scholarly journals Condensin Loaded onto the Replication Fork Barrier Site in the rRNA Gene Repeats during S Phase in a FOB1-Dependent Fashion To Prevent Contraction of a Long Repetitive Array in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (6) ◽  
pp. 2226-2236 ◽  
Author(s):  
Katsuki Johzuka ◽  
Masahiro Terasawa ◽  
Hideyuki Ogawa ◽  
Tomoko Ogawa ◽  
Takashi Horiuchi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Copy Number ◽  
Replication Fork ◽  
Tandem Repeats ◽  
S Phase ◽  
Rrna Gene ◽  
Nontranscribed Spacer ◽  
Condensin Complex ◽  
Unknown System ◽  
Average Copy

ABSTRACT 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 Δfob1 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 Δfob1 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.

scholarly journals The S-Phase Cyclin Clb5 Promotes rRNA Gene (rDNA) Stability by Maintaining Replication Initiation Efficiency in rDNA

2021 ◽  
Vol 41 (5) ◽  
Author(s):  
Mayuko Goto ◽  
Mariko Sasaki ◽  
Takehiko Kobayashi
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Tandem Repeats ◽  
Genome Instability ◽  
S Phase ◽  
Replication Initiation ◽  
Rrna Gene ◽  
Replication Origins ◽  
Dna Double Strand Breaks ◽  
Replication Fork Arrest

ABSTRACT Regulation of replication origins is important for complete duplication of the genome, but the effect of origin activation on the cellular response to replication stress is poorly understood. The budding yeast rRNA gene (rDNA) forms tandem repeats and undergoes replication fork arrest at the replication fork barrier (RFB), inducing DNA double-strand breaks (DSBs) and genome instability accompanied by copy number alterations. Here, we demonstrate that the S-phase cyclin Clb5 promotes rDNA stability. Absence of Clb5 led to reduced efficiency of replication initiation in rDNA but had little effect on the number of replication forks arrested at the RFB, suggesting that arrival of the converging fork is delayed and forks are more stably arrested at the RFB. Deletion of CLB5 affected neither DSB formation nor its repair at the RFB but led to homologous recombination-dependent rDNA instability. Therefore, arrested forks at the RFB may be subject to DSB-independent, recombination-dependent rDNA instability. The rDNA instability in clb5Δ was not completely suppressed by the absence of Fob1, which is responsible for fork arrest at the RFB. Thus, Clb5 establishes the proper interval for active replication origins and shortens the travel distance for DNA polymerases, which may reduce Fob1-independent DNA damage.

scholarly journals Saccharomyces cerevisiae Rrm3p DNA Helicase Promotes Genome Integrity by Preventing Replication Fork Stalling: Viability of rrm3 Cells Requires the Intra-S-Phase Checkpoint and Fork Restart Activities

2004 ◽  
Vol 24 (8) ◽  
pp. 3198-3212 ◽  
Author(s):  
Jorge Z. Torres ◽  
Sandra L. Schnakenberg ◽  
Virginia A. Zakian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Replication Fork ◽  
Opportunity To Learn ◽  
Normal Growth ◽  
Dna Helicase ◽  
S Phase ◽  
Exogenous Dna ◽  
Fork Stalling ◽  
S Phase Checkpoint

ABSTRACT Rrm3p is a 5′-to-3′ DNA helicase that helps replication forks traverse protein-DNA complexes. Its absence leads to increased fork stalling and breakage at over 1,000 specific sites located throughout the Saccharomyces cerevisiae genome. To understand the mechanisms that respond to and repair rrm3-dependent lesions, we carried out a candidate gene deletion analysis to identify genes whose mutation conferred slow growth or lethality on rrm3 cells. Based on synthetic phenotypes, the intra-S-phase checkpoint, the SRS2 inhibitor of recombination, the SGS1/TOP3 replication fork restart pathway, and the MRE11/RAD50/XRS2 (MRX) complex were critical for viability of rrm3 cells. DNA damage checkpoint and homologous recombination genes were important for normal growth of rrm3 cells. However, the MUS81/MMS4 replication fork restart pathway did not affect growth of rrm3 cells. These data suggest a model in which the stalled and broken forks generated in rrm3 cells activate a checkpoint response that provides time for fork repair and restart. Stalled forks are converted by a Rad51p-mediated process to intermediates that are resolved by Sgs1p/Top3p. The rrm3 system provides a unique opportunity to learn the fate of forks whose progress is impaired by natural impediments rather than by exogenous DNA damage.

scholarly journals Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Christophe de La Roche Saint-André ◽  
Vincent Géli
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
Genetic Material ◽  
S Phase ◽  
Replication Forks ◽  
H3k4 Methylation ◽  
Origin Firing ◽  
Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

scholarly journals Replication fork rate and origin activation during the S phase of Saccharomyces cerevisiae.

1980 ◽  
Vol 85 (1) ◽  
pp. 108-115 ◽  
Author(s):  
C J Rivin ◽  
W L Fangman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cycle Length ◽  
Replication Fork ◽  
Nitrogen Sources ◽  
S Phase ◽  
Phase Duration ◽  
Yeast Saccharomyces Cerevisiae ◽  
Origin Activation ◽  
Cell Cycle Length

When the growth rate of the yeast Saccharomyces cerevisiae is limited with various nitrogen sources, the duration of the S phase is proportional to cell cycle length over a fourfold range of growth rates (C.J. Rivin and W. L. Fangman, 1980, J. Cell Biol. 85:96-107). Molecular parameters of the S phases of these cells were examined by DNA fiber autoradiography. Changes in replication fork rate account completely for the changes in S-phase duration. No changes in origin-to-origin distances were detected. In addition, it was found that while most adjacent replication origins are activated within a few minutes of each other, new activations occur throughout the S phase.

scholarly journals A gene with specific and global effects on recombination of sequences from tandemly repeated genes in Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 711-718 ◽  
Author(s):  
R L Keil ◽  
A D McWilliams
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Copy Number ◽  
Tandem Repeats ◽  
Chromosomal Location ◽  
Specific Factor ◽  
Yeast Saccharomyces Cerevisiae ◽  
Naturally Occurring ◽  
Type Information ◽  
Repeated Genes

Abstract The preservation of sequence homogeneity and copy number of tandemly repeated genes may require specific mechanisms or regulation of recombination. We have identified mutations that specifically affect recombination among natural repetitions in the yeast Saccharomyces cerevisiae. The rrm3 mutation stimulates mitotic recombination in the naturally occurring tandem repeats of the rDNA and copper chelatin (CUP1) genes. This mutation does not affect recombination of several other types of repeated genes tested including Ty elements, mating type information and duplications created by transformation. In addition to stimulating exchange among the multiple CUP1 repeats at their natural chromosomal location, rrm3 also increases recombination of a duplication of CUP1 units present at his4. This suggests that the RRM3 gene may encode a sequence-specific factor that contributes to a global suppression of mitotic exchange in sequences that can be maintained as tandem arrays.

scholarly journals Contrasting Roles of Checkpoint Proteins as Recombination Modulators at Fob1-Ter Complexes with or without Fork Arrest

2009 ◽  
Vol 8 (4) ◽  
pp. 487-495 ◽  
Author(s):  
Bidyut K. Mohanty ◽  
Narendra K. Bairwa ◽  
Deepak Bastia
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Dna ◽  
Replication Fork ◽  
Intergenic Spacer ◽  
Sensor Kinase ◽  
Adapter Proteins ◽  
Widespread Occurrence ◽  
Checkpoint Proteins ◽  
Nontranscribed Spacer ◽  
Replication Terminus

ABSTRACT The replication terminator protein Fob1 of Saccharomyces cerevisiae specifically interacts with two tandem Ter sites (replication fork barriers) located in the nontranscribed spacer of ribosomal DNA (rDNA) to cause polar fork arrest. The Fob1-Ter complex is multifunctional and controls other DNA transactions such as recombination by multiple mechanisms. Here, we report on the regulatory roles of the checkpoint proteins in the initiation and progression of recombination at Fob1-Ter complexes. The checkpoint adapter proteins Tof1 and Csm3 either positively or negatively controlled recombination depending on whether it was provoked by polar fork arrest or by transcription, respectively. The absolute requirements for these proteins for inducing recombination at an active replication terminus most likely masked their negative modulatory role at a later step of the process. Other checkpoint proteins of the checkpoint adapter/mediator class such as Mrc1 and Rad9, which channel signals from the sensor to the effector kinase, tended to suppress recombination at Fob1-Ter complexes regardless of how it was initiated. We have also discovered that the checkpoint sensor kinase Mec1 and the effector Rad53 were positive modulators of recombination initiated by transcription but had little effect on recombination at Ter. The work also showed that the two pathways were Rad52 dependent but Rad51 independent. Since Ter sites occur in the intergenic spacer of rDNA from yeast to humans, the mechanism is likely to be of widespread occurrence.

scholarly journals The S-phase Cyclin Clb5 Promotes rDNA Stability by Maintaining Replication Initiation Efficiency in rDNA

2020 ◽  
Author(s):  
Mayuko Goto ◽  
Mariko Sasaki ◽  
Takehiko Kobayashi
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Tandem Repeats ◽  
Genome Instability ◽  
S Phase ◽  
Replication Initiation ◽  
Replication Origins ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Replication Fork Arrest

ABSTRACTRegulation of replication origins is important for complete duplication of the genome, but the effect of origin activation on the cellular response to replication stress is poorly understood. The budding yeast ribosomal RNA gene (rDNA) forms tandem repeats and undergoes replication fork arrest at the replication fork barrier (RFB), inducing DNA double-strand breaks (DSBs) and genome instability accompanied by copy number alterations. Here we demonstrate that the S-phase cyclin Clb5 promotes rDNA stability. Absence of Clb5 led to reduced efficiency of replication initiation in rDNA but had little effect on the amount of replication forks arrested at the RFB, suggesting that arrival of the converging fork is delayed and forks are more stably arrested at the RFB. Deletion of CLB5 affected neither DSB formation nor its repair at the RFB, but led to an accumulation of recombination intermediates. Therefore, arrested forks at the RFB may be subject to DSB-independent, recombination-dependent rDNA instability. The rDNA instability in clb5Δ was not completely suppressed by the absence of Fob1, which is responsible for fork arrest at the RFB. Thus, Clb5 establishes the proper interval for active replication origins and shortens the travel distance for DNA polymerases, which may reduce Fob1-independent DNA damage.

scholarly journals Fast and accurate average genome size and 16S rRNA gene average copy number computation in metagenomic data

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Emiliano Pereira-Flores ◽  
Frank Oliver Glöckner ◽  
Antonio Fernandez-Guerra
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Genome Size ◽  
Copy Number ◽  
Metagenomic Data ◽  
Rrna Gene ◽  
Average Copy Number ◽  
Average Copy

scholarly journals Ribosomal DNA Replication Fork Barrier and HOT1Recombination Hot Spot: Shared Sequences but Independent Activities

2000 ◽  
Vol 20 (13) ◽  
pp. 4948-4957 ◽  
Author(s):  
Teresa R. Ward ◽  
Margaret L. Hoang ◽  
Reeta Prusty ◽  
Corine K. Lau ◽  
Ralph L. Keil ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Simple Model ◽  
Ribosomal Rna ◽  
Ribosomal Dna ◽  
Replication Fork ◽  
Hot Spot ◽  
Replication Forks ◽  
Nontranscribed Spacer ◽  
A Site ◽  
Replication Fork Arrest

ABSTRACT In the ribosomal DNA of Saccharomyces cerevisiae, sequences in the nontranscribed spacer 3′ of the 35S ribosomal RNA gene are important to the polar arrest of replication forks at a site called the replication fork barrier (RFB) and also to thecis-acting, mitotic hyperrecombination site calledHOT1. We have found that the RFB and HOT1activity share some but not all of their essential sequences. Many of the mutations that reduce HOT1 recombination also decrease or eliminate fork arrest at one of two closely spaced RFB sites, RFB1 and RFB2. A simple model for the juxtaposition of RFB andHOT1 sequences is that the breakage of strands in replication forks arrested at RFB stimulates recombination. Contrary to this model, we show here that HOT1-stimulated recombination does not require the arrest of forks at the RFB. Therefore, whileHOT1 activity is independent of replication fork arrest,HOT1 and RFB require some common sequences, suggesting the existence of a common trans-acting factor(s).

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