Siz2 Prevents Ribosomal DNA Recombination by Modulating Levels of Tof2 in Saccharomyces cerevisiae.
ABSTRACT: 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: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:Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.
Project description:The ribosomal DNA (rDNA) genes of Saccharomyces cerevisiae are located in a tandem array of about 150 repeats. Using a diploid with markers flanking and within the rDNA array, we showed that low levels of DNA polymerase alpha elevate recombination between both homologues and sister chromatids, about five-fold in mitotic cells and 30-fold in meiotic cells. This stimulation is independent of Fob1p, a protein required for the programmed replication fork block (RFB) in the rDNA. We observed that the fob1 mutation alone significantly increased meiotic, but not mitotic, rDNA recombination, suggesting a meiosis-specific role for this protein. We found that meiotic cells with low polymerase alpha had decreased Sir2p binding and increased Spo11p-catalyzed double-strand DNA breaks in the rDNA. Furthermore, meiotic crossover interference in the rDNA is absent. These results suggest that the hyper-Rec phenotypes resulting from low levels of DNA polymerase alpha in mitosis and meiosis reflect two fundamentally different mechanisms: the increased mitotic recombination is likely due to increased double-strand DNA breaks (DSBs) resulting from Fob1p-independent stalled replication forks, whereas the hyper-Rec meiotic phenotype results from increased levels of Spo11-catalyzed DSBs in the rDNA.
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: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:Protein-mediated "chromosome kissing" between two DNA sites in trans (or in cis) is known to facilitate three-dimensional control of gene expression and DNA replication. However, the mechanisms of regulation of the long-range interactions are unknown. Here, we show that the replication terminator protein Fob1 of Saccharomyces cerevisiae promoted chromosome kissing that initiated rDNA recombination and controlled the replicative life span (RLS). Oligomerization of Fob1 caused synaptic (kissing) interactions between pairs of terminator (Ter) sites that initiated recombination in rDNA. Fob1 oligomerization and Ter-Ter kissing were regulated by intramolecular inhibitory interactions between the C-terminal domain (C-Fob1) and the N-terminal domain (N-Fob1). Phosphomimetic substitutions of specific residues of C-Fob1 counteracted the inhibitory interaction. A mutation in either N-Fob1 that blocked Fob1 oligomerization or C-Fob1 that blocked its phosphorylation antagonized chromosome kissing and recombination and enhanced the RLS. The results provide novel insights into a mechanism of regulation of Fob1-mediated chromosome kissing.
Project description:Homologous recombination is believed to play important roles in processing stalled/blocked replication forks in eukaryotes. In accordance with this, recombination is induced by replication fork barriers (RFBs) within the rDNA locus. However, the rDNA locus is a specialised region of the genome, and therefore the action of recombinases at its RFBs may be atypical. We show here for the first time that direct repeat recombination, dependent on Rad22 and Rhp51, is induced by replication fork blockage at a site-specific RFB (RTS1) within a 'typical' genomic locus in fission yeast. Importantly, when the RFB is positioned between the direct repeat, conservative gene conversion events predominate over deletion events. This is consistent with recombination occurring without breakage of the blocked fork. In the absence of the RecQ family DNA helicase Rqh1, deletion events increase dramatically, which correlates with the detection of one-sided DNA double-strand breaks at or near RTS1. These data indicate that Rqh1 acts to prevent blocked replication forks from collapsing and thereby inducing deletion events.
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: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:The ribosomal RNA genes (rDNA) comprise a highly repetitive gene cluster. The copy number of genes at this locus can readily change and is therefore one of the most unstable regions of the genome. DNA damage in rDNA occurs after binding of the replication fork blocking protein Fob1 in S phase, which triggers unequal sister chromatid recombination. However, the precise mechanisms by which such DNA double-strand breaks (DSBs) are repaired is not well understood. Here, we demonstrate that the conserved protein kinase Tel1 maintains rDNA stability after replication fork arrest. We show that rDNA associates with nuclear pores, which is dependent on DNA damage checkpoint kinases Mec1/Tel1 and replisome component Tof1. These findings suggest that rDNA-nuclear pore association is due to a replication fork block and subsequent DSB. Indeed, quantitative microscopy revealed that rDNA is relocated to the nuclear periphery upon induction of a DSB. Finally, rDNA stability was reduced in strains where this association with the nuclear envelope was prevented, which suggests its importance for avoiding improper recombination repair that could induce repeat instability.