scholarly journals Sir2p suppresses recombination of replication forks stalled at the replication fork barrier of ribosomal DNA in Saccharomyces cerevisiae

2003 ◽  
Vol 31 (3) ◽  
pp. 893-898 ◽  
Author(s):  
A. Benguria
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Dna ◽  
Replication Fork ◽  
Replication Forks

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).

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 Organization of replication of ribosomal DNA in Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (11) ◽  
pp. 4927-4935 ◽  
Author(s):  
M H Linskens ◽  
J A Huberman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Dna ◽  
Repeat Unit ◽  
Replication Forks ◽  
Sequence Element ◽  
Autonomously Replicating Sequence ◽  
Nontranscribed Spacer ◽  
Precursor Rna ◽  
Mapping Techniques ◽  
Region Ii

Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the nontranscribed spacer region, (ii) only a fraction of the potential origins are utilized in a single S phase, and (iii) the replication forks moving counter to the direction of transcription of the 37S precursor RNA stop at or near the termination site of transcription. Consequently, most ribosomal DNA is replicated unidirectionally by forks moving in the direction of transcription and most replicons are larger than the repeat unit. The significance of this finding for the replication of abundantly transcribed genes is discussed.

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 Type I elements mediate replication fork pausing at conserved upstream sites in the Tetrahymena thermophila ribosomal DNA minichromosome.

1997 ◽  
Vol 17 (8) ◽  
pp. 4517-4525 ◽  
Author(s):  
D M MacAlpine ◽  
Z Zhang ◽  
G M Kapler
Keyword(s):  
Ribosomal Dna ◽  
Replication Fork ◽  
Tetrahymena Thermophila ◽  
Replication Initiation ◽  
Rrna Genes ◽  
Type I ◽  
Replication Forks ◽  
Coding Region ◽  
Two Dimensional Gel Electrophoresis ◽  
Directional Bias

Two-dimensional gel electrophoresis was used to study replication of the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome. During vegetative growth, the rDNA is replicated exclusively from origins in the 5' nontranscribed spacer (NTS). Whereas replication fork movement through the rest of the chromosome appears to be continuous, movement through the 5' NTS is not. Replication forks arrest transiently at three prominent replication fork pausing sites (RFPs) located in or immediately adjacent to nucleosome-free regions of the 5' NTS. Pausing at these sites is dramatically diminished during replication in Escherichia coli, suggesting that chromatin organization or Tetrahymena-specific proteins may be required. A conserved tripartite sequence was identified at each pausing site. Mutations in type I elements diminish pausing at proximal RFPs. Hence, type I elements, previously shown to control replication initiation, also regulate elongation of existing replication forks. Studies with rDNA transformants revealed a strong directional bias for fork pausing. Strong pausing only occurred in forks moving toward the rRNA-coding region. We propose that fork pausing in the 5' NTS evolved to synchronize replication and transcription of the downstream rRNA genes.

Organization of replication of ribosomal DNA in Saccharomyces cerevisiae

1988 ◽  
Vol 8 (11) ◽  
pp. 4927-4935
Author(s):  
M H Linskens ◽  
J A Huberman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Dna ◽  
Repeat Unit ◽  
Replication Forks ◽  
Sequence Element ◽  
Autonomously Replicating Sequence ◽  
Nontranscribed Spacer ◽  
Precursor Rna ◽  
Mapping Techniques ◽  
Region Ii

Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the nontranscribed spacer region, (ii) only a fraction of the potential origins are utilized in a single S phase, and (iii) the replication forks moving counter to the direction of transcription of the 37S precursor RNA stop at or near the termination site of transcription. Consequently, most ribosomal DNA is replicated unidirectionally by forks moving in the direction of transcription and most replicons are larger than the repeat unit. The significance of this finding for the replication of abundantly transcribed genes is discussed.

scholarly journals Mechanism of Regulation of Intrachromatid Recombination and Long-Range Chromosome Interactions in Saccharomyces cerevisiae

2016 ◽  
Vol 36 (10) ◽  
pp. 1451-1463 ◽  
Author(s):  
Shamsu Zaman ◽  
Malay Choudhury ◽  
James C. Jiang ◽  
Pankaj Srivastava ◽  
Bidyut K. Mohanty ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Histone Deacetylase ◽  
Long Range ◽  
Ribosomal Dna ◽  
Replication Fork ◽  
Regulatory Mechanisms ◽  
Content Type ◽  
Nontranscribed Spacer ◽  
Rdna Array

The NAD-dependent histone deacetylase Sir2 controls ribosomal DNA (rDNA) silencing by inhibiting recombination and RNA polymerase II-catalyzed transcription in the rDNA ofSaccharomyces cerevisiae. Sir2 is recruited to nontranscribed spacer 1 (NTS1) of the rDNA array by interaction between the RENT (regulation ofnucleolarsilencing andtelophase 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 intransare downregulated by Sir2. These regulatory mechanisms control intrachromatid recombination and the replicative life span (RLS).

scholarly journals Actualización en caracterización molecular de levaduras de interés industrial

2016 ◽  
Vol 18 (2) ◽  
pp. 129 ◽  
Author(s):  
Jorge Alberto Vásquez C ◽  
Mauricio Ramirez Castrillón ◽  
Zulma Isabel Monsalve F
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Dna

Las levaduras, además de ser un modelo de la investigación biomédica, tienen diversas aplicaciones en la industria alimentaria, en agricultura y la producción de etanol combustible. Dado que la calidad y la cantidad del producto dependen de la dinámica y la frecuencia de los microorganismos presentes en la fermentación, el uso de herramientas de caracterización molecular se ha incrementado y popularizado en las industrias que emplean levaduras. Estas técnicas se basan en la amplificación o análisis por enzimas de restricción de una porción del ADN genómico de levadura y se clasifican de acuerdo a su capacidad de resolución taxonómica para discriminar a nivel inter o intra-específica. La primera parte de la revisión incluye pruebas interespecíficas tales como, análisis de restricción o RFLP para las regiones ITS2, ITS1-5.8, D1 / D2 de los genes 26S ribosomal DNA. La segunda parte incluye, pruebas de uso común para caracterización nivel de cepa, tales como: la amplificación aleatoria del ADN polimórfico (RAPD), análisis cromosómico por electroforesis en gel de campo pulsado (PFGE), análisis de restricción del ADN mitocondrial (ADNmt- RFLP) análisis por mini / micro satélites y la huella genética de ADN por amplificación de regiones interdelta de los transposones Ty. Esta revisión describe y discute los detalles técnicos de los métodos más utilizados para la caracterización molecular de las levaduras y algunos ejemplos de sus aplicaciones en el contexto industrial.Palabras clave: Levaduras, caracterización molecular, identificación intraespecífica especies, Saccharomyces cerevisiae.

scholarly journals Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

2014 ◽  
Vol 25 (12) ◽  
pp. 1916-1924 ◽  
Author(s):  
David Öling ◽  
Rehan Masoom ◽  
Kristian Kvint
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Life Span ◽  
Rna Polymerase Ii ◽  
Ribosomal Dna ◽  
Genetic Evidence ◽  
Wild Type ◽  
Replicative Life Span ◽  
Unequal Recombination

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

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