cis-acting components in the replication origin from ribosomal DNA of Saccharomyces cerevisiae.

The ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae contain an autonomously replicating sequence (ARS) that colocalizes with a chromosomal origin of replication. We show that a minimal sequence necessary for full ARS function corresponds to a 107-bp rDNA fragment which contains three 10-of-11-bp matches to the ARS consensus sequence. Point mutations in only one of the 10-of-11-bp matches, GTTTAT GTTTT, inactivate the rDNA ARS, indicating that this consensus sequence is essential. A perfect match to a revised ARS consensus is present but not essential. Sequences up to 9 bp 5' from the essential consensus are dispensable. A broad DNA region directly 3' to the essential consensus is required and is easily unwound as indicated by: (i) hypersensitivity to nicking of an approximately 100-bp region by mung bean nuclease in a negatively supercoiled plasmid and (ii) helical instability determined by thermodynamic analysis of the nucleotide sequence. A correlation between DNA helical instability and replication efficiency of wild-type and mutated ribosomal ARS derivatives suggests that a broad region 3' to the essential ARS consensus functions as a DNA unwinding element. Certain point mutations that do not stabilize the DNA helix in the 3' region but reduce ARS efficiency reveal an element distinct from, but overlapping, the DNA unwinding element. The nucleotide sequence of the functionally important constituents in the ARS appears to be conserved among the rDNA repeats in the chromosome.     

Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin.

Strains of Saccharomyces cerevisiae harboring M1-dsRNA, the determinant of type 1 killer and immunity phenotypes, secrete a dimeric 19-kd toxin that kills sensitive yeast cells by the production of cation-permeable pores in the cytoplasmic membrane. The preprotoxin, an intracellular precursor to toxin, has the domain sequence delta-alpha-gamma-beta where alpha and beta are the 9.5-and 9.0-kd subunits of secreted toxin. Plasmids containing a partial cDNA copy of M1, in which alpha, gamma, and beta are fused to the PH05 promoter and signal peptide, have previously been shown to express phosphate-repressible toxin production and immunity. Here the construction of a complete DNA copy of the preprotoxin gene and its mutagenesis are described. Analysis of the expression of these mutants from the PH05 promoter elucidates the functions of the preprotoxin domains. delta acts as a leader peptide and efficiently mediates the secretion, glycosylation and maturation of killer toxin. Mutations within the beta subunit indicate it to be essential for binding of toxin to and killing of whole cells but unnecessary for the killing of spheroplasts. Mutations within the putative active site of alpha prevent killing of both cells and spheroplasts. The probable role of beta is therefore recognition and binding to the cell wall receptor whereas alpha is the active ionophore. Mutations within alpha causing loss of toxicity also cause loss of immunity, while the mutants described within gamma and beta retain partial or complete immunity. Expression of gamma without alpha or beta confers no phenotype. The immunity determinant may minimally consist of the alpha domain and the N-terminal portion of gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mutational analysis reveals new functional interactions between domains of the Oxa1 protein in Saccharomyces cerevisiae

The Oxa1/YidC/Alb3 family plays a key role in the biogenesis of the respiratory and photosynthetic complexes in bacteria and organelles. In Saccharomyces cerevisiae, Oxa1 mediates the co‐translational insertion of mitochondrially encoded subunits of the three respiratory complexes III, IV and V within the inner membrane and also controls a late step in complex V assembly. No crystal structure of YidC or Oxa1 is available and little is known about the respective role of each transmembrane segment (TM) and hydrophilic loop of this polytopic protein on the biogenesis of the three complexes. Here, we have generated a collection of random point mutations located in the hydrophobic and hydrophilic domains of the protein and characterized their effects on the assembly of the three respiratory complexes. Our results show mutant‐dependent differential effects, particularly on complex V. In order to identify tertiary interactions within Oxa1, we have also isolated revertants carrying second‐site compensatory mutations able to restore respiration. This analysis reveals the existence of functional interactions between TM2 and TM5, TM4 and TM5 as well as between TM4 and loop 2, highlighting the key position of TM4 and TM5 in the Oxa1 protein.     

Localization of a DNA replication origin and termination zone on chromosome III of Saccharomyces cerevisiae.

Two-dimensional gel electrophoretic replicon mapping techniques were used to identify all functional DNA replication origins and termini in a 26.5-kbp stretch in the left arm of yeast chromosome III. Only one origin was detected; it coincided with an ARS element (ARS306), as have all previously mapped yeast origins. A replication termination region was identified in a 4.3-kbp stretch at the telomere-proximal end of the investigated region, between the origin identified in this paper and the neighboring, previously mapped, ARS305-associated origin (previously called the A6C origin). Termination does not occur at a specific site; instead, it appears to be the consequence of replication forks converging in a stretch of DNA of at least 4.3 kbp.     

Protein-DNA interactions in soluble telosomes from Saccharomyces cerevisiae.

Telomeric DNA in Saccharomyces is organized into a non-nucleosomal chromatin structure called the telosome that can be released from chromosome ends in soluble form by nuclease digestion (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210). The protein-DNA interactions of soluble telosomes were investigated by monitoring isolated telomeric DNA fragments for the retention of bound protein using both gel mobility shift and nitrocellulose filter-binding assays. Telosomal proteins remained associated with telomeric DNA at concentrations of ethidium bromide that dissociated nucleosomes. The protein-DNA interactions in the yeast telosome were also disrupted by much lower salt concentrations than those known to disrupt either the interactions of ciliate terminus-binding proteins with telomeric DNA or the interactions of histones with DNA in nucleosomes. Taken together, these data corroborate previously published nuclease mapping data indicating that telosomes are distinct in structure from conventional nucleosomes. These data also indicate that yeast do not possess telomere binding proteins similar to those detected in ciliates that remain tightly bound to telomeric DNA even in high salt. In addition, the characteristic gel mobility shift of soluble telosomes could be mimicked by complexes formed in vitro with yeast telomeric DNA and recombinant Rap1p suggesting that Rap1p, a known component of soluble yeast telosomes (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210; Conrad, M. N., Wright, J. H., Wolf, A. J. and Zakian, V. A. (1990) Cell 63, 739-750), is likely to be the major structural protein bound directly to yeast telomeric DNA.     

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.     

Mutational analysis of Saccharomyces cerevisiae U4 small nuclear RNA identifies functionally important domains.

U4 small nuclear RNA (snRNA) is essential for pre-mRNA splicing, although its role is not yet clear. On the basis of a model structure (C. Guthrie and B. Patterson, Annu. Rev. Genet. 22:387-419, 1988), the molecule can be thought of as having six domains: stem II, 5' stem-loop, stem I, central region, 3' stem-loop, and 3'-terminal region. We have carried out extensive mutagenesis of the yeast U4 snRNA gene (SNR14) and have obtained information on the effect of mutations at 105 of its 160 nucleotides. Fifteen critical residues in the U4 snRNA have been identified in four domains: stem II, the 5' stem-loop, stem I, and the 3'-terminal region. These domains have been shown previously to be insensitive to oligonucleotide-directed RNase H cleavage (Y. Xu, S. Petersen-Bjørn, and J. D. Friesen, Mol. Cell. Biol. 10:1217-1225, 1990), suggesting that they are involved in intra- or intermolecular interactions. Stem II, a region that base pairs with U6 snRNA, is the most sensitive to mutation of all U4 snRNA domains. In contrast, stem I is surprisingly insensitive to mutational change, which brings into question its role in base pairing with U6 snRNA. All mutations in the putative Sm site of U4 snRNA yield a lethal or conditional-lethal phenotype, indicating that this region is important functionally. Only two nucleotides in the 5' stem-loop are sensitive to mutation; most of this domain can tolerate point mutations or small deletions. The 3' stem-loop, while essential, is very tolerant of change. A large portion of the central domain can be removed or expanded with only minor effects on phenotype, suggesting that it has little function of its own. Analysis of conditional mutations in stem II and stem I indicates that although these single-base changes do not have a dramatic effect on U4 snRNA stability, they are defective in RNA splicing in vivo and in vitro, as well as in spliceosome assembly. These results are discussed in the context of current knowledge of the interactions involving U4 snRNA.     

The functional origin of bacteriophage f1 DNA replication. Its signals and domains

The origin of DNA replication of bacteriophage f1 functions as a signal, not only for initiation of viral strand synthesis, but also for its termination. Viral (plus) strand synthesis initiates and terminates at a specific site (plus origin) that is recognized and nicked by the viral gene II protein. Mutational analysis of the 5' side (upstream) of the origin of plus strand replication of phage f1 led us to postulate the existence of a set of overlapping functional domains. These included ones for strand nicking, and initiation and termination of DNA synthesis. Mutational analysis of the 3' side (downstream) of the origin has verified the existence of these domains and determined their extent. The results indicate that the f1 "functional origin" can be divided into two domains: (1) a "core region", about 40 nucleotides long, that is absolutely required for plus strand synthesis and contains three distinct but partially overlapping signals, (a) the gene II protein recognition sequence, which is necessary both for plus strand initiation and termination, (b) the termination signal, which extends for eight more nucleotides on the 5' side of the gene II protein recognition sequence, (c) the initiation signal that extends for about ten more nucleotides on the 3' side of the gene II protein recognition sequence; (2) a "secondary region", 100 nucleotides long, required exclusively for plus strand initiation. Disruption of the secondary region does not completely abolish the functionality of the f1 origin but does drastically reduce it (1% residual biological activity). We discuss a possible explanation of the fact that this region can be interrupted (e.g. f1, M13 cloning vectors) by large insertions of foreign DNA without significantly affecting replication.     

Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae

During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.     

p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains.

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.     

Control of Saccharomyces cerevisiae 2microN DNA replication by cell division cycle genes that control nuclear DNA replication.

The replication of the 2 μm DNA of Saccharomyces cerevisiae has been examined in cell division cycle (cdc) mutants. The 2 μm DNA does not replicate at the restrictive temperature in cells bearing the cdc28, cdc4, and cdc7 mutations which prevent passage of cells from the G1 phase into S phase. Plasmid replication also is prevented in a mating-type cells by α factor, a mating hormone which prevents cells from completing an event early in G1 phase. The 2 μm DNA ceases replication at 36 °C in a mutant harboring the cdc8 mutation, a defect in the elongation reactions of nuclear DNA replication. Plasmid replication continues at the restrictive temperature for approximately one generation in a cdc13 mutant defective in nuclear division. These results show that 2 μm DNA replication is controlled by the same genes that control the initiation and completion of nuclear DNA replication.     

Functional analysis of the domains of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae

The LAT1 gene encoding the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase (PDH) complex from Saccharomyces cerevisiae was disrupted, and the lat1 null mutant was used to analyze the structure and function of the domains of E2. Disruption of LAT1 did not affect the viability of the cells. Apparently, flux through the PDH complex is not required for growth of S. cerevisiae under the conditions tested. The wild-type and mutant PDH complexes were purified to near-homogeneity and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme assays. Mutant cells transformed with LAT1 on a unit-copy plasmid produced a PDH complex very similar to that of the wild-type PDH complex. Deletion of most of the putative lipoyl domain (residues 8-84) resulted in loss of about 85% of the overall activity, but did not affect the acetyltransferase activity of E2 or the binding of pyruvate dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), and protein X to the truncated E2. Similar results were obtained by deleting the lipoyl domain plus the first hinge region (residues 8-145) and by replacing lysine-47, the putative site of covalent attachment of the lipoyl moiety, by arginine. Although the lipoyl domain of E2 and/or its covalently bound lipoyl moiety were removed, the mutant complexes retained 12-15% of the overall activity of the wild-type PDH complex. Replacement of both lysine-47 in E2 and the equivalent lysine-43 in protein X by arginine resulted in complete loss of overall activity of the mutant PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae.

Previous studies have demonstrated that bent DNA is a conserved property of Saccharomyces cerevisiae autonomously replicating sequences (ARSs). Here we showed that bending elements are contained within ARS subdomains identified by others as replication enhancers. To provide a direct test for the function of this unusual structure, we analyzed the ARS activity of plasmids that contained synthetic bent DNA substituted for the natural bending element in yeast ARS1. The results demonstrated that deletion of the natural bending locus impaired ARS activity which was restored to a near wild-type level with synthetic bent DNA. Since the only obvious common features of the natural and synthetic bending elements are the sequence patterns that give rise to DNA bending, the results suggest that the bent structure per se is crucial for ARS function.     

Prediction of Saccharomyces cerevisiae replication origins

Background  

Autonomously replicating sequences (ARSs) function as replication origins in Saccharomyces cerevisiae. ARSs contain the 17 bp ARS consensus sequence (ACS), which binds the origin recognition complex. The yeast genome contains more than 10,000 ACS matches, but there are only a few hundred origins, and little flanking sequence similarity has been found. Thus, identification of origins by sequence alone has not been possible.     

Centromeric DNA from Saccharomyces uvarum is functional in Saccharomyces cerevisiae

Previous comparisons of centromeric DNA sequences in laboratory strains of Saccharomyces cerevisiae have revealed conserved sequences within 120 base pairs (bp) which appear to be essential for centromere function. We wanted to find out whether centromeric DNA in Saccharomyces strains with different degrees of DNA sequence divergence carry the same conserved sequences or not. Bam HI DNA fragments from two S. cerevisiae strains and one Saccharomyces uvarum strain were cloned into a centromere selection vector and tested for centromere function in a S. cerevisiae laboratory strain. Fragments having centromere function were obtained at approximately equal frequencies from all three strains. Two of the S. uvarum centromeric DNAs and two of the S. cerevisiae centromeric DNAs were sequenced and shown to carry in a 120 bp region sequences essentially like those of centromeric DNA in S. cerevisiae laboratory strains. DNA hybridization to separated chromosomal DNAs revealed that the two newly determined S. cerevisiae centromeric DNA sequences belong to chromosomes V and XIII, respectively. On leave from: Department of Cell and Tumor Biology, Roswell Park Memorial Institute, Buffalo, NY 14263, USA; On leave from: The Biological Laboratories, University of Leiden, The Netherlands     

Post-translational regulation of Saccharomyces cerevisiae proteins tagged with the hormone-binding domains of mammalian nuclear receptors

In the post-genome sequencing era the functional analysis of newly discovered proteins becomes more and more important. In this report we describe a genetic approach to the post-translational regulation of protein function in Saccharomyces cerevisiae by creating conditional lethal mutants. The yeast ORFs YDL139c, YDL147w, ERG3 and ERG11 were tagged with sequences encoding the hormone-binding domains of mammalian steroid receptors by PCR-mediated, targeted integration into the yeast genome. We found that the function of the chimeric proteins is regulated in a hormone-dependent way. This technique provides another important tool for the functional analysis of the yeast proteome.