The effect on chromosome stability of deleting replication origins

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.10.5346-5355.1991 ◽

1991 ◽

Vol 11 (10) ◽

pp. 5346-5355

Author(s):

D D Dubey ◽

L R Davis ◽

S A Greenfeder ◽

L Y Ong ◽

J G Zhu ◽

...

Keyword(s):

Mating Type ◽

Transcriptional Repression ◽

Replication Origins ◽

Chromosomal Dna ◽

Chromosomal Replication ◽

Type Locus ◽

Ars Elements ◽

Dna Fragment ◽

Dna Replication Origins ◽

Chromosome Iii

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.10.5346 ◽

1991 ◽

Vol 11 (10) ◽

pp. 5346-5355 ◽

Cited By ~ 83

Author(s):

D D Dubey ◽

L R Davis ◽

S A Greenfeder ◽

L Y Ong ◽

J G Zhu ◽

...

Keyword(s):

Mating Type ◽

Transcriptional Repression ◽

Replication Origins ◽

Chromosomal Dna ◽

Chromosomal Replication ◽

Type Locus ◽

Ars Elements ◽

Dna Fragment ◽

Dna Replication Origins ◽

Chromosome Iii

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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Method of mapping DNA replication origins

Molecular and Cellular Biology ◽

10.1128/mcb.5.1.85-92.1985 ◽

1985 ◽

Vol 5 (1) ◽

pp. 85-92

Author(s):

L D Spotila ◽

J A Huberman

Keyword(s):

Dna Replication ◽

Simian Virus 40 ◽

Simian Virus ◽

Replication Forks ◽

Replication Origins ◽

Chromosomal Dna ◽

Dna Restriction Fragments ◽

Dna Restriction ◽

Dna Replication Origins

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.

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DNA Replication Origins Fire Stochastically in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e05-07-0657 ◽

2006 ◽

Vol 17 (1) ◽

pp. 308-316 ◽

Cited By ~ 133

Author(s):

Prasanta K. Patel ◽

Benoit Arcangioli ◽

Stephen P. Baker ◽

Aaron Bensimon ◽

Nicholas Rhind

Keyword(s):

Dna Replication ◽

Fission Yeast ◽

Replication Initiation ◽

Epigenetic Mechanism ◽

Replication Origins ◽

Origin Firing ◽

Cell Cycles ◽

Initiation Of Replication ◽

Dna Combing ◽

Dna Replication Origins

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.

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Sir2 mitigates an intrinsic imbalance in origin licensing efficiency between early and late replicating euchromatin

10.1101/2020.01.24.918862 ◽

2020 ◽

Author(s):

Timothy Hoggard ◽

Carolin A. Müller ◽

Conrad A. Nieduszynski ◽

Michael Weinreich ◽

Catherine A. Fox

Keyword(s):

Saccharomyces Cerevisiae ◽

Spatial Distribution ◽

Dna Replication ◽

Genome Stability ◽

Chromatin Modification ◽

Replication Origins ◽

Chromosomal Dna ◽

Eukaryotic Chromosome ◽

Origin Licensing ◽

Dna Replication Origins

AbstractA eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled on chromosomal DNA in G1-phase (a.k.a. origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing established the normal spatial distribution of origins across yeast chromosomes required for normal genome duplication.Significance statementIn eukaryotes, multiple DNA replication origins, the sites where new DNA synthesis begins during the process of cell division, must be adequately distributed across chromosomes to maintain normal cell proliferation and genome stability. This study describes a repressive chromatin-mediated mechanism that acts at the level of individual origins to attenuate the efficiency of origin formation. This attenuation is essential for achieving the normal spatial distribution of origins across the chromosomes of the eukaryotic microbe Saccharomyces cerevisiae. While the importance of chromosomal origin distribution to cellular fitness is now widely acknowledged, this study is the first to define a specific chromatin modification that establishes the normal spatial distribution of origins across a eukaryotic genome.

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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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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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