Identification of a site required for DNA replication fork blocking activity in the rRNA gene cluster in Saccharomyces cerevisiae

Kobayashi, Takehiko; Hidaka, Masaaki; Nishizawa, Masafumi; Horiuchi, Takashi

doi:10.1007/bf00265431

Cited by 114 publications

(121 citation statements)

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“…The 35S coding regions are separated by a nontranscribed spacer (NTS), which is divided by the 5S gene into NTS1 and NTS2. Solid bars indicate the replication fork block region (RFB) and the Pol I transcription initiator region (TIR; Elion andWarner 1984, 1986;Brewer and Fangman 1988;Kobayashi et al 1992) the effect of deleting SIR2 on H3 acetylation levels for rDNA regions corresponding to the 25S rRNA, NTS1, and NTS2/18S. We observed an increase in H3 acetylation at each of the above rDNA regions in the range of threefold to fivefold in sir2⌬ compared with SIR2 + cells but no effect on H3 acetylation at the control CUP1 locus ( Fig.…”

Section: Net1 and Sir2 Are Preferentially Associated With Two Regionsmentioning

confidence: 83%

“…Interestingly, recombination rates are significantly lower than would be expected for such a large and repetitive locus, suggesting that recombination is somehow suppressed (Petes 1980). Furthermore, recombination levels are reduced despite the presence of several sequence elements within rDNA that can stimulate recombination (Keil and Roeder 1984;Voelkel-Meiman et al 1987;Brewer and Fangman 1988;Kobayashi et al 1992Huang and Keil 1995;Gruber et al 2000;Ward et al 2000). These findings suggest that both positive and negative regulatory mechanisms have evolved to properly control recombination levels of rDNA.…”

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confidence: 99%

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Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing

Huang¹,

Moazed²

2003

Genes Dev.

210

322

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Silencing within the yeast rDNA repeats inhibits hyperrecombination, represses transcription from foreign promoters, and extends replicative life span. rDNA silencing is mediated by a Sir2-containing complex called RENT (regulator of nucleolar silencing and telophase exit). We show that the Net1 (also called Cfi1) and Sir2 subunits of RENT localize primarily to two distinct regions within rDNA: in one of the nontranscribed spacers (NTS1) and around the Pol I promoter, extending into the 35S rRNA coding region. Binding to NTS1 overlaps the recombination hotspot and replication fork barrier elements, which have been shown previously to require the Fob1 protein for their activities. In cells lacking Fob1, silencing and the association of RENT subunits are abolished specifically at NTS1, while silencing and association at the Pol I promoter region are unaffected or increased. We find that Net1 and Sir2 are physically associated with Fob1 and subunits of RNA polymerase I. Together with the localization data, these results suggest the existence of two distinct modes for the recruitment of the RENT complex to rDNA and reveal a role for Fob1 in rDNA silencing and in the recruitment of the RENT complex. Furthermore, the Fob1-dependent associations of Net1 and Sir2 with the recombination hotspot region strongly suggest that Sir2 acts directly at this region to carry out its inhibitory effect on rDNA recombination and accelerated aging.[Keywords: Gene silencing; chromatin; Sir2; recombination; Fob1; rDNA] Supplemental material is available at http://www.genesdev.org.

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Section: Net1 and Sir2 Are Preferentially Associated With Two Regionsmentioning

confidence: 83%

mentioning

confidence: 99%

Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing

Huang¹,

Moazed²

2003

Genes Dev.

210

322

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“…In S. cerevisiae, a central component of rDNA maintenance is the Fob1 protein that plays an important role in the control of the expansion and contraction of the rDNA copy number (Kobayashi et al, 1992(Kobayashi et al, , 1998Kobayashi and Horiuchi, 1996;Defossez et al, 1999). Fob1 binds at the polar Replication Fork Barrier (RFB) where it blocks RFs from moving into an adjacent rDNA repeat in a direction opposite to that of rDNA transcription.…”

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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“…Sequence-specific replication fork barriers have also been observed in the nontranscribed spacer regions of ribosomal DNAs of Saccharomyces cerevisiae (4,5), plants (6), Xenopus (7), and human (8).…”

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confidence: 96%

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Mohanty

Sahoo

Bastia

1998

Journal of Biological Chemistry

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Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.The chromosomes of Escherichia coli and Bacillus subtilis, and some plasmids like R6K, initiate replication from specific origins and the replication forks moving either bi-or unidirectionally, are arrested at sequence-specific replication termini (1-3). Sequence-specific replication fork barriers have also been observed in the nontranscribed spacer regions of ribosomal DNAs of Saccharomyces cerevisiae (4, 5), plants (6), Xenopus (7), and human (8).The replication termini are sequence-specific and bind to cognate terminator proteins, and the protein-DNA complex arrests replication forks in an orientation-dependent manner (9, 10). DnaB (11,12,19). Detailed biochemical and biophysical analyses have been made possible in both the systems by the determination of the crystal structures of RTP at 2.6 Å (21) and of Tus-Ter () complex at 2.7 Å (22). The DNA binding (23), dimer-dimer interaction (24), and DnaB interaction domains (25) of RTP have been determined by genetic and biochemical analyses that used the crystal structure as a guide. Similarly, the DNA binding domain of Tus has been determined with the help of crystal structure and genetic analysis (22).The impact of transcription on the initiation (26, 27) and elongation stages of DNA replication have been reported (28,29). We have reported previously that Tus and RTP can block RNA chain elongation catalyzed by several prokaryotic RN...

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Identification of a site required for DNA replication fork blocking activity in the rRNA gene cluster in Saccharomyces cerevisiae

Cited by 114 publications

References 35 publications

Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing

Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing

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

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

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