Smc5/6 in the rDNA modulates lifespan independently of Fob1.
ABSTRACT: The ribosomal DNA (rDNA) in Saccharomyces cerevisiae is in one tandem repeat array on Chromosome XII. Two regions within each repetitive element, called intergenic spacer 1 (IGS1) and IGS2, are important for organizing the rDNA within the nucleolus. The Smc5/6 complex localizes to IGS1 and IGS2. We show that Smc5/6 has a function in the rDNA beyond its role in homologous recombination (HR) at the replication fork barrier (RFB) located in IGS1. Fob1 is required for optimal binding of Smc5/6 at IGS1 whereas the canonical silencing factor Sir2 is required for its optimal binding at IGS2, independently of Fob1. Through interdependent interactions, Smc5/6 stabilizes Sir2 and Cohibin at both IGS and its recovery at IGS2 is important for nucleolar compaction and transcriptional silencing, which in turn supports rDNA stability and lifespan.
Project description:Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA) array. Each rDNA repeat contains a programmed replication fork barrier (RFB) established by the Fob1 protein. Fob1 removal improves rDNA replication in Smc5/6 depleted cells, implicating Smc5/6 in the management of programmed fork pausing. A similar improvement is achieved by removing the DNA helicase Mph1 whose recombinogenic activity can be inhibited by Smc5/6 under DNA damage conditions. DNA 2D gel analyses further show that Smc5/6 loss increases recombination structures at RFB regions; moreover, mph1? and fob1? similarly reduce this accumulation. These findings point to an important mitotic role for Smc5/6 in restraining recombination events when protein barriers in rDNA stall replication forks. As rDNA maintenance influences multiple essential cellular processes, Smc5/6 likely links rDNA stability to overall mitotic growth.
Project description:RNA polymerase II (Pol II)-transcribed genes embedded within the yeast rDNA locus are repressed through a Sir2-dependent process called 'rDNA silencing'. Sir2 is recruited to the rDNA promoter through interactions with RNA polymerase I (Pol I), and to a pair of DNA replication fork block sites (Ter1 and Ter2) through interaction with Fob1. We utilized a reporter gene (mURA3) integrated adjacent to the leftmost rDNA gene to investigate localized Pol I and Fob1 functions in silencing. Silencing was attenuated by loss of Pol I subunits or insertion of an ectopic Pol I terminator within the adjacent rDNA gene. Silencing left of the rDNA array is naturally attenuated by the presence of only one intact Fob1 binding site (Ter2). Repair of the 2nd Fob1 binding site (Ter1) dramatically strengthens silencing such that it is no longer impacted by local Pol I transcription defects. Global loss of Pol I activity, however, negatively affects Fob1 association with the rDNA. Loss of Ter2 almost completely eliminates localized silencing, but is restored by artificially targeting Fob1 or Sir2 as Gal4 DNA binding domain fusions. We conclude that Fob1 and Pol I make independent contributions to establishment of silencing, though Pol I also reinforces Fob1-dependent silencing.
Project description:The NAD-dependent histone deacetylase Sir2 controls ribosomal DNA (rDNA) silencing by inhibiting recombination and RNA polymerase II-catalyzed transcription in the rDNA of Saccharomyces cerevisiae Sir2 is recruited to nontranscribed spacer 1 (NTS1) of the rDNA array by interaction between the RENT ( RE: gulation of N: ucleolar S: ilencing and T: elophase exit) complex and the replication terminator protein Fob1. The latter binds to its cognate sites, called replication termini (Ter) or replication fork barriers (RFB), that are located in each copy of NTS1. This work provides new mechanistic insights into the regulation of rDNA silencing and intrachromatid recombination by showing that Sir2 recruitment is stringently regulated by Fob1 phosphorylation at specific sites in its C-terminal domain (C-Fob1), which also regulates long-range Ter-Ter interactions. We show further that long-range Fob1-mediated Ter-Ter interactions in trans are downregulated by Sir2. These regulatory mechanisms control intrachromatid recombination and the replicative life span (RLS).
Project description:The replication terminator protein Fob1 of Saccharomyces cerevisiae is multifunctional, and it not only promotes polar replication fork arrest at the tandem Ter sites located in the intergenic spacer region of rDNA but also loads the NAD-dependent histone deacetylase Sir2 at Ter sites via a protein complex called RENT (regulator of nucleolar silencing and telophase exit). Sir2 is a component of the RENT complex, and its loading not only silences intrachromatid recombination in rDNA but also RNA polymerase II-catalyzed transcription. Here, we present three lines of evidence showing that the two aforementioned activities of Fob1 are independent of each other as well as functionally separable. First, a Fob1 ortholog of Saccharomyces bayanus expressed in a fob1Delta strain of S. cerevisiae restored polar fork arrest at Ter but not rDNA silencing. Second, a mutant form (I407T) of S. cerevisiae Fob1 retained normal fork arresting activity but was partially defective in rDNA silencing. We further show that the silencing defect of S. bayanus Fob1 and the Iota407Tau mutant of S. cerevisiae Fob1 were caused by the failure of the proteins to interact with two members of the S. cerevisiae RENT complex, namely S. cerevisiae Sir2 and S. cerevisiae Net1. Third, deletions of the intra-S phase checkpoint proteins Tof1 and Csm3 abolished fork arrest by Fob1 at Ter without causing loss of silencing. Taken together, the data support the conclusion that unlike some other functions of Fob1, rDNA silencing at Ter is independent of fork arrest.
Project description:An average of 200 copies of the rRNA gene (rDNA) is clustered in a long tandem array in Saccharomyces cerevisiae. FOB1 is known to be required for expansion/contraction of the repeats by stimulating recombination, thereby contributing to the maintenance of the average copy number. In Deltafob1 cells, the repeats are still maintained without any fluctuation in the copy number, suggesting that another, unknown system acts to prevent repeat contraction. Here, we show that condensin acts together with FOB1 in a functionally complemented fashion to maintain the long tandem repeats. Six condensin mutants possessing severely contracted rDNA repeats were isolated in Deltafob1 cells but not in FOB1+ cells. We also found that the condensin complex associated with the nontranscribed spacer region of rDNA with a major peak coincided with the replication fork barrier (RFB) site in a FOB1-dependent fashion. Surprisingly, condensin association with the RFB site was established during S phase and was maintained until anaphase. These results indicate that FOB1 plays a novel role in preventing repeat contraction by regulating condensin association and suggest a link between replication termination and chromosome condensation and segregation.
Project description:Fob1 protein plays an important role in aging and maintains genomic stability by avoiding clashes between the replication and transcription machinery. It facilitates polar arrest by binding to replication fork barrier (RFB) sites, present within the nontranscribed spacer region of the ribosomal DNA. Here, we investigate the mechanism of unidirectional arrest by creating multiple prosthetic forks within the RFB, with fluorescent adenine analogue 2-aminopurine incorporated site-specifically in both the "permissible" and "nonpermissible" directions. The motional dynamics of the RFB-Fob1 complexes analyzed by fluorescence lifetime and fluorescence anisotropy decay kinetics shows that Fob1 adopts a clamp-lock model of arrest and causes stronger perturbation with the bases in the double-stranded region of the nonpermissible-directed forks over those of the permissible directed ones, thereby creating a polar barrier. Corroborative thermal melting studies reveal a skewed distribution of GC content within the RFB sequence that potentially assists in Fob1-mediated arrest.
Project description:Ribosomal DNA (rDNA) recombination in budding yeast is regulated by multiple converging processes, including posttranslational modifications such as SUMOylation. In this study, we report that the absence of a SUMO E3 ligase, Siz2, results in increased unequal rDNA exchange. We show that Siz2 is enriched at the replication fork barrier (RFB) in the rDNA and also controls the homeostasis of Tof2 protein. siz2? resulted in increased accumulation of total Tof2 in the cell and a consequent increase in the enrichment of Tof2 at the rDNA. Overproducing Tof2 ectopically or conditional overexpression of Tof2 also resulted in higher levels of rDNA recombination, suggesting a direct role for Tof2. Additionally, our chromatin immunoprecipitation (ChIP) data indicate that the accumulation of Tof2 in a siz2? mutant resulted in an enhanced association of Fob1, an RFB binding protein at the rDNA at the RFB. This increased Fob1 association at the RFB may have resulted in the elevated rDNA recombination. Our study thus demonstrates that the Tof2 levels modulate recombination at the rDNA.IMPORTANCE The genes that encode rRNA in Saccharomyces cerevisiae are organized as multiple repeats. The repetitive nature and heavy transcription of this region make it prone to DNA breaks. DNA breaks could lead to recombination, which could result in either loss or gain of repeats with detrimental consequences to the cell. Multiple mechanisms operate to maintain the stability of rDNA. Earlier studies reported that the absence of Ulp2, a deSUMOylase, resulted in declining levels of Tof2 and thereby disrupted rDNA silencing. In contrast, our findings suggest that accumulation of Tof2 can also result in increased rDNA recombination, through a mechanism that involves Fob1, an RFB-bound protein. While our study has examined only Tof2, rDNA recombination could be regulated by other proteins through a mechanism similar to this.
Project description:The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1? sir2? double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2?. We find, in addition, that the viability of rif1? cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1? cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.
Project description:Purpose: This study was designed to determine the mechanism of condensin and Sir2 recruitment to the RDT1 promoter region of S. cerevisiae chromosome III, and to test whether preventing condensin recruitment by deleting the key factors, Fob1 and Lrs4, significantly alters the conformation of chromosome III. Conclusions: Conformational changes in chromosome III and loss of CEN12-rDNA looping in chromosome XII observed in lrs4? and fob1? mutants.
Project description:In eukaryotic cells, ribosomal DNA (rDNA) forms the basis of the nucleolus. In Saccharomyces cerevisiae, 100-200 copies of a 9.1-kb rDNA repeat exist as a tandem array on chromosome XII. The stability of this highly repetitive array is maintained through silencing. However, the precise mechanisms that regulate rDNA silencing are poorly understood. Here, we report that S. cerevisiae Ydr026c, which we name NTS1 silencing protein 1 (Nsi1), plays a significant role in rDNA silencing. By studying the subcellular localization of 159 nucleolar proteins, we identified 11 proteins whose localization pattern is similar to that of Net1, a well-established rDNA silencing factor. Among these proteins is Nsi1, which is associated with the NTS1 region of rDNA and is required for rDNA silencing at NTS1. In addition, Nsi1 physically interacts with the known rDNA silencing factors Net1, Sir2 and Fob1. The loss of Nsi1 decreases the association of Sir2 with NTS1 and increases histone acetylation at NTS1. Furthermore, Nsi1 contributes to the longevity of yeast cells. Taken together, our findings suggest that Nsi1 is a new rDNA silencing factor that contributes to rDNA stability and lifespan extension in S. cerevisiae.