The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication

Versini, Gwennaëlle; Comet, Itys; Wu, Michelle; Hoopes, Laura L. Mays; Schwob, Étienne; Pasero, Philippe

doi:10.1093/emboj/cdg180

Cited by 95 publications

(93 citation statements)

References 62 publications

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“…Importantly, DNA combing studies also revealed the presence of large inactive regions in HU-treated cells (Tourriere et al 2005;Versini et al 2003). These domains are absent in checkpoint mutants, indicating that they correspond to latereplicating regions repressed by checkpoint kinases (Tourriere et al 2005).…”

Section: Single-molecule Analysis: Lessons From Yeastmentioning

confidence: 96%

“…In this approach, an asynchronous population of cells is pulsed-labeled for a short period of time with halogenated nucleotides to determine the position of replication origins (arrowheads). DNA fibers can be counterstained with anti-ssDNA antibodies (blue) (Versini et al 2003), and specific regions can be analyzed by FISH (Lebofsky et al 2006); c DNA combing analysis of densely spaced origins in Xenopus egg extracts (Marheineke and Hyrien 2001). Initiation sites are labeled with DIG-dUTP (red) to mark replication origins (arrowheads) after release from an aphidicolin block and biotin-dUTP (green) is added at variable times until completion of DNA replication; d DNA combing analysis of interorigin distances (IODs) in yeast.…”

Section: Single-molecule Analysis Of Dna Replication Profilesmentioning

confidence: 99%

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Defining replication origin efficiency using DNA fiber assays

2009

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The timely duplication of eukaryotic genomes depends on the coordinated activation of thousands of replication origins distributed along the chromosomes. Origin activation follows a temporal program that is imposed by the chromosomal context and is under the control of S-phase checkpoints. Although the general mechanisms regulating DNA replication are now well-understood at the level of individual origins, little is known on the coordination of thousands of initiation events at a genome-wide level. Recent studies using DNA combing and other single-molecule assays have shown that eukaryotic genomes contain a large excess of replication origins. Most of these origins remain "dormant" in normal growth conditions but are activated when fork progression is impeded. In this review, we discuss how DNA fiber technologies have changed our view of eukaryotic replication programs and how origin redundancy contributes to the maintenance of genome integrity in eukaryotic cells.

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Section: Single-molecule Analysis: Lessons From Yeastmentioning

confidence: 96%

Section: Single-molecule Analysis Of Dna Replication Profilesmentioning

confidence: 99%

Defining replication origin efficiency using DNA fiber assays

2009

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“…In the absence of Sgs1, the stalled forks could be rescued through a recombinogenic mechanism that may proceed in a similar way to that described above. Recently, Versini et al (2003) showed that DNA replication is specifically retarded at the rDNA locus in sgs1 cells and suggested that this could be due to their inability to prevent recombination at stalled forks, abundant in that region. It remains to be seen whether these proposed mechanisms of rDNA maintenance can be extrapolated to S. pombe and other eukaryotes.…”

Section: Slx1-slx4 Endonucleasementioning

confidence: 99%

Slx1-Slx4 Are Subunits of a Structure-specific Endonuclease That Maintains Ribosomal DNA in Fission Yeast

Coulon

Gaillard

Chahwan

et al. 2004

MBoC

109

173

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In most eukaryotes, genes encoding ribosomal RNAs (rDNA) are clustered in long tandem head-to-tail repeats. Studies of Saccharomyces cerevisiae have indicated that rDNA copy number is maintained through recombination events associated with site-specific blockage of replication forks (RFs). Here, we describe two Schizosaccharomyces pombe proteins, homologs of S. cerevisiae Slx1 and Slx4, as subunits of a novel type of endonuclease that maintains rDNA copy number. The Slx1-Slx4 -dependent endonuclease introduces single-strand cuts in duplex DNA on the 3 side of junctions with single-strand DNA. Deletion of Slx1 or Rqh1 RecQ-like DNA helicase provokes rDNA contraction, whereas simultaneous elimination of Slx1-Slx4 endonuclease and Rqh1 is lethal. Slx1 associates with chromatin at two foci characteristic of the two rDNA repeat loci in S. pombe. We propose a model in which the Slx1-Slx4 complex is involved in the control of the expansion and contraction of the rDNA loci by initiating recombination events at stalled RFs. INTRODUCTIONAccurate duplication of a eukaryotic genome depends on highly proficient DNA replication machinery acting in conjunction with DNA repair and checkpoint signaling pathways Osborn et al., 2002). Repair and checkpoint systems are vital because replisomes stall when they encounter DNA damage, protein complexes, or torsional stress. Arrested replication forks (RFs) are prone to rearrangement or collapse. Studies of bacteria have shown that stalled forks can be rescued through either recombinogenic or nonrecombinogenic pathways (Seigneur et al., 1998;Cox et al., 2000;Seigneur et al., 2000;Cox, 2001;McGlynn and Lloyd, 2002).Recent studies have indicated that eukaryotes rescue stalled forks by mechanisms similar to those proposed to act in prokaryotes (Doe et al., 2000;Cox, 2001). A conserved family of eukaryotic DNA helicases related to Escherichia coli RecQ helicase seems to play a central role in the rescue of stalled forks through a nonrecombinogenic mechanism (Doe et al., 2000;Cox, 2001;Hickson et al., 2001). The crucial role of these RecQ-related helicases in maintenance of genome integrity is underscored by the pleiotropic phenotypes of the human Bloom, Werner, and Rothmund-Thomson syndromes associated with defects in BLM, WRN, and RecQ4 proteins, respectively. These hereditary disorders are characterized by high genomic instability, cancer predisposition and, in the case of Werner syndrome, also premature aging (Shen and Loeb, 2000). In budding yeast, Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe, mutations in the genes encoding the RecQ-related helicases Sgs1 and Rqh1, respectively, are associated with hyper-recombination phenotypes. The sgs1 phenotype is characterized by an increase in intra-and interchromosomal recombination, especially at the tandem repeated ribosomal DNA (rDNA) loci (Watt et al., 1995;Sinclair and Guarente, 1997), whereas rqh1 mutants are unable to segregate their chromosomes properly under conditions that stall replication (Stewart et al., 1997...

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“…We used the single-molecule method of DNA combing (13)(14)(15) and genomic microarrays (16) to probe coordination of DNA replication. By using DNA combing, we found that in individual Escherichia coli chromosomes the relative progress of the two replisomes was highly variable; most of the time, the leftward replisome was farther from oriC.…”

mentioning

confidence: 99%

Independence of replisomes in Escherichia coli chromosomal replication

Breier

Cozzarelli

2005

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

103

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In Escherichia coli DNA replication is carried out by the coordinated action of the proteins within a replisome. After replication initiation, the two bidirectionally oriented replisomes from a single origin are colocalized into higher-order structures termed replication factories. The factory model postulated that the two replisomes are also functionally coupled. We tested this hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA surrounding oriC in single, combed chromosomes showed instead that one replisome, usually the leftward one, was significantly ahead of the other 70% of the time. We next used microarrays to follow replication throughout the genome by measuring DNA copy number. We found in multiple E. coli strains that the replisomes are independent, with the leftward replisome ahead of the rightward one. The size of the bias was strain-specific, varying from 50 to 130 kb in the array results. When we artificially blocked one replisome, the other continued unabated, again demonstrating independence. We suggest an improved version of the factory model that retains the advantages of threading DNA through colocalized replisomes at about equal rates, but allows the cell flexibility to overcome obstacles encountered during elongation.DNA combing ͉ microarrays ͉ initiation ͉ replication origin ͉ factory model C ellular DNA replication is almost always initiated in a bidirectional manner. Therefore, initiation of replication requires the recruitment of four polymerases and assorted auxiliary proteins at a replication origin (1). Before 1983, the dominant model was that the leading and lagging polymerases continually and repeatedly sped past each other (Fig. 1A) because of the antiparallel nature of duplex DNA. It was difficult to see how the polymerases could be coordinated to achieve synthesis of both DNA strands at about the same time. Alberts et al. (2) suggested an elegant solution, the trombone model, in which one strand is looped so that the two polymerases are colocalized and point in the same direction (Fig. 1B), forming a replisome that additionally contains the auxiliary proteins. Subsequent work confirmed that sister polymerases are physically attached to one another (1). This colocalization is accompanied by coordination; blocking leading-strand replication stops the whole replisome (3-5).A colocalization of the replisomes themselves was proposed in the factory model of DNA replication (6) and demonstrated cytologically (7). Instead of the left and right replisomes racing away from each other until they meet at the terminus (Fig. 1C), this model proposed that the replisomes have stationary positions in the center of the cell (7). Much as in the trombone model, colocalization is facilitated through looping of the DNA, which is fed through the factory (Fig. 1D). This model organizes replication to the cell's advantage (8,9). Extruding the two daughter chromosomes in opposite directions should greatly facilitate chromosome partitioning (10-12). Furthermore, resources such as dNT...

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The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication

Cited by 95 publications

References 62 publications

Defining replication origin efficiency using DNA fiber assays

Defining replication origin efficiency using DNA fiber assays

Slx1-Slx4 Are Subunits of a Structure-specific Endonuclease That Maintains Ribosomal DNA in Fission Yeast

Independence of replisomes in Escherichia coli chromosomal replication

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