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

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.

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

2013 ◽  
Vol 24 (15) ◽  
pp. 2431-2441 ◽  
Author(s):  
Yu-Hung Chen ◽  
Barnabas Szakal ◽  
Federica Castellucci ◽  
Dana Branzei ◽  
Xiaolan Zhao
Keyword(s):  
Dna Damage ◽  
Chronic Stress ◽  
Replication Stress ◽  
Dna Helicase ◽  
Dna Damage Checkpoint ◽  
Recombinational Repair ◽  
Two Phase ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Chromosomal Segregation

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.

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 DNA Repair Protein Rad55 Is a Terminal Substrate of the DNA Damage Checkpoints

2000 ◽  
Vol 20 (12) ◽  
pp. 4393-4404 ◽  
Author(s):  
Vladimir I. Bashkirov ◽  
Jeff S. King ◽  
Elena V. Bashkirova ◽  
Jacqueline Schmuckli-Maurer ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cellular Response ◽  
Time Course ◽  
Gamma Ray ◽  
Genotoxic Stress ◽  
Dna Damage Checkpoint ◽  
Recombinational Repair ◽  
Checkpoint Control ◽  
Repair Protein

ABSTRACT Checkpoints, which are integral to the cellular response to DNA damage, coordinate transient cell cycle arrest and the induced expression of DNA repair genes after genotoxic stress. DNA repair ensures cellular survival and genomic stability, utilizing a multipathway network. Here we report evidence that the two systems, DNA damage checkpoint control and DNA repair, are directly connected by demonstrating that the Rad55 double-strand break repair protein of the recombinational repair pathway is a terminal substrate of DNA damage and replication block checkpoints. Rad55p was specifically phosphorylated in response to DNA damage induced by the alkylating agent methyl methanesulfonate, dependent on an active DNA damage checkpoint. Rad55p modification was also observed after gamma ray and UV radiation. The rapid time course of phosphorylation and the recombination defects identified in checkpoint-deficient cells are consistent with a role of the DNA damage checkpoint in activating recombinational repair. Rad55p phosphorylation possibly affects the balance between different competing DNA repair pathways.

scholarly journals Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis

2019 ◽  
Author(s):  
Hiroyuki Sasanuma ◽  
Hana Subhan M. Sakurai ◽  
Yuko Furihata ◽  
Kiran Challa ◽  
Lira Palmer ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromosome Segregation ◽  
Dna Recombination ◽  
Dna Helicase ◽  
Aggregate Formation ◽  
Late Prophase ◽  
Prophase I ◽  
Early Protein ◽  
Srs2 Helicase ◽  
Yeast Meiosis

AbstractProper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase-I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci or among the foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphase-I and -II, suggesting the absence of DNA damage-induced cell-cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase-I.

scholarly journals Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11

2013 ◽  
Vol 110 (51) ◽  
pp. 20605-20610 ◽  
Author(s):  
C. Chen ◽  
L. Zhang ◽  
N.-J. Huang ◽  
B. Huang ◽  
S. Kornbluth
Keyword(s):  
Dna Damage ◽  
Dna Damage Checkpoint ◽  
Checkpoint Signaling

scholarly journals Direct Kinase-to-Kinase Signaling Mediated by the FHA Phosphoprotein Recognition Domain of the Dun1 DNA Damage Checkpoint Kinase

2003 ◽  
Vol 23 (4) ◽  
pp. 1441-1452 ◽  
Author(s):  
Vladimir I. Bashkirov ◽  
Elena V. Bashkirova ◽  
Edwin Haghnazari ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Dna Damage ◽  
Target Genes ◽  
Genotoxic Stress ◽  
Dna Damage Checkpoint ◽  
Kinase Signaling ◽  
M Cell ◽  
Fha Domain ◽  
Checkpoint Pathway

ABSTRACT The serine-threonine kinase Dun1 contains a forkhead-associated (FHA) domain and functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. It belongs to the Chk2 family of checkpoint kinases, which includes S. cerevisiae Rad53 and Mek1, Schizosaccharomyces pombe Cds1, and human Chk2. Dun1 is required for DNA damage-induced transcription of certain target genes, transient G2/M arrest after DNA damage, and DNA damage-induced phosphorylation of the DNA repair protein Rad55. Here we report that the FHA phosphoprotein recognition domain of Dun1 is required for direct phosphorylation of Dun1 by Rad53 kinase in vitro and in vivo. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G2/M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.

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

2007 ◽  
Vol 27 (19) ◽  
pp. 6852-6862 ◽  
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.

scholarly journals The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Alex Bronstein ◽  
Lihi Gershon ◽  
Gilad Grinberg ◽  
Elisa Alonso-Perez ◽  
Martin Kupiec
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
Dna Helicase ◽  
Main Role ◽  
Helicase Domain ◽  
Helicase Activity ◽  
C Terminus ◽  
Srs2 Helicase

ABSTRACTHomologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. TheSaccharomyces cerevisiaeyeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2’s helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a newSRS2allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of thesrs2(1-850) allele suggest a role for Srs2’s helicase activity in sister chromatid cohesion. Our results also indicate that Srs2’s function becomes more central in diploid cells.IMPORTANCEHomologous recombination (HR) is a key mechanism that repairs damaged DNA. However, this process has to be tightly regulated; failure to regulate it can lead to genome instability. The Srs2 helicase is considered a regulator of HR; it was shown to be able to evict the recombinase Rad51 from DNA. Cells lacking Srs2 exhibit sensitivity to DNA-damaging agents, and in some cases, they display defects in DNA replication. The relative roles of the helicase and Rad51 removal activities of Srs2 in genome stability remain unclear. To address this question, we created a new Srs2 mutant which has only the DNA helicase domain. Our study shows that only the DNA helicase domain is needed to deal with DNA damage and assist in DNA replication during vegetative growth and in meiosis. Thus, our findings shift the view on the role of Srs2 in the maintenance of genome integrity.

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