DNA damage checkpoint and recombinational repair differentially affect the replication stress tolerance of smc6 mutants

DNA damage checkpoint and recombinational repair are both important for cell survival of replication stress. Because these two processes influence each other, isolation of their respective contributions is challenging. Research in budding yeast shows that removal of the DNA helicase Mph1 improves survival of cells with defective Smc5/6 complex under replication stress. mph1∆ is known to reduce the levels of recombination intermediates in smc6 mutants. Here, we show that mph1∆ also hyperactivates the Mec1 checkpoint. We dissect the effects of recombination regulation and checkpoint hyperactivation by altering the checkpoint circuitry to enhance checkpoint signaling without reducing recombination intermediate levels. We show that these approaches, similar to mph1∆, lead to better survival of smc6 cells upon transient replication stress, likely by ameliorating replication and chromosomal segregation defects. Unlike mph1∆, however, they do not suppress smc6 sensitivity to chronic stress. Conversely, reducing the checkpoint response does not impair survival of smc6 mph1∆ mutants under chronic stress. These results suggest a two-phase model in which smc6 mutant survival upon transient replication stress can be improved by enhancing Mec1 checkpoint signaling, whereas smc6 sensitivity to chronic stress can be overcome by reducing recombination intermediates.

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The Srs2 helicase dampens DNA damage checkpoint by recycling RPA from chromatin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020185118 ◽

2021 ◽

Vol 118 (8) ◽

pp. e2020185118

Author(s):

Nalini Dhingra ◽

Sahiti Kuppa ◽

Lei Wei ◽

Nilisha Pokhrel ◽

Silva Baburyan ◽

...

Keyword(s):

Dna Damage ◽

Genotoxic Stress ◽

Dna Helicase ◽

Dna Damage Checkpoint ◽

Recombinational Repair ◽

Checkpoint Signaling ◽

Cellular Survival ◽

Moderate Reduction ◽

Srs2 Helicase ◽

Harmful Effects

The DNA damage checkpoint induces many cellular changes to cope with genotoxic stress. However, persistent checkpoint signaling can be detrimental to growth partly due to blockage of cell cycle resumption. Checkpoint dampening is essential to counter such harmful effects, but its mechanisms remain to be understood. Here, we show that the DNA helicase Srs2 removes a key checkpoint sensor complex, RPA, from chromatin to down-regulate checkpoint signaling in budding yeast. The Srs2 and RPA antagonism is supported by their numerous suppressive genetic interactions. Importantly, moderate reduction of RPA binding to single-strand DNA (ssDNA) rescues hypercheckpoint signaling caused by the loss of Srs2 or its helicase activity. This rescue correlates with a reduction in the accumulated RPA and the associated checkpoint kinase on chromatin insrs2mutants. Moreover, our data suggest that Srs2 regulation of RPA is separable from its roles in recombinational repair and critically contributes to genotoxin resistance. We conclude that dampening checkpoint by Srs2-mediated RPA recycling from chromatin aids cellular survival of genotoxic stress and has potential implications in other types of DNA transactions.

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Undamaged DNA Transmits and Enhances DNA Damage Checkpoint Signals in Early Embryos

Molecular and Cellular Biology ◽

10.1128/mcb.00195-07 ◽

2007 ◽

Vol 27 (19) ◽

pp. 6852-6862 ◽

Cited By ~ 15

Author(s):

Aimin Peng ◽

Andrea L. Lewellyn ◽

James L. Maller

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Nuclear Dna ◽

Cell Cycle Checkpoint ◽

Dna Damage Checkpoint ◽

Checkpoint Signaling ◽

Checkpoint Response ◽

Egg Extracts ◽

Cycle Checkpoint ◽

Damaged Dna

ABSTRACT In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1/Chk2 phosphorylation and release from nuclear DNA. These responses on physically separate threshold DNA require γ-H2AX and are triggered by an ATM-dependent soluble signal initiated by damaged DNA. The signal persists in egg extracts even after damaged DNA is removed from the system, indicating that the absence of damaged DNA is not sufficient to end the checkpoint response. The results identify a novel mechanism by which undamaged DNA enhances checkpoint signaling and provide an example of how the transition to cell cycle checkpoint activation during development is accomplished by maternally programmed increases in the DNA-to-cytoplasm ratio.

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Esc2 and Sgs1 Act in Functionally Distinct Branches of the Homologous Recombination Repair Pathway inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e08-08-0877 ◽

2009 ◽

Vol 20 (6) ◽

pp. 1683-1694 ◽

Cited By ~ 41

Author(s):

Hocine W. Mankouri ◽

Hien-Ping Ngo ◽

Ian D. Hickson

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Structural Similarity ◽

S Phase ◽

Homologous Recombination Repair ◽

Dna Damage Checkpoint ◽

Checkpoint Signaling ◽

Checkpoint Response ◽

Recombination Repair ◽

Anemia Group

Esc2 is a member of the RENi family of SUMO-like domain proteins and is implicated in gene silencing in Saccharomyces cerevisiae. Here, we identify a dual role for Esc2 during S-phase in mediating both intra-S-phase DNA damage checkpoint signaling and preventing the accumulation of Rad51-dependent homologous recombination repair (HRR) intermediates. These roles are qualitatively similar to those of Sgs1, the yeast ortholog of the human Bloom's syndrome protein, BLM. However, whereas mutation of either ESC2 or SGS1 leads to the accumulation of unprocessed HRR intermediates in the presence of MMS, the accumulation of these structures in esc2 (but not sgs1) mutants is entirely dependent on Mph1, a protein that shows structural similarity to the Fanconi anemia group M protein (FANCM). In the absence of both Esc2 and Sgs1, the intra-S-phase DNA damage checkpoint response is compromised after exposure to MMS, and sgs1esc2 cells attempt to undergo mitosis with unprocessed HRR intermediates. We propose a model whereby Esc2 acts in an Mph1-dependent process, separately from Sgs1, to influence the repair/tolerance of MMS-induced lesions during S-phase.

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

eLife ◽

10.7554/elife.29953 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 7

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.

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The DNA damage checkpoint response to replication stress: A Game of Forks

Frontiers in Genetics ◽

10.3389/fgene.2013.00026 ◽

2013 ◽

Vol 4 ◽

Cited By ~ 43

Author(s):

Rachel Jossen ◽

Rodrigo Bermejo

Keyword(s):

Dna Damage ◽

Replication Stress ◽

Dna Damage Checkpoint ◽

Checkpoint Response

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Regulation of Septum Formation in Aspergillus nidulans by a DNA Damage Checkpoint Pathway

Genetics ◽

10.1093/genetics/148.3.1055 ◽

1998 ◽

Vol 148 (3) ◽

pp. 1055-1067

Author(s):

Steven D Harris ◽

Peter R Kraus

Keyword(s):

Dna Damage ◽

Aspergillus Nidulans ◽

Cellular Response ◽

Dna Damage Checkpoint ◽

Temperature Sensitive ◽

Dna Metabolism ◽

Chromosomal Dna ◽

Checkpoint Response ◽

Septum Formation ◽

Checkpoint Pathway

Abstract In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before the formation of the first septum. Previous characterization of temperature-sensitive sepB and sepJ mutations showed that although they block septation, they also cause moderate defects in chromosomal DNA metabolism. Results presented here demonstrate that a variety of other perturbations of chromosomal DNA metabolism also delay septum formation, suggesting that this is a general cellular response to the presence of sublethal DNA damage. Genetic evidence is provided that suggests that high levels of cyclin-dependent kinase (cdk) activity are required for septation in A. nidulans. Consistent with this notion, the inhibition of septum formation triggered by defects in chromosomal DNA metabolism depends upon Tyr-15 phosphorylation of the mitotic cdk p34nimX. Moreover, this response also requires elements of the DNA damage checkpoint pathway. A model is proposed that suggests that the DNA damage checkpoint response represents one of multiple sensory inputs that modulates p34nimX activity to control the timing of septum formation.

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