scholarly journals Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint

2019 ◽  
Author(s):  
Esther Herruzo ◽  
Beatriz Santos ◽  
Raimundo Freire ◽  
Jesús A. Carballo ◽  
Pedro A. San-Segundo
Keyword(s):  
Meiotic Recombination ◽  
Cell Cycle Progression ◽  
Nucleolar Localization ◽  
Chromosomal Distribution ◽  
Checkpoint Activation ◽  
Checkpoint Response ◽  
Aaa Atpase ◽  
In The Wild ◽  
Functional Relevance ◽  
Recombination Checkpoint

ABSTRACTThe meiotic recombination checkpoint blocks meiotic cell cycle progression in response to synapsis and/or recombination defects to prevent aberrant chromosome segregation. The evolutionarily-conserved budding yeast Pch2TRIP13 AAA+ ATPase participates in this pathway by supporting phosphorylation of the Hop1HORMAD adaptor at T318. In the wild type, Pch2 localizes to synapsed chromosomes and to the unsynapsed rDNA region (nucleolus), excluding Hop1. In contrast, in synaptonemal complex (SC)-defective zip1Δ mutants, which undergo checkpoint activation, Pch2 is detected only on the nucleolus. Alterations in some epigenetic marks that lead to Pch2 dispersion from the nucleolus suppress zip1Δ-induced checkpoint arrest. These observations have led to the notion that Pch2 nucleolar localization could be important for the meiotic recombination checkpoint. Here we investigate how Pch2 chromosomal distribution impacts on checkpoint function. We have generated and characterized several mutations that alter Pch2 localization pattern resulting in aberrant Hop1 distribution and compromised meiotic checkpoint response. Besides the AAA+ signature, we have identified a basic motif in the extended N-terminal domain critical for Pch2’s checkpoint function and localization. We have also examined the functional relevance of the described Orc1-Pch2 interaction. Both proteins colocalize in the rDNA, and Orc1 depletion during meiotic prophase prevents Pch2 targeting to the rDNA allowing unwanted Hop1 accumulation on this region. However, Pch2 association with SC components remains intact in the absence of Orc1. We finally show that checkpoint activation is not affected by the lack of Orc1 demonstrating that, in contrast to previous hypotheses, nucleolar localization of Pch2 is actually dispensable for the meiotic checkpoint.

scholarly journals Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm

2021 ◽  
Author(s):  
Esther Herruzo ◽  
Ana Lago-Maciel ◽  
Sara Baztán ◽  
Beatriz Santos ◽  
Jesús A. Carballo ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Cell Cycle Progression ◽  
Nuclear Accumulation ◽  
Axial Component ◽  
Chromosomal Distribution ◽  
Checkpoint Activation ◽  
Checkpoint Response ◽  
Aaa Atpase ◽  
Recombination Checkpoint ◽  
Cellular Compartments

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic NES or NLS sequences or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.

scholarly journals Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm

PLoS Genetics ◽  
2021 ◽  
Vol 17 (7) ◽  
pp. e1009560
Author(s):  
Esther Herruzo ◽  
Ana Lago-Maciel ◽  
Sara Baztán ◽  
Beatriz Santos ◽  
Jesús A. Carballo ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Nuclear Export ◽  
Cell Cycle Progression ◽  
Nuclear Accumulation ◽  
Axial Component ◽  
Chromosomal Distribution ◽  
Checkpoint Activation ◽  
Checkpoint Response ◽  
Aaa Atpase ◽  
Recombination Checkpoint

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic Nuclear Export (NES) or Nuclear Localization (NLS) sequences, or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.

scholarly journals Functional impact of the H2A.Z histone variant during meiosis in Saccharomyces cerevisiae

2018 ◽  
Author(s):  
Sara González-Arranz ◽  
Santiago Cavero ◽  
Macarena Morillo-Huesca ◽  
Eloisa Andújar ◽  
Mónica Pérez-Alegre ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Cell Cycle Progression ◽  
Histone Variants ◽  
Histone Variant ◽  
Dependent Manner ◽  
Additional Function ◽  
Chromatin Dynamics ◽  
Checkpoint Response ◽  
Meiotic Chromosomes ◽  
Recombination Checkpoint

AbstractAmong the collection of chromatin modifications that influence its function and structure, the substitution of canonical histones by the so-called histone variants is one of the most prominent actions. Since crucial meiotic transactions are modulated by chromatin, here we investigate the functional contribution of the H2A.Z histone variant during both unperturbed meiosis and upon challenging conditions where the meiotic recombination checkpoint is triggered in budding yeast by the absence of the synaptonemal complex component Zip1. We have found that H2A.Z localizes to meiotic chromosomes in an SWR1-dependent manner. Although meiotic recombination is not substantially altered, the htz1 mutant (lacking H2A.Z) shows inefficient meiotic progression, impaired sporulation and reduced spore viability. These phenotypes are likely accounted for by the misregulation of meiotic gene expression landscape observed in htz1. In the zip1 mutant, the absence of H2A.Z results in a tighter meiotic arrest imposed by the meiotic recombination checkpoint. We have found that Mec1-dependent Hop1-T318 phosphorylation and the ensuing Mek1 activation are not significantly altered in zip1 htz1; however, downstream checkpoint targets, such as the meiosis I-promoting factors Ndt80, Cdc5 and Clb1, are drastically down-regulated. The study of the checkpoint response in zip1 htz1 has also allowed us to reveal the existence of an additional function of the Swe1 kinase, independent of CDK inhibitory phosphorylation, which is relevant to restrain meiotic cell cycle progression. In summary, our study shows that the H2A.Z histone variant impacts various aspects of meiotic development adding further insight into the relevance of chromatin dynamics for accurate gametogenesis.

scholarly journals The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Takeshi Wakida ◽  
Masae Ikura ◽  
Kenji Kuriya ◽  
Shinji Ito ◽  
Yoshiharu Shiroiwa ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Cell Cycle Progression ◽  
Genotoxic Stress ◽  
Dna Damage Checkpoint ◽  
Proliferating Cells ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Promote Cell Proliferation

Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive. We show that human Cyclin-Dependent-Kinases (CDKs) target the RAD9 subunit of the 9-1-1 checkpoint clamp on Thr292, to modulate DNA damage checkpoint activation. Thr292 phosphorylation on RAD9 creates a binding site for Polo-Like-Kinase1 (PLK1), which phosphorylates RAD9 on Thr313. These CDK-PLK1-dependent phosphorylations of RAD9 suppress checkpoint activation, therefore maintaining high DNA synthesis rates during DNA replication stress. Our results suggest that CDK locally initiates a PLK1-dependent signaling response that antagonizes the ability of the DNA damage checkpoint to detect DNA damage. These findings provide a mechanism for the suppression of DNA damage checkpoint signaling, to promote cell proliferation under genotoxic stress conditions.

scholarly journals The budding yeast polo-like kinase Cdc5 regulates the Ndt80 branch of the meiotic recombination checkpoint pathway

2011 ◽  
Vol 22 (18) ◽  
pp. 3478-3490 ◽  
Author(s):  
Isabel Acosta ◽  
David Ontoso ◽  
Pedro A. San-Segundo
Keyword(s):  
Meiotic Recombination ◽  
Meiotic Prophase ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Checkpoint Control ◽  
Checkpoint Adaptation ◽  
Expression Of Genes ◽  
Checkpoint Pathway ◽  
Meiotic Prophase Arrest ◽  
Recombination Checkpoint

Defects in chromosome synapsis and/or meiotic recombination activate a surveillance mechanism that blocks meiotic cell cycle progression to prevent anomalous chromosome segregation and formation of aberrant gametes. In the budding yeast zip1 mutant, which lacks a synaptonemal complex component, the meiotic recombination checkpoint is triggered, resulting in extremely delayed meiotic progression. We report that overproduction of the polo-like kinase Cdc5 partially alleviates the meiotic prophase arrest of zip1, leading to the formation of inviable meiotic products. Unlike vegetative cells, we demonstrate that Cdc5 overproduction does not stimulate meiotic checkpoint adaptation because the Mek1 kinase remains activated in zip1 2μ-CDC5 cells. Inappropriate meiotic divisions in zip1 promoted by high levels of active Cdc5 do not result from altered function of the cyclin-dependent kinase (CDK) inhibitor Swe1. In contrast, CDC5 overexpression leads to premature induction of the Ndt80 transcription factor, which drives the expression of genes required for meiotic divisions, including CLB1. We also show that depletion of Cdc5 during meiotic prophase prevents the production of Ndt80 and that CDK activity contributes to the induction of Ndt80 in zip1 cells overexpressing CDC5. Our results reveal a role for Cdc5 in meiotic checkpoint control by regulating Ndt80 function.

scholarly journals Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

2016 ◽  
Vol 6 (12) ◽  
pp. 3869-3881
Author(s):  
Nicole A Najor ◽  
Layne Weatherford ◽  
George S Brush
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Protein Kinase ◽  
Meiotic Recombination ◽  
Mitotic Cell ◽  
Mitotic Cell Cycle ◽  
Cyclin Dependent Kinase ◽  
Checkpoint Response ◽  
Recombination Checkpoint ◽  
Dna Rereplication

Abstract In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs) are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control.

scholarly journals The N-Terminal Region of the Polo Kinase Cdc5 is Required for Downregulation of the Meiotic Recombination Checkpoint

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2561
Author(s):  
Sara González-Arranz ◽  
Isabel Acosta ◽  
Jesús A. Carballo ◽  
Beatriz Santos ◽  
Pedro A. San-Segundo
Keyword(s):  
Cell Cycle ◽  
Meiotic Recombination ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Terminal Region ◽  
Cycle Progression ◽  
Protein Levels ◽  
Additional Layer ◽  
C Complex ◽  
Recombination Checkpoint

During meiosis, the budding yeast polo-like kinase Cdc5 is a crucial driver of the prophase I to meiosis I (G2/M) transition. The meiotic recombination checkpoint restrains cell cycle progression in response to defective recombination to ensure proper distribution of intact chromosomes to the gametes. This checkpoint detects unrepaired DSBs and initiates a signaling cascade that ultimately inhibits Ndt80, a transcription factor required for CDC5 gene expression. Previous work revealed that overexpression of CDC5 partially alleviates the checkpoint-imposed meiotic delay in the synaptonemal complex-defective zip1Δ mutant. Here, we show that overproduction of a Cdc5 version (Cdc5-ΔN70), lacking the N-terminal region required for targeted degradation of the protein by the APC/C complex, fails to relieve the zip1Δ-induced meiotic delay, despite being more stable and reaching increased protein levels. However, precise mutation of the consensus motifs for APC/C recognition (D-boxes and KEN) has no effect on Cdc5 stability or function during meiosis. Compared to the zip1Δ single mutant, the zip1Δ cdc5-ΔN70 double mutant exhibits an exacerbated meiotic block and reduced levels of Ndt80 consistent with persistent checkpoint activity. Finally, using a CDC5-inducible system, we demonstrate that the N-terminal region of Cdc5 is essential for its checkpoint erasing function. Thus, our results unveil an additional layer of regulation of polo-like kinase function in meiotic cell cycle control.

scholarly journals TRIP13PCH-2 promotes Mad2 localization to unattached kinetochores in the spindle checkpoint response

2015 ◽  
Vol 211 (3) ◽  
pp. 503-516 ◽  
Author(s):  
Christian R. Nelson ◽  
Tom Hwang ◽  
Pin-Hsi Chen ◽  
Needhi Bhalla
Keyword(s):  
Cell Division ◽  
Caenorhabditis Elegans ◽  
Spindle Checkpoint ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Aaa Atpase ◽  
C Elegans ◽  
Anaphase Onset ◽  
Data Support

The spindle checkpoint acts during cell division to prevent aneuploidy, a hallmark of cancer. During checkpoint activation, Mad1 recruits Mad2 to kinetochores to generate a signal that delays anaphase onset. Yet, whether additional factors contribute to Mad2’s kinetochore localization remains unclear. Here, we report that the conserved AAA+ ATPase TRIP13PCH-2 localizes to unattached kinetochores and is required for spindle checkpoint activation in Caenorhabditis elegans. pch-2 mutants effectively localized Mad1 to unattached kinetochores, but Mad2 recruitment was significantly reduced. Furthermore, we show that the C. elegans orthologue of the Mad2 inhibitor p31(comet)CMT-1 interacts with TRIP13PCH-2 and is required for its localization to unattached kinetochores. These factors also genetically interact, as loss of p31(comet)CMT-1 partially suppressed the requirement for TRIP13PCH-2 in Mad2 localization and spindle checkpoint signaling. These data support a model in which the ability of TRIP13PCH-2 to disassemble a p31(comet)/Mad2 complex, which has been well characterized in the context of checkpoint silencing, is also critical for spindle checkpoint activation.

scholarly journals Genetic Control of Recombination Partner Preference in Yeast Meiosis: Isolation and Characterization of Mutants Elevated for Meiotic Unequal Sister-Chromatid Recombination

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 621-641 ◽  
Author(s):  
Dawn A Thompson ◽  
Franklin W Stahl
Keyword(s):  
Sister Chromatid ◽  
Meiotic Recombination ◽  
Class Ii ◽  
Class Iii ◽  
Viability Analysis ◽  
Isolation And Characterization ◽  
Recombination Checkpoint ◽  
Recombination Pathway ◽  
Checkpoint Genes ◽  
Sister Chromatid Recombination

AbstractMeiotic exchange occurs preferentially between homologous chromatids, in contrast to mitotic recombination, which occurs primarily between sister chromatids. To identify functions that direct meiotic recombination events to homologues, we screened for mutants exhibiting an increase in meiotic unequal sister-chromatid recombination (SCR). The msc (meiotic sister-chromatid recombination) mutants were quantified in spo13 meiosis with respect to meiotic unequal SCR frequency, disome segregation pattern, sporulation frequency, and spore viability. Analysis of the msc mutants according to these criteria defines three classes. Mutants with a class I phenotype identified new alleles of the meiosis-specific genes RED1 and MEK1, the DNA damage checkpoint genes RAD24 and MEC3, and a previously unknown gene, MSC6. The genes RED1, MEK1, RAD24, RAD17, and MEC1 are required for meiotic prophase arrest induced by a dmc1 mutation, which defines a meiotic recombination checkpoint. Meiotic unequal SCR was also elevated in a rad17 mutant. Our observation that meiotic unequal SCR is elevated in meiotic recombination checkpoint mutants suggests that, in addition to their proposed monitoring function, these checkpoint genes function to direct meiotic recombination events to homologues. The mutants in class II, including a dmc1 mutant, confer a dominant meiotic lethal phenotype in diploid SPO13 meiosis in our strain background, and they identify alleles of UBR1, INP52, BUD3, PET122, ELA1, and MSC1-MSC3. These results suggest that DMC1 functions to bias the repair of meiosis-specific double-strand breaks to homologues. We hypothesize that the genes identified by the class II mutants function in or are regulators of the DMC1-promoted interhomologue recombination pathway. Class III mutants may be elevated for rates of both SCR and homologue exchange.

scholarly journals A Role for MMS4 in the Processing of Recombination Intermediates During Meiosis in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1511-1525 ◽  
Author(s):  
Teresa de los Santos ◽  
Josef Loidl ◽  
Brittany Larkin ◽  
Nancy M Hollingsworth
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Biochemical Data ◽  
Dna Metabolism ◽  
Spore Viability ◽  
Vegetative Cells ◽  
Recombination Checkpoint

Abstract The MMS4 gene of Saccharomyces cerevisiae was originally identified due to its sensitivity to MMS in vegetative cells. Subsequent studies have confirmed a role for MMS4 in DNA metabolism of vegetative cells. In addition, mms4 diploids were observed to sporulate poorly. This work demonstrates that the mms4 sporulation defect is due to triggering of the meiotic recombination checkpoint. Genetic, physical, and cytological analyses suggest that MMS4 functions after the single end invasion step of meiotic recombination. In spo13 diploids, red1, but not mek1, is epistatic to mms4 for sporulation and spore viability, suggesting that MMS4 may be required only when homologs are capable of undergoing synapsis. MMS4 and MUS81 are in the same epistasis group for spore viability, consistent with biochemical data that show that the two proteins function in a complex. In contrast, MMS4 functions independently of MSH5 in the production of viable spores. We propose that MMS4 is required for the processing of specific recombination intermediates during meiosis.

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