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 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 Managing Single-Stranded DNA during Replication Stress in Fission Yeast

2021 ◽  
Author(s):  
Sarah A. Sabatinos ◽  
Susan L. Forsburg
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Single Stranded Dna ◽  
Replication Checkpoint ◽  
Primary Signal ◽  
Fork Stalling ◽  
Chromosome Integrity

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

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 Managing Single-Stranded DNA during Replication Stress in Fission Yeast

2021 ◽  
Author(s):  
Sarah A. Sabatinos ◽  
Susan L. Forsburg
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Single Stranded Dna ◽  
Replication Checkpoint ◽  
Primary Signal ◽  
Fork Stalling ◽  
Chromosome Integrity

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

scholarly journals Unscheduled DNA replication in G1 causes genome instability through head-to-tail replication fork collisions

2021 ◽  
Author(s):  
Karl-Uwe Reusswig ◽  
Julia Bittmann ◽  
Martina Peritore ◽  
Michael Wierer ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Replication Fork ◽  
Genome Instability ◽  
S Phase ◽  
Replication Initiation ◽  
Dna Replication Initiation ◽  
Chromosome Breaks ◽  
Naturally Occurring ◽  
Identical Response

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1-phase of the cell cycle. Unscheduled G1 replication initiated at canonical S-phase origins across the genome. We quantified differences in replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se did not trigger cellular checkpoints. Subsequent replication during S-phase, however, resulted in over-replication and led to chromosome breaks via head-to-tail replication fork collisions that are marked by chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA. Low-level, sporadic induction of G1 replication induced an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation by G1/S deregulation.

scholarly journals TIP60 K430 SUMOylation attenuates its interaction with DNA-PKcs in S-phase cells: Facilitating homologous recombination and emerging target for cancer therapy

2020 ◽  
Vol 6 (28) ◽  
pp. eaba7822 ◽  
Author(s):  
Shan-Shan Gao ◽  
Hua Guan ◽  
Shuang Yan ◽  
Sai Hu ◽  
Man Song ◽  
...  
Keyword(s):  
Cancer Therapy ◽  
Homologous Recombination ◽  
Cellular Response ◽  
Parp Inhibitors ◽  
S Phase ◽  
Tumor Growth Inhibition ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Pathway Activation ◽  
Pathway Choice

Nonhomologous end joining (NHEJ) and homologous recombination (HR) are major repair pathways of DNA double-strand breaks (DSBs). The pathway choice of HR and NHEJ is tightly regulated in cellular response to DNA damage. Here, we demonstrate that the interaction of TIP60 with DNA-PKcs is attenuated specifically in S phase, which facilitates HR pathway activation. SUMO2 modification of TIP60 K430 mediated by PISA4 E3 ligase blocks its interaction with DNA-PKcs, whereas TIP60 K430R mutation recovers its interaction with DNA-PKcs, which results in abnormally increased phosphorylation of DNA-PKcs S2056 in S phase and marked inhibition of HR efficiency, but barely affects NHEJ activity. TIP60 K430R mutant cancer cells are more sensitive to radiation and PARP inhibitors in cancer cell killing and tumor growth inhibition. Collectively, coordinated regulation of TIP60 and DNA-PKcs facilitates HR pathway choice in S-phase cells. TIP60 K430R mutant is a potential target of radiation and PARPi cancer therapy.

scholarly journals Activation of the DNA Damage Checkpoint in Mutants Defective in DNA Replication Initiation

2008 ◽  
Vol 19 (10) ◽  
pp. 4374-4382 ◽  
Author(s):  
Ling Yin ◽  
Alexandra Monica Locovei ◽  
Gennaro D'Urso
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
S Phase ◽  
Replication Initiation ◽  
Dna Damage Checkpoint ◽  
Origin Firing ◽  
Dna Replication Initiation ◽  
Fork Stalling ◽  
S Phase Checkpoint

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.

scholarly journals POLD replicates both strands of small kilobase-long replication bubbles initiated at a majority of human replication origins

2017 ◽  
Author(s):  
Artem V. Artemov ◽  
Maria A. Andrianova ◽  
Georgii A. Bazykin ◽  
Vladimir B. Seplyarskiy
Keyword(s):  
Dna Sequencing ◽  
Replication Fork ◽  
Additional Data ◽  
Replication Initiation ◽  
Mutational Bias ◽  
Replication Origins ◽  
Induced Mutations ◽  
Mutator Phenotype ◽  
New Model ◽  
Polymerase Epsilon

AbstractError-prone mutants of polymerase epsilon (POLE*) or polymerase delta (POLD1*) induce a mutator phenotype in human cancers. Here we show that the rate of mutations introduced by POLD1* is elevated by 50%, while the rate of POLE*-induced mutations is decreased twofold, within one kilobase from replication origins. These results support a model in which POLD1 replicates both the leading and the lagging strands within a kilobase from an origin. The magnitude of the mutational bias suggests that the probability of an individual origin to initiate replication exceeds 50%, which is much higher than previous estimates. Using additional data from nascent DNA sequencing and Okazaki fragments sequencing (OK-seq) experiments, we showed that a majority of origins are firing at each replication round, but the initiated replication fork does not propagate further than 1Kb in both directions. Analyses based on mutational data and on OK-seq data concordantly suggest that only approximately a quarter of fired origins result in a processive replication fork. Taken together, our results provide a new model of replication initiation.

scholarly journals New Faces of old Friends: Emerging new Roles of RNA-Binding Proteins in the DNA Double-Strand Break Response

2021 ◽  
Vol 8 ◽  
Author(s):  
Julie A. Klaric ◽  
Stas Wüst ◽  
Stephanie Panier
Keyword(s):  
Cellular Response ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genome Instability ◽  
Strand Break ◽  
Dna Lesions ◽  
Cell Cycle Checkpoints ◽  
Dna Double Strand Breaks ◽  
Dna Strand

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. To protect genomic stability and ensure cell homeostasis, cells mount a complex signaling-based response that not only coordinates the repair of the broken DNA strand but also activates cell cycle checkpoints and, if necessary, induces cell death. The last decade has seen a flurry of studies that have identified RNA-binding proteins (RBPs) as novel regulators of the DSB response. While many of these RBPs have well-characterized roles in gene expression, it is becoming increasingly clear that they also have non-canonical functions in the DSB response that go well beyond transcription, splicing and mRNA processing. Here, we review the current understanding of how RBPs are integrated into the cellular response to DSBs and describe how these proteins directly participate in signal transduction, amplification and repair at damaged chromatin. In addition, we discuss the implications of an RBP-mediated DSB response for genome instability and age-associated diseases such as cancer and neurodegeneration.

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