Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast

Takahashi, Tatsuro; Ohara, Eri; Nishitani, Hideo; Masukata, Hisao

doi:10.1093/emboj/cdg079

Cited by 57 publications

(67 citation statements)

References 45 publications

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“…Thus, the overall affinity of SpOrc4 for such a sequence would be a complex function of the number of possible binding modes and the binding affinity of each mode and would be expected to increase with AT content and length. Consistent with these ideas, it has been demonstrated experimentally that SpOrc4 has significant affinity for multiple sites within a single ars element (41)(42)(43)46). Moreover, genetic studies have shown that sequences capable of functioning as origins contain multiple redundant AT-rich elements that contribute to activity (14-16, 18, 38).…”

Section: Discussionmentioning

confidence: 88%

DNA replication origins in the Schizosaccharomyces pombe genome

Dai

Chuang

Kelly

2004

Proc. Natl. Acad. Sci. U.S.A.

121

135

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Origins of DNA replication in Schizosaccharomyces pombe lack a specific consensus sequence analogous to the Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus, raising the question of how they are recognized by the replication machinery. Because all well characterized S. pombe origins are located in intergenic regions, we analyzed the sequence properties and biological activity of such regions. The AT content of intergenes is very high (Ϸ70%), and runs of A's or T's occur with a significantly greater frequency than expected. Additionally, the two DNA strands in intergenes display compositional asymmetry that strongly correlates with the direction of transcription of flanking genes. Importantly, the sequence properties of known S. pombe origins of DNA replication are similar to those of intergenes in general. In functional studies, we assayed the in vivo origin activity of 26 intergenes in a 68-kb region of S. pombe chromosome 2. We also assayed the origin activity of sets of randomly chosen intergenes with the same length or AT content. Our data demonstrate that at least half of intergenes have potential origin activity and that the relative ability of an intergene to function as an origin is governed primarily by AT content and length. We propose a stochastic model for initiation of DNA replication in the fission yeast. In this model, the number of AT tracts in a given sequence is the major determinant of its probability of binding SpORC and serving as a replication origin. A similar model may explain some features of origins of DNA replication in metazoans. T he replicon model postulates that initiation of DNA replication takes place at specific chromosomal sequence elements (replicators) that are recognized by regulatory proteins (initiators) (1). This model was originally proposed to explain features of the replication of prokaryotic cells and viruses and has been validated in such systems by a large body of evidence (2). In eukaryotic cells, DNA replication is initiated from hundreds to thousands of different chromosomal sites in each cell cycle, raising the question of whether the replicon model provides a valid description of the initiation process. Although early genetic studies indicated that DNA replication in the budding yeast Saccharomyces cerevisiae conforms to the main features of the model, it is not yet clear that this is the case for most other eukaryotic species.Origins of DNA replication in S. cerevisiae were identified as sequence elements that conferred the property of autonomous replication on extrachromosomal plasmids (3). Genetic analysis demonstrated that such autonomously replicating sequences (ARS) were Ϸ100 bp in length and contained a common 11-bp consensus sequence essential for origin activity, as well as other sequences that augmented origin activity (3-7). The characterization of S. cerevisiae ARS elements led to the identification of the yeast origin recognition complex (ScORC), the initiator protein that recognizes the ARS-consensus sequence (8). The spe...

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Section: Discussionmentioning

confidence: 88%

DNA replication origins in the Schizosaccharomyces pombe genome

Dai

Chuang

Kelly

2004

Proc. Natl. Acad. Sci. U.S.A.

121

135

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“…ars2004, located in the left arm of chromosome II (Fig. 1A), is a well-characterized early origin (Okuno et al 1999;Takahashi et al 2003) where initiation factors assemble efficiently at the onset of S phase (Yamada et al 2004;Yabuuchi et al 2006). AT2088/ARS745 (Maundrell et al 1988;Segurado et al 2003) is a late/dormant origin located in the right arm of chromosome II (Fig.…”

Section: Identification Of the Dna Element That Controls Replication mentioning

confidence: 99%

Telomere-binding protein Taz1 controls global replication timing through its localization near late replication origins in fission yeast

et al. 2012

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In eukaryotes, the replication of chromosome DNA is coordinated by a replication timing program that temporally regulates the firing of individual replication origins. However, the molecular mechanism underlying the program remains elusive. Here, we report that the telomere-binding protein Taz1 plays a crucial role in the control of replication timing in fission yeast. A DNA element located proximal to a late origin in the chromosome arm represses initiation from the origin in early S phase. Systematic deletion and substitution experiments demonstrated that two tandem telomeric repeats are essential for this repression. The telomeric repeats recruit Taz1, a counterpart of human TRF1 and TRF2, to the locus. Genome-wide analysis revealed that Taz1 regulates about half of chromosomal late origins, including those in subtelomeres. The Taz1-mediated mechanism prevents Dbf4-dependent kinase (DDK)-dependent Sld3 loading onto the origins. Our results demonstrate that the replication timing program in fission yeast uses the internal telomeric repeats and binding of Taz1.

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“…We used primers previously designed by Ogawa et al, 1999 to amplify sequences specific to origin DNA (ars2004 or ars3002) and to nonARS DNA, located ϳ25 kb centromere-proximal to ars2004 on chromosome II (Ogawa et al, 1999). Similar primers have been successfully used for the analysis of Orc1 and Mcm binding to origin DNA (Ogawa et al, 1999;Takahashi and Masukata, 2001;Takahashi et al, 2003). These primer sets were used in quantitative PCR on DNA prepared either from immunoprecipitated chromatin fractions or from total DNA isolated from whole cell soluble extracts.…”

mentioning

confidence: 99%

Schizosacchromyces pombeDpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

Feng

Rodriguez-Menocal

Tolun

et al. 2003

MBoC

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Genetic evidence suggests that DNA polymerase epsilon (Pol ⑀) has a noncatalytic essential role during the early stages of DNA replication initiation. Herein, we report the cloning and characterization of the second largest subunit of Pol ⑀ in fission yeast, called Dpb2. We demonstrate that Dpb2 is essential for cell viability and that a temperature-sensitive mutant of dpb2 arrests with a 1C DNA content, suggesting that Dpb2 is required for initiation of DNA replication. Using a chromatin immunoprecipitation assay, we show that Dpb2, binds preferentially to origin DNA at the beginning of S phase. We also show that the C terminus of Pol ⑀ associates with origin DNA at the same time as Dpb2. We conclude that Dpb2 is an essential protein required for an early step in DNA replication. We propose that the primary function of Dpb2 is to facilitate assembly of the replicative complex at the start of S phase. These conclusions are based on the novel cell cycle arrest phenotype of the dpb2 mutant, on the previously uncharacterized binding of Dpb2 to replication origins, and on the observation that the essential function of Pol ⑀ is not dependent on its DNA synthesis activity. INTRODUCTIONChromosomal DNA replication in eukaryotic cells requires the activity of at least three DNA polymerases: ␣, ␦, and ⑀ (Waga and Stillman, 1998;Hubscher et al., 2000;Bell and Dutta, 2002). Pol ␣ is tightly associated with a primase activity capable of de novo DNA synthesis, suggesting that this enzyme is responsible for initiation of both leading and lagging strands (Waga and Stillman, 1998). Pol ␣/primase can only synthesize short primers that must then be extended by the activity of a processive DNA polymerase(s). Biochemical analysis of simian virus 40 (SV40) DNA replication, which has been extensively used as a model system for eukaryotic DNA replication, has shown that primers synthesized by Pol ␣ can be extended by Pol ␦ and that these two polymerases are sufficient for SV40 replication in vitro (Weinberg et al., 1990;Waga et al., 1994).A second processive DNA polymerase, Pol ⑀, is required for cell viability and chromosomal DNA replication in both fission (D'Urso and Nurse, 1997) and budding yeast (Morrison et al., 1990;Araki et al., 1992;Budd and Campbell, 1993). Pol ⑀ has also been implicated in both DNA repair (Jessberger et al., 1993;Wang et al., 1993;Shivji et al., 1995;Holmes, 1999) and cell cycle checkpoint control in eukaryotic cells (Navas et al., 1995). Recently, we have shown that the DNA polymerase and exonuclease domains of Pol ⑀ are dispensable for cell viability in fission yeast (Feng and D'Urso, 2001). Similar observations have been made for the evolutionarily distant yeast, Saccharomyces cerevisiae (Kesti et al., 1999;Dua et al., 2000). These findings have raised important questions regarding the essential role of this replicative enzyme in DNA synthesis. Although the N-terminal catalytic domains are dispensable, the C-terminal half of the enzyme is essential for cell viability and chromosomal replication in fission...

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Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast

Cited by 57 publications

References 45 publications

DNA replication origins in the Schizosaccharomyces pombe genome

DNA replication origins in the Schizosaccharomyces pombe genome

Telomere-binding protein Taz1 controls global replication timing through its localization near late replication origins in fission yeast

Schizosacchromyces pombeDpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

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