Abstract:In Saccharomyces cerevisiae the FOB1 gene affects replication fork blocking activity at the replication fork block (RFB) sequences and promotes recombination events within the rDNA cluster. Using in vivo footprinting assays we mapped two in vivo Fob1p-binding sites, RFB1 and RFB3, located in the rDNA enhancer region and coincident with those previously reported to be in vitro binding sites. We previously provided evidences that DNA topoisomerase I is able to cleave two sites within this region. The results rep… Show more
“…Figure 5.Function of Top1cc at the rRFB and link with previously reported DSBs. ( A ) Map of one rDNA unit and close-up on the DNA nicks ( a , b , d , f and g) observed at the rRFB on the upper strand (13), a and d showing Top1 dependency (41). ( B ) Top1 contributes to the DSB signal on 2D-gels.…”
Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA.
“…Figure 5.Function of Top1cc at the rRFB and link with previously reported DSBs. ( A ) Map of one rDNA unit and close-up on the DNA nicks ( a , b , d , f and g) observed at the rRFB on the upper strand (13), a and d showing Top1 dependency (41). ( B ) Top1 contributes to the DSB signal on 2D-gels.…”
Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA.
“…1C, 1D). Instead, the many roles Fob1p plays as a general facilitator of nucleolar organization 23,42,46,56,57 may potentially explain why it is required for proper condensin targeting to the nucleolus.…”
ACKNOWLEDGEMENTSWe thank M. Nomura, T. Kobayashi, S. Roeder and S. Liebman for strains and plasmids; Micheal Lichten and A. Hinnebusch for critical reading of the manuscript; K. Bloom and A. Arnaoutov for time-lapse microscopy advice; R. Mehta and J. Panebianco for technical help. This research was supported by the Intramural Research Program of the NIH at the NICHD and at the Center for Cancer Research, NCI.
NOTESupplemental information can be found at: http://www.landesbioscience.com/journals/cc/ supplement/wangCC5-19-sup.pdf. http://www.landesbioscience.com/journals/cc/ supplement/wangCC5-16-sup_2A.mov. http://www.landesbioscience.com/journals/cc/ supplement/wangCC5-16-sup_2B.mov.
Report
Condensin Function in Mitotic Nucleolar Segregation is Regulated by rDNA Transcription
ABSTRACTChromosome condensation is established and maintained by the condensin complex. The mechanisms governing loading of condensin onto specific chromosomal sites remain unknown. To elucidate the molecular pathways that determine condensin binding to the nucleolar organizer, a key condensin binding site, we analyzed the properties of condensin-bound sites within the rDNA repeat in budding yeast and demonstrated that the bulk of mitotic condensin binding to rDNA is reduced or eliminated when Pol I transcription is elevated. Conversely, when Pol I transcription is repressed either by rapamycin treatment or by promoter shut-off, condensin binding to rDNA is increased. This novel potential role for constitutive and/or periodic repression of Pol I transcription in rDNA condensin loading is an important factor in determining the segregation proficiency of NOR-containing chromosomes.
“…Fob1 arrests replication forks at Ter sites and induces DNA bending, and the bent DNA presumably recruits topoisomerase I (Topo I) to generate DNA breaks that provoke recombination (14). However, maintenance of a certain level of topoisomerase activity prevents recombination at rDNA (15,16).…”
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 (regulation of nucleolar silencing and telophase 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).
The ribosomal DNA (rDNA) of Saccharomyces cerevisiae is present as ϳ200 tandem repeats in chromosome XII (1). In order to prevent physiologically unwarranted recombination provoked by such a large number of tandem repeats, and the consequent risk of rDNA copy number instability, yeast cells have evolved a mechanism that suppresses intrachromatid recombination and transcription by RNA polymerase II (Pol II) but not by RNA Pol I and Pol III (2). The suppression occurs by loading of the NAD-dependent histone deacetylase Sir2 onto each of the rDNA repeats at nontranscribed sequence 1 (NTS1) by interactions between Fob1 and the Net1 protein of the RENT (regulation of nucleolar silencing and telophase exit) complex (3). One of the two RENT loading sites in rDNA is located at or near the Ter sites in NTS1, and the second one is located at or near the promoter of the 35S precursor rRNA (4). The RENT complex consists of Net1 (a scaffold protein), Cdc14 (phosphatase), which is needed for escape from mitosis (5), and Sir2 (6, 7). An alternative pathway to RENT for loading of Sir2 comprises Tof2, Csm1, and Lrs4, which are components of the monopolin complex, and two inner nuclear membrane proteins called Heh1 and Nur1. Tof2 physically interacts with Fob1 (8, 9).The Fob1 (fork blockage) protein specifically binds to the Ter sites and causes polar arrest of replication forks (10, 11). It has been suggested that Fob1-Ter interaction prevents collision between the rDNA transcription and replication forks approaching from the opposite direction (12).The primary silencing activity of Sir2 is effected through repression of a bidirectional promoter called Epro, located in NTS1 (13). Transcription initiated at Epro is believed to displace cohesin from NTS1, thereby making the region recombinogenic (13).Fob1 arrests replication forks at Ter sites and induces DNA bending, and the bent DNA presumably recruits topoisomerase I (Topo I) to generate DNA breaks that provoke recombination (14). How...
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