A model of the replication fork blocking protein Fob1p based on the catalytic core domain of retroviral integrases

The replication fork blocks are common in both prokaryotes and eukaryotes. In most cases, these blocks are associated with increased levels of mitotic recombination. One of the best-characterized replication fork blocks in eukaryotes is found in ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae. It has been shown that the replication fork blocking protein Fob1p regulates the recombination rate and the number of rDNA copies in S. cerevisiae, but the mechanistic aspects of these events are still poorly understood. Sequence profile searches revealed that Fob1p is related to retroviral integrases. Subsequently, the catalytic domain of HIV-1 integrase was used as a template to build a reliable three-dimensional model of Fob1p. Structural insights from this study may be useful in explaining Fob1p-mediated formation of extrachromosomal rDNA circles that accelerate aging in yeast and recombination events that lead to expansion or contraction of rDNA.     

The replication fork barrier site forms a unique structure with Fob1p and inhibits the replication fork

The replication fork barrier site (RFB) is an approximately 100-bp DNA sequence located near the 3' end of the rRNA genes in the yeast Saccharomyces cerevisiae. The gene FOB1 is required for this RFB activity. FOB1 is also necessary for recombination in the ribosomal DNA (rDNA), including increase and decrease of rDNA repeat copy number, production of extrachromosomal rDNA circles, and possibly homogenization of the repeats. Despite the central role that Foblp plays in both replication fork blocking and rDNA recombination, the molecular mechanism by which Fob1p mediates these activities has not been determined. Here, I show by using chromatin immunoprecipitation, gel shift, footprinting, and atomic force microscopy assays that Fob1p directly binds to the RFB. Fob1p binds to two separated sequences in the RFB. A predicted zinc finger motif in Fob1p was shown to be essential for the RFB binding, replication fork blocking, and rDNA recombination activities. The RFB seems to wrap around Fob1p, and this wrapping structure may be important for function in the rDNA repeats.     

Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.     

The replication fork block protein Fob1 functions as a negative regulator of the FEAR network

BACKGROUND: The protein phosphatase Cdc14 is a key regulator of exit from mitosis in budding yeast. Its activation during anaphase is characterized by dissociation from its inhibitor Cfi1/Net1 in the nucleolus and is controlled by two regulatory networks. The Cdc14 early anaphase release (FEAR) network promotes activation of the phosphatase during early anaphase, whereas the mitotic exit network (MEN) activates Cdc14 during late stages of anaphase. RESULTS: Here we investigate how the FEAR network component Spo12 regulates Cdc14 activation. We identify the replication fork block protein Fob1 as a Spo12-interacting factor. Inactivation of FOB1 leads to premature release of Cdc14 from the nucleolus in metaphase-arrested cells. Conversely, high levels of FOB1 delay the release of Cdc14 from the nucleolus. Fob1 associates with Cfi1/Net1, and consistent with this observation, we find that the bulk of Cdc14 localizes to the Fob1 binding region within the rDNA repeats. Finally, we show that Spo12 phosphorylation is cell cycle regulated and affects its binding to Fob1. CONCLUSIONS: Fob1 functions as a negative regulator of the FEAR network. We propose that Fob1 helps to prevent the dissociation of Cdc14 from Cfi1/Net1 prior to anaphase and that Spo12 activation during early anaphase promotes the release of Cdc14 from its inhibitor by antagonizing Fob1 function.     

Low levels of DNA polymerase alpha induce mitotic and meiotic instability in the ribosomal DNA gene cluster of Saccharomyces cerevisiae

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.     

Sir2-independent life span extension by calorie restriction in yeast

Calorie restriction slows aging and increases life span in many organisms. In yeast, a mechanistic explanation has been proposed whereby calorie restriction slows aging by activating Sir2. Here we report the identification of a Sir2-independent pathway responsible for a majority of the longevity benefit associated with calorie restriction. Deletion of FOB1 and overexpression of SIR2 have been previously found to increase life span by reducing the levels of toxic rDNA circles in aged mother cells. We find that combining calorie restriction with either of these genetic interventions dramatically enhances longevity, resulting in the longest-lived yeast strain reported thus far. Further, calorie restriction results in a greater life span extension in cells lacking both Sir2 and Fob1 than in cells where Sir2 is present. These findings indicate that Sir2 and calorie restriction act in parallel pathways to promote longevity in yeast and, perhaps, higher eukaryotes.     

hpr1Δ Affects Ribosomal DNA Recombination and Cell Life Span in Saccharomyces cerevisiae

Multiple genetic pathways have been shown to regulate life span and aging in the yeast Saccharomyces cerevisiae. Here we show that loss of a component of the RNA polymerase II complex, Hpr1p, results in a decreased life span. Although hpr1Delta mutants have an increased rate of recombination within the ribosomal DNA (rDNA) array, this is not accompanied by an increase in extrachromosomal rDNA circles (ERCs). Analyses of mutants that affect replication of the rDNA array and suppressors that reverse the phenotypes of the hpr1Delta mutant show that the reduced life span is associated with increased genomic instability but not with increased ERC formation. The hpr1Delta mutant acts in a pathway distinct from previously described mutants that reduce life span.     

Repeat expansion in the budding yeast ribosomal DNA can occur independently of the canonical homologous recombination machinery

Major eukaryotic genomic elements, including the ribosomal DNA (rDNA), are composed of repeated sequences with well-defined copy numbers that must be maintained by regulated recombination. Although mechanisms that instigate rDNA recombination have been identified, none are directional and they therefore cannot explain precise repeat number control. Here, we show that yeast lacking histone chaperone Asf1 undergo reproducible rDNA repeat expansions. These expansions do not require the replication fork blocking protein Fob1 and are therefore independent of known rDNA expansion mechanisms. We propose the existence of a regulated rDNA repeat gain pathway that becomes constitutively active in asf1Δ mutants. Cells lacking ASF1 accumulate rDNA repeats with high fidelity in a processive manner across multiple cell divisions. The mechanism of repeat gain is dependent on highly repetitive sequence but, surprisingly, is independent of the homologous recombination proteins Rad52, Rad51 and Rad59. The expansion mechanism is compromised by mutations that decrease the processivity of DNA replication, which leads to progressive loss of rDNA repeats. Our data suggest that a novel mode of break-induced replication occurs in repetitive DNA that is dependent on high homology but does not require the canonical homologous recombination machinery.     

Calorie restriction reduces rDNA recombination independently of rDNA silencing

Calorie restriction (CR) extends lifespan in yeast, worms, flies and mammals, suggesting that it acts via a conserved mechanism. In yeast, activation of the NAD‐dependent histone deacetylase, Sir2, by CR is thought to increase silencing at the ribosomal DNA, thereby reducing the recombination‐induced generation of extrachromosomal rDNA circles, hence increasing replicative lifespan. Although accumulation of extrachromosomal rDNA circles is specific to yeast aging, it is thought that Sirtuin activation represents a conserved longevity mechanism through which the beneficial effects of CR are mediated in various species. We show here that growing yeast on 0.05 or 0.5% glucose (severe and moderate CR, respectively) does not increase silencing at either sub‐telomeric or rDNA loci compared with standard (2% glucose) media. Furthermore, rDNA silencing was unaffected in the hxk2Δ, sch9Δ and tor1Δ genetic mimics of CR, but inhibited by FOB1 deletion. All these interventions extend lifespan in multiple yeast backgrounds, revealing a poor correlation between rDNA silencing and longevity. In contrast, CR and deletion of the FOB1, HXK2, SCH9 and TOR1 genes, all significantly reduced rDNA recombination. This silencing‐independent mechanism for suppressing rDNA recombination may therefore contribute to CR‐mediated lifespan extension.     

Binding of the replication terminator protein Fob1p to the Ter sites of yeast causes polar fork arrest

Fob1p protein has been implicated in the termination of replication forks at the two tandem termini present in the non-transcribed spacer region located between the sequences encoding the 35 S and the 5 S RNAs of Saccharomyces cerevisiae. However, the biochemistry and mode of action of this protein were previously unknown. We have purified the Fob1p protein to near-homogeneity, and we developed a novel technique to show that it binds specifically to the Ter1 and Ter2 sequences. Interestingly, the two sequences share no detectable homology. We present two lines of evidence showing that the interaction of the Fob1p with the Ter sites causes replication termination. First, a mutant of FOB1, L104S, that significantly reduced the binding of the mutant form of the protein to the tandem Ter sites, also failed to promote replication termination in vivo. The mutant did not diminish nucleolar transport, and interaction of the mutant form of Fob1p with itself and with another protein encoded in the locus YDR026C suggested that the mutation did not cause global misfolding of the protein. Second, DNA site mutations in the Ter sequences that separately and specifically abolished replication fork arrest at Ter1 or Ter2 also eliminated sequence-specific binding of the Fob1p to the two sites. The work presented here definitively established Ter DNA-Fob1p interaction as an important step in fork arrest.     

Transcription of ribosomal genes can cause nondisjunction

Mitotic disjunction of the repetitive ribosomal DNA (rDNA) involves specialized segregation mechanisms dependent on the conserved phosphatase Cdc14. The reason behind this requirement is unknown. We show that rDNA segregation requires Cdc14 partly because of its physical length but most importantly because a fraction of ribosomal RNA (rRNA) genes are transcribed at very high rates. We show that cells cannot segregate rDNA without Cdc14 unless they undergo genetic rearrangements that reduce rDNA copy number. We then demonstrate that cells with normal length rDNA arrays can segregate rDNA in the absence of Cdc14 as long as rRNA genes are not transcribed. In addition, our study uncovers an unexpected role for the replication barrier protein Fob1 in rDNA segregation that is independent of Cdc14. These findings demonstrate that highly transcribed loci can cause chromosome nondisjunction.     

A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics.     

Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication

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 rif1Δ sir2Δ 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 sir2Δ. We find, in addition, that the viability of rif1Δ 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 rif1Δ 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.     

Enforcement of a lifespan‐sustaining distribution of Sir2 between telomeres,mating‐type loci,and rDNA repeats by Rif1

Telomere dysfunction is linked with genome instability and premature aging. Roles for sirtuin proteins at telomeres are thought to promote lifespan in yeast and mammals. However, replicative lifespan of the budding yeast Saccharomyces cerevisiae shortens upon deletion of Rif1, a protein that limits the recruitment of the sirtuin histone deacetylase Sir2 to telomeres. Here we show that Rif1 maintains replicative lifespan by ultimately stabilizing another age‐related chromosomal domain harboring the ribosomal DNA (rDNA) repeats. Deletion of Rif1 increases Sir2 localization to telomeres and the silent mating‐type loci, while releasing a pool of the histone deacetylase from the intergenic spacer 1 (IGS1) of rDNA. This is accompanied by a disruption of IGS1 silent chromatin assembly and increases in aberrant recombination within rDNA repeats. Lifespan defects linked with Rif1 deletion are abolished if rDNA repeats are forcibly stabilized via deletion of the replication fork‐blocking protein Fob1. In addition, Sir2 overexpression prevents Rif1 deletion from disrupting Sir2 at IGS1 and shortening lifespan. Moreover, subjecting cells lacking Rif1 to caloric restriction increases IGS1 histone deacetylation and lifespan, while uncovering novel genetic interactions between RIF1 and SIR2. Our data indicate that Rif1 maintains lifespan‐sustaining levels of Sir2 at rDNA by preventing excessive recruitment of the histone deacetylase to telomeric and silent mating‐type loci. As sirtuin histone deacetylases, such as Sir2 or mammalian SIRT6, each operate at multiple age‐related loci, we propose that factors limiting the localization of sirtuins to certain age‐related loci can promote lifespan‐sustaining roles of these sirtuins elsewhere in the genome.     

Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53

The yeast checkpoint factors Mrc1p and Tof1p travel with the replication fork and mediate the activation of the Rad53p kinase in response to a replication stress. We show here that both proteins are required for normal fork progression but play different roles at stalled forks. Tof1p is critical for the activity of the rDNA replication fork barrier (RFB) but plays a minor role in the replication checkpoint. In contrast, Mrc1p is not necessary for RFB activity but is essential to mediate the replication stress response. Interestingly, stalled forks did not collapse in mrc1Delta cells exposed to hydroxyurea (HU) as they do in rad53 mutants. However, forks failed to restart when mrc1Delta cells were released from the block. The critical role of Mrc1p in HU is therefore to promote fork recovery in a Rad53p-independent manner, presumably through the formation of a stable fork-pausing complex.     

rDNA enhancer affects replication initiation and mitotic recombination: Fob1 mediates nucleolytic processing independently of replication

To investigate the influence of the ribosomal DNA enhancer on initiation of replication and recombination at the ribosomal array, we used yeast S. cerevisiae strains with adjacent, tagged rRNA genes. We found that the enhancer is an absolute requirement for replication fork barrier function, while it only modulates initiation of replication. Moreover, the formation of monomeric extrachromosomal ribosomal circles depends on this element. Our data indicate that DNA double-strand breaks occur at specific sites in the parental leading arm of replication forks stalled at the replication fork barrier. Additionally, nicks upstream of the replication fork barrier were visualized by nucleotide-resolution mapping. They coincide with essential sequences of the mitotic hyperrecombination site HOT1, which previously has been determined at ectopic sites. Interestingly, these nicks are strictly dependent on the replication fork blocking-protein (Fob1), but are replication independent, suggesting that intrachromosomal ribosomal DNA recombination may occur outside of S phase.     

Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination

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.