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 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 Replication foci dynamics: replication patterns are modulated by S-phase checkpoint kinases in fission yeast

2007 ◽  
Vol 26 (5) ◽  
pp. 1315-1326 ◽  
Author(s):  
Peter Meister ◽  
Angela Taddei ◽  
Aaron Ponti ◽  
Giuseppe Baldacci ◽  
Susan M Gasser
Keyword(s):  
Fission Yeast ◽  
S Phase ◽  
Checkpoint Kinases ◽  
Replication Foci ◽  
S Phase Checkpoint

scholarly journals Replisome Stability at Defective DNA Replication Forks Is Independent of S Phase Checkpoint Kinases

2012 ◽  
Vol 45 (5) ◽  
pp. 696-704 ◽  
Author(s):  
Giacomo De Piccoli ◽  
Yuki Katou ◽  
Takehiko Itoh ◽  
Ryuichiro Nakato ◽  
Katsuhiko Shirahige ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Replication Forks ◽  
Checkpoint Kinases ◽  
S Phase Checkpoint

scholarly journals Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint

2012 ◽  
Vol 41 (1) ◽  
pp. 229-241 ◽  
Author(s):  
Elisa Zucca ◽  
Federica Bertoletti ◽  
Ursula Wimmer ◽  
Elena Ferrari ◽  
Giuliano Mazzini ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Replication Stress ◽  
S Phase ◽  
Human Dna ◽  
Dna Polymerase Λ ◽  
Synthetically Lethal ◽  
S Phase Checkpoint

scholarly journals Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

Oncogenesis ◽  
2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Yannick P. Kok ◽  
Sergi Guerrero Llobet ◽  
Pepijn M. Schoonen ◽  
Marieke Everts ◽  
Arkajyoti Bhattacharya ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Checkpoint Inhibitors ◽  
Replication Stress ◽  
Underlying Mechanism ◽  
Dna Lesions ◽  
Mitotic Catastrophe ◽  
Replication Checkpoint ◽  
Cyclin E1 ◽  
Checkpoint Kinases ◽  
Increased Sensitivity

Abstract Oncogene-induced replication stress, for instance as a result of Cyclin E1 overexpression, causes genomic instability and has been linked to tumorigenesis. To survive high levels of replication stress, tumors depend on pathways to deal with these DNA lesions, which represent a therapeutically actionable vulnerability. We aimed to uncover the consequences of Cyclin E1 or Cdc25A overexpression on replication kinetics, mitotic progression, and the sensitivity to inhibitors of the WEE1 and ATR replication checkpoint kinases. We modeled oncogene-induced replication stress using inducible expression of Cyclin E1 or Cdc25A in non-transformed RPE-1 cells, either in a TP53 wild-type or TP53-mutant background. DNA fiber analysis showed Cyclin E1 or Cdc25A overexpression to slow replication speed. The resulting replication-derived DNA lesions were transmitted into mitosis causing chromosome segregation defects. Single cell sequencing revealed that replication stress and mitotic defects upon Cyclin E1 or Cdc25A overexpression resulted in genomic instability. ATR or WEE1 inhibition exacerbated the mitotic aberrancies induced by Cyclin E1 or Cdc25A overexpression, and caused cytotoxicity. Both these phenotypes were exacerbated upon p53 inactivation. Conversely, downregulation of Cyclin E1 rescued both replication kinetics, as well as sensitivity to ATR and WEE1 inhibitors. Taken together, Cyclin E1 or Cdc25A-induced replication stress leads to mitotic segregation defects and genomic instability. These mitotic defects are exacerbated by inhibition of ATR or WEE1 and therefore point to mitotic catastrophe as an underlying mechanism. Importantly, our data suggest that Cyclin E1 overexpression can be used to select patients for treatment with replication checkpoint inhibitors.

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.

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