Site-Specific Fluorescence Dynamics To Probe Polar Arrest by Fob1 in Replication Fork Barrier Sequences.
ABSTRACT: 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: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: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:In eukaryotes, ribosomal genes (rDNA) are organized in tandem repeats localized in one or a few clusters. Each repeat encompasses a transcription unit and a non-transcribed spacer. Replication forks moving in the direction opposite to transcription are blocked at specific sites called replication fork barriers (rRFBs) in the non-transcribed spacer close to the 3' end of the transcription unit. Here, we investigated and quantified the efficiency of rRFBs in Saccharomyces cerevisiae and to this end transfected budding yeast cells that express dissimilar quantities of Fob1 with circular minichromosomes containing different copies of the minimal 20-bp DNA segment that bind Fob1. To identify fork stalling we used high-resolution 2D agarose gel electrophoresis. The results obtained indicated that neighbor DNA sequences and the relative abundance of Fob1 modulate the efficiency of rRFBs to stall replication forks.
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:In S phase, the replication and transcription of genomic DNA need to accommodate each other, otherwise their machineries collide, with chromosomal instability as a possible consequence. Here, we characterized the human replication fork barrier (RFB) that is present downstream from the 47S pre-rRNA gene (ribosomal DNA [rDNA]). We found that the most proximal transcription terminator, Sal box T1, acts as a polar RFB, while the other, Sal box T4/T5, arrests replication forks bidirectionally. The fork-arresting activity at these sites depends on polymerase I (Pol I) transcription termination factor 1 (TTF-1) and a replisome component, TIMELESS (TIM). We also found that the RFB activity was linked to rDNA copies with hypomethylated CpG and coincided with the time that actively transcribed rRNA genes are replicated. Failed fork arrest at RFB sites led to a slowdown of fork progression moving in the opposite direction to rRNA transcription. Chemical inhibition of transcription counteracted this deceleration of forks, indicating that rRNA transcription impedes replication in the absence of RFB activity. Thus, our results reveal a role of RFB for coordinating the progression of replication and transcription activity in highly transcribed rRNA genes.
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: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:Termination of replication forks at the natural termini of the rDNA of Saccharomyces cerevisiae is controlled in a sequence-specific and polar mode by the interaction of the Fob1p replication terminator protein with the tandem Ter sites located in the nontranscribed spacers. Here we show, by both 2D gel analyses and chromatin immunoprecipitations (ChIP), that there exists a second level of global control mediated by the intra-S-phase checkpoint protein complex of Tof1p and Csm3p that protect stalled forks at Ter sites against the activity of the Rrm3p helicase ("sweepase"). The sweepase tends to release arrested forks presumably by the transient displacement of the Ter-bound Fob1p. Consistent with this mechanism, very few replication forks were arrested at the natural replication termini in the absence of the two checkpoint proteins. In the absence of the Rrm3p helicase, there was a slight enhancement of fork arrest at the Ter sites. Simultaneous deletions of the TOF1 (or CSM3), and the RRM3 genes restored fork arrest by removing both the fork-releasing and fork-protection activities. Other genes such as MRC1, WSS1, and PSY2 that are also involved in the MRC1 checkpoint pathway were not involved in this global control. This observation suggests that Tof1p-Csm3p function differently from MRC1 and the other above-mentioned genes. This mechanism is not restricted to the natural Ter sites but was also observed at fork arrest caused by the meeting of a replication fork with transcription approaching from the opposite direction.
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.