Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins

The silent mating-type loci of Saccharomyces cerevisiae, HML and HMR, are flanked by transcriptional silencers that have ARS activity (i.e., they function as replication origins when in plasmids). To test whether these ARS elements are chromosomal origins, we mapped origins near HML (close to the left telomere of chromosome III). Our results indicate that the HML-associated ARS elements either do not function as chromosomal replication origins or do so at a frequency below our detection level, suggesting that replication from a silencer-associated origin in each S phase is not essential for the maintenance of transcriptional repression at HML. Our results also imply that the ability of a DNA fragment to function as an ARS element in a plasmid does not ensure its ability to function as an efficient chromosomal replication origin. Telomere proximity is not responsible for inactivating these ARS elements, because they are not detectably functional as chromosomal origins even in genetically modified strains in which they are far from the telomere.

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Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

Molecular Biology of the Cell ◽

10.1091/mbc.12.11.3317 ◽

2001 ◽

Vol 12 (11) ◽

pp. 3317-3327 ◽

Cited By ~ 54

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.3524-3534.1994 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.3524 ◽

1994 ◽

Vol 14 (5) ◽

pp. 3524-3534 ◽

Cited By ~ 37

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.

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Conservation of ARS Elements and Chromosomal DNA Replication Origins on Chromosomes III of Saccharomyces cerevisiae and S. carlsbergensis

Genetics ◽

10.1093/genetics/152.3.933 ◽

1999 ◽

Vol 152 (3) ◽

pp. 933-941 ◽

Cited By ~ 3

Author(s):

Chen Yang ◽

James F Theis ◽

Carol S Newlon

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Open Reading Frames ◽

Replication Origins ◽

Chromosomal Dna ◽

Brewing Yeast ◽

Ars Elements ◽

Intergenic Regions ◽

Dna Replication Origins ◽

Chromosome Iii

AbstractDNA replication origins, specified by ARS elements in Saccharomyces cerevisiae, play an essential role in the stable transmission of chromosomes. Little is known about the evolution of ARS elements. We have isolated and characterized ARS elements from a chromosome III recovered from an alloploid Carlsberg brewing yeast that has diverged from its S. cerevisiae homeologue. The positions of seven ARS elements identified in this S. carlsbergensis chromosome are conserved: they are located in intergenic regions flanked by open reading frames homologous to those that flank seven ARS elements of the S. cerevisiae chromosome. The S. carlsbergensis ARS elements were active both in S. cerevisiae and S. monacensis, which has been proposed to be the source of the diverged genome present in brewing yeast. Moreover, their function as chromosomal replication origins correlated strongly with the activity of S. cerevisiae ARS elements, demonstrating the conservation of ARS activity and replication origin function in these two species.

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The effect on chromosome stability of deleting replication origins

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.391-398.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 391-398

Author(s):

A Dershowitz ◽

C S Newlon

Keyword(s):

Ring Chromosome ◽

High Rate ◽

Chromosome Stability ◽

Replication Origins ◽

Chromosomal Dna ◽

Circular Chromosome ◽

Cell Cycles ◽

Highly Active ◽

Dna Replication Origins ◽

Chromosome Iii

The observed spacing between chromosomal DNA replication origins in Saccharomyces cerevisiae is at least four times shorter than should be necessary to ensure complete replication of chromosomal DNA during the S phase. To test whether all replication origins are required for normal chromosome stability, the loss rates of derivatives of chromosome III from which one or more origins had been deleted were measured. In the case of a 61-kb circular derivative of the chromosome that has two highly active origins and one origin that initiates only 10 to 20% of the time, deletion of either highly active origin increased its rate of loss two- to fourfold. Deletion of both highly active origins caused the ring chromosome to be lost in approximately 20% of cell divisions. This very high rate of loss demonstrates that there are no efficient cryptic origins on the ring chromosome that are capable of ensuring its replication in the absence of the origins that are normally used. Deletion of the same two origins from the full-length chromosome III, which contains more than six replication origins, had no effect on its rate of loss. These results suggest that the increase in the rate of loss of the small circular chromosome from which a single highly active origin was deleted was caused by the failure of the remaining highly active origin to initiate replication in a small fraction (approximately 0.003) of cell cycles.

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The replication of yeast chromosomes: lessons fromSaccharomyces cerevisiaechromosome III

Canadian Journal of Botany ◽

10.1139/b95-248 ◽

1995 ◽

Vol 73 (S1) ◽

pp. 208-214 ◽

Cited By ~ 1

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.

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The effect on chromosome stability of deleting replication origins.

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.391 ◽

1993 ◽

Vol 13 (1) ◽

pp. 391-398 ◽

Cited By ~ 49

Author(s):

A Dershowitz ◽

C S Newlon

Keyword(s):

Ring Chromosome ◽

High Rate ◽

Chromosome Stability ◽

Replication Origins ◽

Chromosomal Dna ◽

Circular Chromosome ◽

Cell Cycles ◽

Highly Active ◽

Dna Replication Origins ◽

Chromosome Iii

The observed spacing between chromosomal DNA replication origins in Saccharomyces cerevisiae is at least four times shorter than should be necessary to ensure complete replication of chromosomal DNA during the S phase. To test whether all replication origins are required for normal chromosome stability, the loss rates of derivatives of chromosome III from which one or more origins had been deleted were measured. In the case of a 61-kb circular derivative of the chromosome that has two highly active origins and one origin that initiates only 10 to 20% of the time, deletion of either highly active origin increased its rate of loss two- to fourfold. Deletion of both highly active origins caused the ring chromosome to be lost in approximately 20% of cell divisions. This very high rate of loss demonstrates that there are no efficient cryptic origins on the ring chromosome that are capable of ensuring its replication in the absence of the origins that are normally used. Deletion of the same two origins from the full-length chromosome III, which contains more than six replication origins, had no effect on its rate of loss. These results suggest that the increase in the rate of loss of the small circular chromosome from which a single highly active origin was deleted was caused by the failure of the remaining highly active origin to initiate replication in a small fraction (approximately 0.003) of cell cycles.

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Chromosomal replication origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae are functional in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.152.3.983-993.1982 ◽

1982 ◽

Vol 152 (3) ◽

pp. 983-993

Author(s):

N E Harding ◽

J M Cleary ◽

D W Smith ◽

J J Michon ◽

W S Brusilow ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Enterobacter Aerogenes ◽

Replication Origins ◽

Chromosomal Dna ◽

Protein Activity ◽

Chromosomal Replication ◽

E Coli ◽

Dna Replication Origins ◽

Unc Operon

The chromosomal DNA replication origins (oriC) from two members of the family Enterobacteriaceae, Enterobacter aerogenes and Klebsiella pneumoniae, have been isolated as functional replication origins in Escherichia coli. The origins in the SalI restriction fragments of 17.5 and 10.2 kilobase pairs, cloned from E. aerogenes and K. pneumoniae, respectively, were found to be between the asnA and uncB genes, as are the origins of the E. coli and Salmonella typhimurium chromosomes. Plasmids containing oriC from E aerogenes, K. pneumoniae, and S. typhimurium replicate in the E. coli cell-free enzyme system (Fuller, et al., Proc. Natl. Acad. Sci. U.S.A. 78:7370--7374, 1981), and this replication is dependent on dnaA protein activity. These SalI fragments from E. aerogenes and K. pneumoniae carry a region which is lethal to E. coli when many copies are present. We show that this region is also carried on the E. coli 9.0-kilobase-pair EcoRI restriction fragment containing oriC. The F0 genes of the atp or unc operon, when linked to the unc operon promoter, are apparently responsible for the lethality.

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High-Resolution Structural Analysis of Chromatin at Specific Loci: Saccharomyces cerevisiae Silent Mating Type Locus HMLα

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5392 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5392-5403 ◽

Cited By ~ 88

Author(s):

Kerstin Weiss ◽

Robert T. Simpson

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Chromatin Structure ◽

Transcriptional Repression ◽

Order Structure ◽

Histone H4 ◽

Type Locus ◽

Transcription Start Sites ◽

Chromosome Iii ◽

Nucleotide Resolution

ABSTRACT Genetic studies have suggested that chromatin structure is involved in repression of the silent mating type loci in Saccharomyces cerevisiae. Chromatin mapping at nucleotide resolution of the transcriptionally silent HMLα and the activeMATα shows that unique organized chromatin structure characterizes the silent state of HMLα. Precisely positioned nucleosomes abutting the silencers extend over the α1 and α2 coding regions. The HO endonuclease recognition site, nuclease hypersensitive at MATα, is protected atHMLα. Although two precisely positioned nucleosomes incorporate transcription start sites at HMLα, the promoter region of the α1 and α2 genes is nucleosome free and more nuclease sensitive in the repressed than in the transcribed locus. Mutations in genes essential for HML silencing disrupt the nucleosome array near HML-I but not in the vicinity of HML-E, which is closer to the telomere of chromosome III. At the promoter and the HO site, the structure of HMLα in Sir protein and histone H4 N-terminal deletion mutants is identical to that of the transcriptionally active MATα. The discontinuous chromatin structure of HMLα contrasts with the continuous array of nucleosomes found at repressed a-cell-specific genes and the recombination enhancer. Punctuation at HMLα may be necessary for higher-order structure or karyoskeleton interactions. The unique chromatin architecture of HMLα may relate to the combined requirements of transcriptional repression and recombinational competence.

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