scholarly journals Linear Derivatives of Saccharomyces cerevisiae Chromosome III Can Be Maintained in the Absence of Autonomously Replicating Sequence Elements

2007 ◽  
Vol 27 (13) ◽  
pp. 4652-4663 ◽  
Author(s):  
Ann Dershowitz ◽  
Marylynn Snyder ◽  
Mohammed Sbia ◽  
Joan H. Skurnick ◽  
Loke Y. Ong ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Loss Rate ◽  
Replication Initiation ◽  
Replication Origins ◽  
Additional Increase ◽  
Autonomously Replicating Sequence ◽  
Sequence Elements ◽  
Chromosome Fragments ◽  
Chromosome Iii ◽  
The Right

ABSTRACT Replication origins in Saccharomyces cerevisiae are spaced at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all sequences that function as autonomously replicating sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators.

scholarly journals Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

2001 ◽  
Vol 12 (11) ◽  
pp. 3317-3327 ◽  
Author(s):  
Arkadi Poloumienko ◽  
Ann Dershowitz ◽  
Jitakshi De ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Complete Analysis ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Chromosome Iii

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ∼40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements onS. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in ≤10% of cells in the population and two ARS elements active in ≥90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

The replication of yeast chromosomes: lessons fromSaccharomyces cerevisiaechromosome III

1995 ◽  
Vol 73 (S1) ◽  
pp. 208-214 ◽  
Author(s):  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Replication Origins ◽  
Chromosomal Replication ◽  
Position Effects ◽  
Autonomously Replicating Sequence ◽  
Systematic Analysis ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Constant Rate ◽  
Chromosome Iii

To understand how a eukaryotic chromosome is replicated, a systematic analysis of chromosome III of Saccharomyces cerevisiae has been undertaken. Replication origins are specified by autonomously replicating sequence (ARS) elements, whose sequences can be dissected using a simple plasmid assay. Only a subset of ARS elements are active as chromosomal replication origins. Replication origins are required for normal chromosome transmission, but they appear to be redundant; several origins can be deleted without affecting chromosome stability. Replication origin position has been conserved on chromosome III in diverged strains, suggesting that origin position is important for chromosome function. The inability of some ARS elements to function as chromosomal replication origins appears likely to result from chromosomal context or position effects. Replication termination occurs over broad regions between active replication origins. The position of termination can be altered by deleting origins, suggesting that no specific replication termination elements are required. Replication forks appear to move at a relatively constant rate through the chromosome. A replication pause site associated with the centromere results from the kinetochore protein complex that binds the centromere to mediate chromosome segregation. Key words: Saccharomyces cerevisiae, ARS elements, replication origins, replication termination, DNA replication intermediates.

Chromosomal DNA replication initiates at the same origins in meiosis and mitosis

1994 ◽  
Vol 14 (5) ◽  
pp. 3524-3534
Author(s):  
I Collins ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
S Phase ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Chromosome Iii

Autonomously replicating sequence (ARS) elements are identified by their ability to promote high-frequency transformation and extrachromosomal replication of plasmids in the yeast Saccharomyces cerevisiae. Six of the 14 ARS elements present in a 200-kb region of Saccharomyces cerevisiae chromosome III are mitotic chromosomal replication origins. The unexpected observation that eight ARS elements do not function at detectable levels as chromosomal replication origins during mitotic growth suggested that these ARS elements may function as chromosomal origins during premeiotic S phase. Two-dimensional agarose gel electrophoresis was used to map premeiotic replication origins in a 100-kb segment of chromosome III between HML and CEN3. The pattern of origin usage in premeiotic S phase was identical to that in mitotic S phase, with the possible exception of ARS308, which is an inefficient mitotic origin associated with CEN3. CEN3 was found to replicate during premeiotic S phase, demonstrating that the failure of sister chromatids to disjoin during the meiosis I division is not due to unreplicated centromeres. No origins were found in the DNA fragments without ARS function. Thus, in both mitosis and meiosis, chromosomal replication origins are coincident with ARS elements but not all ARS elements have chromosomal origin function. The efficiency of origin use and the patterns of replication termination are similar in meiosis and in mitosis. DNA replication termination occurs over a broad distance between active origins.

scholarly journals Chromosomal DNA replication initiates at the same origins in meiosis and mitosis.

1994 ◽  
Vol 14 (5) ◽  
pp. 3524-3534 ◽  
Author(s):  
I Collins ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
S Phase ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Chromosome Iii

Autonomously replicating sequence (ARS) elements are identified by their ability to promote high-frequency transformation and extrachromosomal replication of plasmids in the yeast Saccharomyces cerevisiae. Six of the 14 ARS elements present in a 200-kb region of Saccharomyces cerevisiae chromosome III are mitotic chromosomal replication origins. The unexpected observation that eight ARS elements do not function at detectable levels as chromosomal replication origins during mitotic growth suggested that these ARS elements may function as chromosomal origins during premeiotic S phase. Two-dimensional agarose gel electrophoresis was used to map premeiotic replication origins in a 100-kb segment of chromosome III between HML and CEN3. The pattern of origin usage in premeiotic S phase was identical to that in mitotic S phase, with the possible exception of ARS308, which is an inefficient mitotic origin associated with CEN3. CEN3 was found to replicate during premeiotic S phase, demonstrating that the failure of sister chromatids to disjoin during the meiosis I division is not due to unreplicated centromeres. No origins were found in the DNA fragments without ARS function. Thus, in both mitosis and meiosis, chromosomal replication origins are coincident with ARS elements but not all ARS elements have chromosomal origin function. The efficiency of origin use and the patterns of replication termination are similar in meiosis and in mitosis. DNA replication termination occurs over a broad distance between active origins.

scholarly journals A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (6) ◽  
pp. 3261-3271 ◽  
Author(s):  
A M Merchant ◽  
Y Kawasaki ◽  
Y Chen ◽  
M Lei ◽  
B K Tye
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Replication Origins ◽  
Minichromosome Maintenance ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Dna Replication Initiation ◽  
Initiation Factors

We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.

A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (11) ◽  
pp. 5648-5659
Author(s):  
F J McNally ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Protein A ◽  
Replication Initiation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Functional Components

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2523-2535
Author(s):  
J H Hegemann ◽  
J H Shero ◽  
G Cottarel ◽  
P Philippsen ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Vector System ◽  
Chromosome Loss ◽  
Wild Type ◽  
Sequence Elements ◽  
Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

scholarly journals Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (1) ◽  
pp. 68-75 ◽  
Author(s):  
A Gaudet ◽  
M Fitzgerald-Hayes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Composition ◽  
Consensus Sequence ◽  
Fold Increase ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Core Element ◽  
Sequence Elements ◽  
Dna Elements ◽  
Chromosome Iii

Centromere DNA from 11 of the 16 chromosomes of the yeast Saccharomyces cerevisiae have been analyzed and reveal three sequence elements common to each centromere, referred to as conserved centromere DNA elements (CDE). The adenine-plus-thymine (A + T)-rich central core element, CDE II, is flanked by two short conserved sequences, CDE I (8 base pairs [bp]) and CDE III (25 bp). Although no consensus sequence exists among the different CDE II regions, they do have three common features of sequence organization. First, the CDE II regions are similar in length, ranging from 78 to 86 bp measured from CDE I to the left boundary of CDE III. Second, the base composition is always greater than 90% A + T. Finally, the A and T residues in these segments are often arranged in runs of A and runs of T residues, sometimes with six or seven bases in a stretch. We constructed insertion, deletion, and replacement mutations in the CDE II region of the centromere from chromosome III, CEN3, designed to investigate the length and sequence requirements for function of the CDE II region of the centromere. We analyzed the effect of these altered centromeres on plasmid and chromosome segregation in S. cerevisiae. Our results show that increasing the length of CDE II from 84 to 154 bp causes a 100-fold increase in chromosome nondisjunction. Deletion mutations removing segments of the A + T-rich CDE II DNA also cause aberrant segregation. In some cases partial function could be restored by replacing the deleted DNA with fragments whose primary sequence or base composition is very different from that of the wild-type CDE II DNA. In addition, we found that identical mutations introduced into different positions in CDE II have very similar effects.

scholarly journals Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

1982 ◽  
Vol 2 (1) ◽  
pp. 11-20 ◽  
Author(s):  
R K Chan ◽  
C A Otte
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Solid Medium ◽  
G1 Arrest ◽  
Alpha Cells ◽  
Opposite Mating Type ◽  
Alpha Factor ◽  
Complementation Groups ◽  
Chromosome Iii ◽  
The Right

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.

scholarly journals Activation of Silent Replication Origins at Autonomously Replicating Sequence Elements near the HML Locus in Budding Yeast

1999 ◽  
Vol 19 (9) ◽  
pp. 6098-6109 ◽  
Author(s):  
Marija Vujcic ◽  
Charles A. Miller ◽  
David Kowalski
Keyword(s):  
Replication Origin ◽  
Budding Yeast ◽  
Transcriptional Silencing ◽  
Growth Conditions ◽  
Replication Origins ◽  
Additional Time ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Active Replication ◽  
Chromosome Iii

ABSTRACT In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302). Another, ARS303, resides betweenHML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303and in the process discovered a new ARS, ARS320. BothARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster withARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene,trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), orcis-acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for theHML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share keycis- and trans-acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at theHML ARS cluster, as well as at ARS301, the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances fromHML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins.

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