scholarly journals DDK/Hsk1 phosphorylates and targets fission yeast histone deacetylase Hst4 for degradation to stabilize stalled DNA replication forks

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Shalini Aricthota ◽  
Devyani Haldar
Keyword(s):  
Stress Response ◽  
Fission Yeast ◽  
Cancer Therapeutics ◽  
Replication Stress ◽  
S Phase ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Fork Protection Complex ◽  
And Function ◽  
Replication Initiator

In eukaryotes, paused replication forks are prone to collapse, which leads to genomic instability, a hallmark of cancer. Dbf4 Dependent Kinase (DDK)/Hsk1Cdc7 is a conserved replication initiator kinase with conflicting roles in replication stress response. Here, we show that fission yeast DDK/Hsk1 phosphorylates sirtuin, Hst4 upon replication stress at C-terminal serine residues. Phosphorylation of Hst4 by DDK marks it for degradation via the ubiquitin ligase SCFpof3. Phosphorylation defective hst4 mutant (4SA-hst4) displays defective recovery from replication stress, faulty fork restart, slow S-phase progression and decreased viability. The highly conserved Fork Protection Complex (FPC) stabilizes stalled replication forks. We found that the recruitment of FPC components, Swi1 and Mcl1 to the chromatin is compromised in the 4SA-hst4 mutant, although whole cell levels increased. These defects are dependent upon H3K56ac and independent of intra S-phase checkpoint activation. Finally, we show conservation of H3K56ac dependent regulation of Timeless, Tipin and And-1 in human cells. We propose that degradation of Hst4 via DDK increases H3K56ac, changing the chromatin state in the vicinity of stalled forks facilitating recruitment and function of FPC. Overall, this study identified a crucial role of DDK and FPC in the regulation of replication stress response with implications in cancer therapeutics.

scholarly journals The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes

2021 ◽  
Vol 118 (24) ◽  
pp. e2019183118
Author(s):  
Yang Liu ◽  
Lu Wang ◽  
Xin Xu ◽  
Yue Yuan ◽  
Bo Zhang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Dna Polymerases ◽  
Replication Stress ◽  
Underlying Mechanism ◽  
S Phase ◽  
Replication Forks ◽  
Helicase Activity ◽  
Replicative Helicase ◽  
Checkpoint Kinases ◽  
S Phase Checkpoint

DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2. However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2-mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.

scholarly journals KDM5A and KDM5B histone-demethylases contribute to HU-induced replication stress response and tolerance

Biology Open ◽  
2021 ◽  
Vol 10 (5) ◽  
Author(s):  
Solenne Gaillard ◽  
Virginie Charasson ◽  
Cyril Ribeyre ◽  
Kader Salifou ◽  
Marie-Jeanne Pillaire ◽  
...  
Keyword(s):  
Stress Response ◽  
Enzymatic Activity ◽  
Stress Tolerance ◽  
Ribonucleotide Reductase ◽  
Replication Stress ◽  
S Phase ◽  
Regulatory Subunit ◽  
Replication Forks ◽  
Histone Demethylases ◽  
Major Player

ABSTRACT KDM5A and KDM5B histone-demethylases are overexpressed in many cancers and have been involved in drug tolerance. Here, we describe that KDM5A, together with KDM5B, contribute to replication stress (RS) response and tolerance. First, they positively regulate RRM2, the regulatory subunit of ribonucleotide reductase. Second, they are required for optimal levels of activated Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. We also found that KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1. Because RRM2 is a major determinant of replication stress tolerance, we developed cells resistant to HU, and show that KDM5A/B proteins are required for both RRM2 overexpression and tolerance to HU. Altogether, our results indicate that KDM5A/B are major players of RS management. They also show that drugs targeting the enzymatic activity of KDM5 proteins may not affect all cancer-related consequences of KDM5A/B overexpression.

scholarly journals Chl1 helicase controls replication fork progression by regulating dNTP pools

2022 ◽  
Vol 5 (4) ◽  
pp. e202101153
Author(s):  
Amandine Batté ◽  
Sophie C van der Horst ◽  
Mireille Tittel-Elmer ◽  
Su Ming Sun ◽  
Sushma Sharma ◽  
...  
Keyword(s):  
Stress Response ◽  
Replication Fork ◽  
Replication Stress ◽  
Stress Conditions ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Replication Fork Progression ◽  
Intracellular Dntp ◽  
Replication Fork Arrest

Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.

scholarly journals KDM5 histone-demethylases contribute to replication stress response and tolerance

2019 ◽  
Author(s):  
Solenne Gaillard ◽  
Virginie Charasson ◽  
Cyril Ribeyre ◽  
Kader Salifou ◽  
Marie-Jeanne Pillaire ◽  
...  
Keyword(s):  
Stress Response ◽  
Enzymatic Activity ◽  
Ribonucleotide Reductase ◽  
Replication Stress ◽  
S Phase ◽  
Regulatory Subunit ◽  
Replication Forks ◽  
Histone Demethylases ◽  
Major Player ◽  
Demethylase Activity

SUMMARYKDM5A and KDM5B histone-demethylases are overexpressed in many cancers and have been involved in drug tolerance. Here, we describe that KDM5A, together with KDM5B, contribute to replication stress (RS) response and tolerance. First, they positively regulate RRM2, the regulatory subunit of Ribonucleotide Reductase. Second, they are required for optimal activation of Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. This role in Chk1 activation is probably direct since KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1. Because RRM2 is a major determinant of replication stress tolerance, we developed cells resistant to HU, and show that KDM5A/B proteins are required for both RRM2 overexpression and tolerance to HU, in a manner that is independent of their demethylase activity. Altogether, our results indicate that KDM5A/B are major players of RS management. They also show that drugs targeting the enzymatic activity of KDM5 proteins may not affect all cancer-related consequences of KDM5A/B overexpression.

scholarly journals DNA2 in Chromosome Stability and Cell Survival—Is It All about Replication Forks?

2021 ◽  
Vol 22 (8) ◽  
pp. 3984
Author(s):  
Jessica J. R. Hudson ◽  
Ulrich Rass
Keyword(s):  
Replication Stress ◽  
Strand Break ◽  
Replication Forks ◽  
Phenotypic Data ◽  
Checkpoint Activation ◽  
Dna Double Strand Break ◽  
Promising Target ◽  
Anti Cancer ◽  
Okazaki Fragment Processing ◽  
Response And Recovery

The conserved nuclease-helicase DNA2 has been linked to mitochondrial myopathy, Seckel syndrome, and cancer. Across species, the protein is indispensable for cell proliferation. On the molecular level, DNA2 has been implicated in DNA double-strand break (DSB) repair, checkpoint activation, Okazaki fragment processing (OFP), and telomere homeostasis. More recently, a critical contribution of DNA2 to the replication stress response and recovery of stalled DNA replication forks (RFs) has emerged. Here, we review the available functional and phenotypic data and propose that the major cellular defects associated with DNA2 dysfunction, and the links that exist with human disease, can be rationalized through the fundamental importance of DNA2-dependent RF recovery to genome duplication. Being a crucial player at stalled RFs, DNA2 is a promising target for anti-cancer therapy aimed at eliminating cancer cells by replication-stress overload.

scholarly journals DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity

2014 ◽  
Vol 204 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Maria M. Magiera ◽  
Elisabeth Gueydon ◽  
Etienne Schwob
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Replication Stress ◽  
S Phase ◽  
Genome Integrity ◽  
Replication Forks ◽  
Mitotic Entry ◽  
Transient Activation ◽  
Spindle Checkpoints ◽  
Spindle Assembly Checkpoints

Deoxyribonucleic acid (DNA) replication and chromosome segregation must occur in ordered sequence to maintain genome integrity during cell proliferation. Checkpoint mechanisms delay mitosis when DNA is damaged or upon replication stress, but little is known on the coupling of S and M phases in unperturbed conditions. To address this issue, we postponed replication onset in budding yeast so that DNA synthesis is still underway when cells should enter mitosis. This delayed mitotic entry and progression by transient activation of the S phase, G2/M, and spindle assembly checkpoints. Disabling both Mec1/ATR- and Mad2-dependent controls caused lethality in cells with deferred S phase, accompanied by Rad52 foci and chromosome missegregation. Thus, in contrast to acute replication stress that triggers a sustained Mec1/ATR response, multiple pathways cooperate to restrain mitosis transiently when replication forks progress unhindered. We suggest that these surveillance mechanisms arose when both S and M phases were coincidently set into motion by a unique ancestral cyclin–Cdk1 complex.

scholarly journals Multi-BRCT Domain Protein Brc1 Links Rhp18/Rad18 and γH2A To Maintain Genome Stability during S Phase

2017 ◽  
Vol 37 (22) ◽  
Author(s):  
Michael C. Reubens ◽  
Sophie Rozenzhak ◽  
Paul Russell
Keyword(s):  
Genome Stability ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Dna Lesions ◽  
Brct Domain ◽  
Replication Forks ◽  
Content Type ◽  
Domain Protein ◽  
Chromosome Integrity

ABSTRACT DNA replication involves the inherent risk of genome instability, since replisomes invariably encounter DNA lesions or other structures that stall or collapse replication forks during the S phase. In the fission yeast Schizosaccharomyces pombe, the multi-BRCT domain protein Brc1, which is related to budding yeast Rtt107 and mammalian PTIP, plays an important role in maintaining genome integrity and cell viability when cells experience replication stress. The C-terminal pair of BRCT domains in Brc1 were previously shown to bind phosphohistone H2A (γH2A) formed by Rad3/ATR checkpoint kinase at DNA lesions; however, the putative scaffold interactions involving the N-terminal BRCT domains 1 to 4 of Brc1 have remained obscure. Here, we show that these domains bind Rhp18/Rad18, which is an E3 ubiquitin protein ligase that has crucial functions in postreplication repair. A missense allele in BRCT domain 4 of Brc1 disrupts binding to Rhp18 and causes sensitivity to replication stress. Brc1 binding to Rhp18 and γH2A are required for the Brc1 overexpression suppression of smc6-74, a mutation that impairs the Smc5/6 structural maintenance of chromosomes complex required for chromosome integrity and repair of collapsed replication forks. From these findings, we propose that Brc1 provides scaffolding functions linking γH2A, Rhp18, and Smc5/6 complex at damaged replication forks.

scholarly journals ATAD5 restricts R-loop formation through PCNA unloading and RNA helicase maintenance at the replication fork

2020 ◽  
Author(s):  
Sangin Kim ◽  
Nalae Kang ◽  
Su Hyung Park ◽  
James Wells ◽  
Taejoo Hwang ◽  
...  
Keyword(s):  
Replication Fork ◽  
Replication Stress ◽  
S Phase ◽  
Replication Forks ◽  
Genomic Integrity ◽  
Loop Formation ◽  
Replication Rate ◽  
Rna Helicases ◽  
Dual Functions

Abstract R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.

scholarly journals Replication stress in early S phase generates apparent micronuclei and chromosome rearrangement in fission yeast

2015 ◽  
Vol 26 (19) ◽  
pp. 3439-3450 ◽  
Author(s):  
Sarah A. Sabatinos ◽  
Nimna S. Ranatunga ◽  
Ji-Ping Yuan ◽  
Marc D. Green ◽  
Susan L. Forsburg
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Chromosome Rearrangement ◽  
Chromosome Instability ◽  
Replication Stress ◽  
Parent Nucleus ◽  
S Phase ◽  
Yeast Mutant ◽  
Mcm Helicase ◽  
Dna Replication Stress

DNA replication stress causes genome mutations, rearrangements, and chromosome missegregation, which are implicated in cancer. We analyze a fission yeast mutant that is unable to complete S phase due to a defective subunit of the MCM helicase. Despite underreplicated and damaged DNA, these cells evade the G2 damage checkpoint to form ultrafine bridges, fragmented centromeres, and uneven chromosome segregations that resembles micronuclei. These micronuclei retain DNA damage markers and frequently rejoin with the parent nucleus. Surviving cells show an increased rate of mutation and chromosome rearrangement. This first report of micronucleus-like segregation in a yeast replication mutant establishes underreplication as an important factor contributing to checkpoint escape, abnormal chromosome segregation, and chromosome instability.

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