Specific interaction between a Saccharomyces cerevisiae protein and a DNA element associated with certain autonomously replicating sequences.

We have isolated a protein from Saccharomyces cerevisiae that binds specifically to a nucleotide sequence associated with the autonomously replicating sequence (ARS) ARS120, located in the telomeric region of a yeast chromosome. "Footprinting" analysis revealed that a 26-base-pair DNA sequence, 5'-CAAGTGCCGTGCATAATGATGTGGGT-3', was protected by this protein from DNase I digestion. A plasmid containing 48 direct tandem repeats of this oligonucleotide was constructed and used to affinity-purify the binding activity. The purified protein, OBF1 (origin binding factor), showed specific binding to ARS120. The 26-base-pair OBF1-protected sequence was sufficient for the recognition and binding of the protein, since the mobility of a DNA fragment containing the synthetic binding site was retarded in agarose gels when incubated with OBF1. By performing competition experiments with a number of different ARSs, we showed that OBF1 binds tightly to some but not all ARSs. Interestingly, OBF1 does not appear to have a discernible affinity for ARS1 or the ARSs associated with mating type loci, HML alpha and HMRa, which are substrates for a DNA-binding activity reported by others. Since OBF1 appears to bind to DNA associated with a number of ARSs, we suggest that this protein may have a function related to ARS activity, perhaps in the initiation of DNA replication at selected ARSs.     

A DNA replication enhancer in Saccharomyces cerevisiae.

We have dissected the autonomously replicating sequence ARS121 using site-directed in vitro mutagenesis. Three domains important for origin function were identified; one of these is essential and contains an 11-base-pair sequence resembling the canonical ARS core consensus; the second region, deletion of which affects the efficiency of the origin, is located 3' to the T-rich strand of the essential sequence and encompasses several elements with near matches to the ARS core consensus; the third region, containing two OBF1 DNA-binding sites and located 5' to the essential sequence, also affects the efficiency of the ARS. Here we demonstrate that a synthetic OBF1 DNA-binding site can substitute for the entire third domain in origin function. A dimer of the synthetic binding site, fused to a truncated origin containing only domains one and two, restored the origin activity to the levels of the wild-type ARS. The stimulation of origin function by the synthetic binding site was relatively orientation independent and could occur at distances as far as 1 kilobase upstream to the essential domain. Based on these results we conclude that the OBF1 DNA-binding site is an enhancer of DNA replication. We suggest that the DNA-binding site and the OBF1 protein are involved in the regulation of the activation of nuclear origins of replication in Saccharomyces cerevisiae.     

Lack of positional requirements for autonomously replicating sequence elements on artificial yeast chromosomes.

In the yeast Saccharomyces cerevisiae, origins of replication (autonomously replicating sequences; ARSs), centromeres, and telomeres have been isolated and characterized. The identification of these structures allows the construction of artificial chromosomes in which the architecture of eukaryotic chromosomes may be studied. A common feature of most, and possibly all, natural yeast chromosomes is that they have an ARS within 2 kilobases of their physical ends. To study the effects of such telomeric ARSs on chromosome maintenance, we introduced artificial chromosomes of approximately 15 and 60 kilobases into yeast cells and analyzed the requirements for telomeric ARSs and the effects of ARS-free chromosomal arms on the stability of these molecules. We find that terminal blocks of telomeric repeats are sufficient to be recognized as telomeres. Moreover, artificial chromosomes containing telomere-associated Y' sequences and telomeric ARSs were no more stable during both mitosis and meiosis than artificial chromosomes lacking terminal ARSs, indicating that yeast-specific blocks of telomeric sequences are the only cis-acting requirement for a functional telomere during both mitotic growth and meiosis. The results also show that there is no requirement for an origin of replication on each arm of the artificial chromosomes, indicating that a replication fork may efficiently move through a functional centromere region.     

Transformation of Phycomyces blakesleeanus to G-418 resistance by an autonomously replicating plasmid.

The fungus, Phycomyces blakesleeanus, shows many well-defined responses to a number of external stimuli. Genetic analysis shows that at least eight genes are involved in Phycomyces sensory transduction. As a first step toward the molecular analysis of these genes and their products, we have developed a transformation protocol for Phycomyces by using a plasmid containing the kanamycin-resistance gene from Tn903 and a Phycomyces DNA fragment capable of supporting autonomous replication in yeast (ARS). Our results demonstrate that the Tn903 gene is expressed in Phycomyces and that the ARS fragment selected in yeast supports autonomous replication in Phycomyces as well.     

Isolation of a condensed, intracellular form of the 2-micrometer DNA plasmid of Saccharomyces cerevisiae.

We have initiated an investigation of the proteins bound in vivo to the 2-micrometer DNA plasmid found in the yeast Saccharomyces cerevisiae by examining its intracellular form. To isolate 2-micrometer DNA without disturbing proteins bound to the plasmid, an extract was prepared from a strain lacking mitochondrial DNA and the nuclear chromatin was removed from the extract by centrifugation. When the DNA in this extract was sedimented through a sucrose gradient containing 0.15 M NaCl, plasmid DNA had a sedimentation coefficient of approximately 70. This S value is greater than the S value of 25 for naked, superhelical 2-micrometer DNA. Cosedimenting with the DNA were proteins of the same size as the histone proteins associated with yeast nuclear chromatin. Digestion of the plasmid DNA with micrococcal nuclease and electrophoresis of the resulting DNA fragments revealed that segments of discrete sizes are protected from degradation. Examination of the plasmid DNA protein complex by electron microscopy showed nucleosome structures. We conclude that 2-micron DNA exists as a condensed chromosome body within the cell.     

The nucleotide sequence of chromosome I from Saccharomyces cerevisiae.

Chromosome I from the yeast Saccharomyces cerevisiae contains a DNA molecule of approximately 231 kbp and is the smallest naturally occurring functional eukaryotic nuclear chromosome so far characterized. The nucleotide sequence of this chromosome has been determined as part of an international collaboration to sequence the entire yeast genome. The chromosome contains 89 open reading frames and 4 tRNA genes. The central 165 kbp of the chromosome resembles other large sequenced regions of the yeast genome in both its high density and distribution of genes. In contrast, the remaining sequences flanking this DNA that comprise the two ends of the chromosome and make up more than 25% of the DNA molecule have a much lower gene density, are largely not transcribed, contain no genes essential for vegetative growth, and contain several apparent pseudogenes and a 15-kbp redundant sequence. These terminally repetitive regions consist of a telomeric repeat called W', flanked by DNA closely related to the yeast FLO1 gene. The low gene density, presence of pseudogenes, and lack of expression are consistent with the idea that these terminal regions represent the yeast equivalent of heterochromatin. The occurrence of such a high proportion of DNA with so little information suggests that its presence gives this chromosome the critical length required for proper function.     

Specificity of mismatch repair following transformation of Saccharomyces cerevisiae with heteroduplex plasmid DNA.

A method is described for genetic detection of mismatch repair products following transformation of Saccharomyces cerevisiae. The method is based on the detection of beta-galactosidase activity in clonal derivatives of cells transformed with heteroduplex plasmid DNA. Heteroduplex plasmid substrates were constructed by insertion of an oligonucleotide heteroduplex into the coding sequence of the Escherichia coli lacZ gene. The plasmid and oligonucleotides were designed so that one strand of the construct would code for a functional beta-galactosidase and the other strand would contain an in-frame nonsense codon. The frequencies of transformed clones containing only Lac+ cells, only Lac- cells, or a mixture of the two Lac phenotypes provided information on the efficiency of the repair reaction. With this method, plasmids carrying single-base substitution mismatches, a single-base frameshift mismatch (T/delta), or a 3-base-pair substitution mismatch (TGA/GAA) were tested. A/C, G/T, G/A, G/G, and T/delta mismatches were repaired with significantly greater efficiencies than C/C, A/A, T/T, and TGA/GAA. T/C was repaired with an intermediate efficiency. The frequencies of products obtained with G/G, G/A, and T/delta mismatches suggested modest inequality of repair in the two possible directions. Strains carrying the repair-deficient pms1-1 mutation were also tested. The efficiencies of repair of A/C, G/T, G/G, and A/A mismatches were reduced in pms1-1 cells compared with wild-type cells. In addition, a change in repair inequality was detected when transformation of the two strains with an A/C mismatch was compared.     

The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae

The origin recognition complex (ORC) is conserved in all eukaryotes. The six proteins of the Saccharomyces cerevisiae ORC that form a stable complex bind to origins of DNA replication and recruit prereplicative complex (pre-RC) proteins, one of which is Cdc6. To further understand the function of ORC we recently determined by single-particle reconstruction of electron micrographs a low-resolution, 3D structure of S. cerevisiae ORC and the ORC–Cdc6 complex. In this article, the spatial arrangement of the ORC subunits within the ORC structure is described. In one approach, a maltose binding protein (MBP) was systematically fused to the N or the C termini of the five largest ORC subunits, one subunit at a time, generating 10 MBP-fused ORCs, and the MBP density was localized in the averaged, 2D EM images of the MBP-fused ORC particles. Determining the Orc1–5 structure and comparing it with the native ORC structure localized the Orc6 subunit near Orc2 and Orc3. Finally, subunit–subunit interactions were determined by immunoprecipitation of ORC subunits synthesized in vitro. Based on the derived ORC architecture and existing structures of archaeal Orc1–DNA structures, we propose a model for ORC and suggest how ORC interacts with origin DNA and Cdc6. The studies provide a basis for understanding the overall structure of the pre-RC.     

Cloning and nucleotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.

A 537-base cDNA encoding a portion of Saccharomyces cerevisiae dihydrolipoamide acetyltransferase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) was isolated from a lambda gt11 yeast cDNA library by immunoscreening. This cDNA was subcloned and used as a probe to screen a lambda gt11 yeast genomic DNA library. Two overlapping clones were used to determine the complete sequence of the acetyltransferase gene. The composite sequence has an open reading frame of 1446 nucleotides encoding a presequence of 28 amino acids and a mature protein of 454 amino acids (Mr = 48,546). The deduced amino acid sequence contains the experimentally determined amino acid sequences of the amino terminus and two internal peptide fragments of the acetyltransferase. Hybridization analysis of yeast genomic DNA showed that the gene has a single copy. A 915-base segment of the acetyltransferase gene hybridized to a yeast mRNA of approximately equal to 1.6 kilobases. Analysis of the deduced amino acid sequence of the dihydrolipoamide acetyltransferase revealed a multidomain structure similar to those reported for the corresponding acetyltransferases from Escherichia coli and rat liver, and extensive sequence similarity among the three enzymes. However, the yeast enzyme contains only one lipoyl domain, in contrast to three lipoyl domains reported for the E. coli enzyme and apparently two for the rat liver enzyme.     

Cloning and nucleotide sequence of the gene for protein X from Saccharomyces cerevisiae.

The gene encoding the protein X component of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been cloned and sequenced. A 487-base fragment of yeast genomic DNA encoding the amino-terminal region of protein X was amplified by the polymerase chain reaction using synthetic oligonucleotide primers based on amino-terminal and internal amino acid sequences. This DNA fragment was used as a probe to select two genomic DNA restriction fragments, which were cloned and sequenced. A 2.1-kilobase insert contains the complete sequence of the protein X gene. This insert has an open reading frame of 1230 nucleotides encoding a presequence of 30 amino acid residues and a mature protein of 380 amino acid residues (Mr, 42,052). Hybridization analysis showed that there is a single copy of the protein X gene and that the size of the mRNA is approximately 1.5 kilobases. Comparison of the deduced amino acid sequences of yeast protein X and dihydrolipoamide acetyltransferase indicates that the two proteins evolved from a common ancestor. The amino-terminal part of protein X (residues 1-195) resembles the acetyltransferase, but the remainder is quite different. There is strong homology between protein X and the acetyltransferase in the amino-terminal region (residues 1-84) that corresponds to the putative lipoyl domain. Protein X lacks the highly conserved sequence His-Xaa-Xaa-Xaa-Asp-Gly near the carboxyl terminus, which is thought to be part of the active site of all dihydrolipoamide acyltransferases.     

Purification of the cdc8 protein of Saccharomyces cerevisiae by complementation in an aphidicolin-sensitive in vitro DNA replication system.

DNA synthesis in vitro in Brij-treated Saccharomyces cerevisiae requires the product of the CDC8 gene (Hereford, L. M. & Hartwell, L. H. (1971) Nature (London) New Biol. 234, 171-172). Extracts of wild-type A364a yeast restore DNA synthesis in Brij-treated cdc8, a mutant containing a thermolabile cdc8 gene product. This constitutes a complementation assay by which the cdc8 gene product can be monitored during purification. A heat-stable protein responsible for this complementation has been partially purified from both wild-type A364a cells and from a cdc8 temperature-sensitive mutant. The complementation activity from the mutant is thermolabile when compared to the wild-type activity, indicating that CDC8 is the structural gene for the protein.     

Isolation and sequence of the gene for actin in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is known to contain the highly conserved and unbiquitous protein actin. We have used cloned actin sequences from Dictyostelium discoideum to identify and clone the actin gene in yeast. Hybridization to genomic fragments of yeast DNA suggest that there is a single actin gene in yeast. We have determined the nucleotide sequence of that gene and its flanking regions. The sequence of the gene reveals an intervening sequence of 309 base pairs in the coding sequences at the 5' end of the gene. The existence and location of the intervening sequence was verified by using the dideoxy chain termination technique to determine the sequence at the 5' terminus of the actin mRNA. The similarity of the splice junction sequences in this gene to those found in higher eukaryotes suggests that yeast must possess a similar splicing enzyme.     

CDC46/MCM5, a yeast protein whose subcellular localization is cell cycle-regulated, is involved in DNA replication at autonomously replicating sequences.

Saccharomyces cerevisiae cells containing mutations in the cell-division-cycle gene CDC46 arrest with a large bud and a single nucleus with unreplicated DNA at the non-permissive temperature. This G1/S arrest, together with the increased rates of mitotic chromosome loss and recombination phenotype, suggests that these mutants are defective in DNA replication. The subcellular localization of the CDC46 protein changes with the cell cycle; it is nuclear between the end of M phase and the G1/S transition but is cytoplasmic in other phases of the cell cycle. Here we show that CDC46 is identical to MCM5, based on complementation analysis of the mcm5-1 and cdc46-1 alleles, complementation of the minichromosome maintenance defect of mcm5-1 by CDC46, and the genetic linkage of these two genes. Like mcm5-1, cdc46-1 and cdc46-5 also show a minichromosome maintenance defect thought to be associated with DNA replication initiation at autonomously replicating sequences. Taken together, these observations suggest that CDC46/MCM5 acts during a very narrow window at the G1/S transition or the beginning of S phase by virtue of its nuclear localization to effect the initiation of DNA replication at autonomously replicating sequences.     

Isolation of the yeast INO1 gene: located on an autonomously replicating plasmid, the gene is fully regulated.

The Saccharomyces cerevisiae gene, INO1 , encoding the highly regulated enzyme, myo-inositol-1-phosphate synthase [1L-myo-inositol-1-phosphate lyase (isomerizing), EC 5.5.1.4], was isolated by genetic complementation. The cloned sequence was shown to complement two independent IN01 alleles ( ino1 -5 and ino1 -13). One of these mutants ( ino1 -5) fails to make any material that is crossreactive with antibody to the wild-type inositol-1-phosphate synthase. The cloned DNA restored not only inositol prototrophy to this mutant but also its ability to make material crossreactive with anti-inositol-1-phosphate synthase antibody. The sequence on an integrative plasmid was also shown to recombine with the INO1 locus, thereby confirming its genetic identity. The DNA was subcloned and used for Southern blot analysis, revealing that the cloned DNA (5.4 kilobases long) represents a unique sequence in the yeast genome. Inositol-1-phosphate synthase was fully regulated when its gene was located extrachromosomally on the autonomously replicating plasmid. In cells ( ino1 -) containing the cloned INO1 gene on a high-copy-number plasmid, the enzyme was fully repressible. Furthermore, the gene product was not expressed when the plasmid was transferred into a strain containing an ino4 mutation, which also prevents expression of chromosomal copies of INO1 . These results establish that the intact structural gene and associated regulatory components have been isolated and that positioning of the gene in its normal chromosomal location is not required for full regulation of inositol-1-phosphate synthase.     

Assembly of the mitochondrial membrane system: partial sequence of a mitochondrial ATPase gene in Saccharomyces cerevisiae.

The nucleotide sequence of mitochondrial DNA of a cytoplasmic "petite" mutant of Saccharomyces cerevisiae is reported. The DNA has a repeat length of 1060 base pairs and contains a genetic marker (oli-1) for the ATPase proteolipid. The nucleotide sequence reveals the presence of part of the structural gene of the subunit-9 proteolipid of the ATPase complex and an extended A+T-rich region adjacent to the carboxyl-terminal end of the gene. The structural gene sequence agrees with the primary structure of the protein. These studies point out the feasibility of using the DNA of appropriately marked "petite" mutants to obtain the sequence of mitochondrial genes.     

Origin of replication of pBR345 plasmid DNA.

A small (approximately 1100 base pairs) ColE1-type plasmid, pBR345, was constructed from plasmid pMB1 by a series of in vitro recombinant manipulations. Approximately 9% of the supercoiled pBR345 DNA obtained from cultures amplified with chloramphenicol appears to be replicative intermediates with replicating "eye" structures of uniform size. Results obtained from electron microscopy and biochemical analyses have enabled us to localize the origin of replication at the same position as that reported for ColE1. A sequence of 420 nucleotides surrounding this origin has been determined. A comparison between this sequence and the one determined for the origin of replication of ColE1 is presented.     

RAD3 protein of Saccharomyces cerevisiae is a DNA helicase.

The Saccharomyces cerevisiae RAD3 gene, which is required for cell viability and excision repair of damaged DNA, encodes an 89-kDa protein that has a single-stranded DNA-dependent ATPase activity. We now show that the RAD3 protein also possesses a helicase activity that unwinds duplex regions in DNA substrates constructed by annealing DNA fragments of 71-851 nucleotides to circular, single-stranded M13 DNA. The DNA helicase activity is dependent on the hydrolysis of ATP, has a pH optimum of approximately 5.6, and is inhibited by antibodies raised against a truncated RAD3 protein produced in Escherichia coli. The RAD3 helicase translocates along single-stranded DNA in the 5'----3' direction. The direction of RAD3 helicase movement is consistent with the possibility that it unwinds DNA duplexes in advance of the replication fork during DNA replication.     

DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication.

A temperature-sensitive DNA topoisomerase II mutant of the yeast Saccharomyces cerevisiae has been identified. Genetic analysis shows that a single recessive nuclear mutation is responsible for both temperature-sensitive growth and enzymatic activity. Thus, topoisomerase II is essential for viability and the mutation is most probably in the structural gene. Experiments with synchronized mutant cells show that at the nonpermissive temperature cells can undergo one, and only one, round of DNA replication. These cells are arrested at medial nuclear division. Analysis of 2-microns plasmid DNA from these cells shows it to be in the form of multiply intertwined catenated dimers. The results suggest that DNA topoisomerase II is necessary for the segregation of chromosomes at the termination of DNA replication.     

Cleavage of cruciform DNA structures by an activity from Saccharomyces cerevisiae.

Protein extracts from Saccharomyces cerevisiae have been fractionated to reveal a nuclease activity that cleaves cruciform structures in DNA. Negatively supercoiled plasmids that contain inverted repeats that are extruded into cruciform structures have been used as DNA substrates. The sites of cleavage of pColIR215 DNA are located within the extruded cruciform stems and are symmetrically opposed to each other across the cruciform junction. Neither relaxed duplex DNA nor single-stranded DNA serve as substrates. The native molecular weight of the activity was estimated to be approximately equal to 200,000 by gel filtration.