved cdc2+/ MPF kinase subunit involved in regulation of mitosis (3).CDC4 is homologous to one subunit of the signal transducing protein, transducin (4). The DNA sequence of the CDC7 gene predicts a protein that has homology to catalytic domains of the protein kinases CDC2/CDC28, NIM1, and a number of kinase-related transforming proteins (5). The CDC7 sequence differs from that of other protein kinases in the so-called phosphate acceptor domain, however (5, 6). We have been interested in the role of CDC7 in the events linking Start and the initiation of DNA replication. Mutations in CDC7 appear to block a precondition for DNA synthesis because cells carrying these lesions cannot start new rounds of DNA replication after a shift from permissive to nonpermissive temperature (7,8). The cd...

The binding sites and consecutive binding constants of alkali metal ions, (M ϩ ϭ Na ϩ , K ϩ , Rb ϩ , and Cs ϩ ), to thrombin-binding aptamer (TBA) DNA were studied by Fouriertransform ion cyclotron resonance spectrometry. TBA-metal complexes were produced by electrospray ionization (ESI) and the ions of interest were mass-selected for further characterization. The structural motif of TBA in an ESI solution was checked by circular dichroism. The metal-binding constants and sites were determined by the titration method and infrared multiphoton dissociation (IRMPD), respectively. The binding constant of potassium is 5-8 times greater than those of other alkali metal ions, and the potassium binding site is different from other metal binding sites. In the 1:1 TBA-metal complex, potassium is coordinated between the bottom G-quartet and two adjacent TT loops of TBA. In the 1:2 TBA-metal complex, the second potassium ion binds at the TGT loop of TBA, which is in line with the antiparallel G-quadruplex structure of TBA. On the other hand, other alkali metal ions bind at the lateral TGT loop in both 1:1 and 1:2 complexes, presumably due to the formation of ion-pair adducts. IRMPD studies of the binding sites in combination with measurements of the consecutive binding constants help elucidate the binding modes of alkali metal ions on DNA aptamer at the molecular level. . The active form of TBA adopts a chair-type intramolecular G-quadruplex (G4), where the two G-tetrads are interconnected through the lateral TT and TGT loops in antiparallel conformation [2][3][4][5][6]. Some metal ions induce the structural transition to the G4 structure upon binding [7][8][9][10]. The binding modes of metal ions on the G4 structure are of fundamental interest in understanding their specific and/or nonspecific interactions. Various methods have been applied to the studies of binding modes and structures of TBA-metal complexes. NMR experiments have shown that potassium induces the chair-type G-quadruplex formation, but sodium does not [11]. Circular dichroism (CD) and UV absorption studies have shown that potassium, strontium, and barium stabilize the G4 structure in solution [12]. Recently, electrospray ionization mass spectrometry (ESI-MS) has confirmed the unimolecular G4 structure of TBA-metal (metal ϭ potassium, strontium and barium) complexes in the gas phase by H/D exchange [13]. Here, we studied the binding modes of alkali metal ions on TBA and determined the consecutive binding constants to unravel the specific and/or non-specific interactions between alkali metal ions and G4 TBA.Most of the structural studies carried out in solution deal with an ensemble of dynamic structures present under ambient conditions. To more firmly establish the binding mode, we need to employ a molecular probe that is selective toward the specific ions of interest. ESI-MS offers a mass-specific means to bring the complex ions present in solution into the gas-phase [14,15] for the studies of noncovalent ligand-nucleic acid interactions. Moreover, ESI-MS h...