ATR and a Chk1-Aurora B pathway coordinate postmitotic genome surveillance with cytokinetic abscission

Aurora B regulates cytokinesis timing and plays a central role in the abscission checkpoint. Cellular events monitored by this checkpoint are beginning to be elucidated, yet signaling pathways upstream of Aurora B in this context remain poorly understood. Here we reveal a new connection between postmitotic genome surveillance and cytokinetic abscission. Underreplicated DNA lesions are known to be transmitted through mitosis and protected in newly formed nuclei by recruitment of 53BP1 and other proteins until repair takes place. We find that this genome surveillance initiates before completion of cytokinesis. Elevating replication stress increases this postmitotic process and delays cytokinetic abscission by keeping the abscission checkpoint active. We further find that ATR activity in midbody-stage cells links postmitotic genome surveillance to abscission timing and that Chk1 integrates this and other signals upstream of Aurora B to regulate when the final step in the physical separation of daughter cells occurs.

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ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins

eLife ◽

10.7554/elife.06547 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 53

Author(s):

Anna Caballe ◽

Dawn M Wenzel ◽

Monica Agromayor ◽

Steven L Alam ◽

Jack J Skalicky ◽

...

Keyword(s):

Nuclear Pore ◽

Aurora B ◽

Physical Separation ◽

Protection Mechanism ◽

Endosomal Sorting ◽

Daughter Cells ◽

Escrt Machinery ◽

A Genome ◽

Biochemical Studies ◽

Genome Protection

The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations.

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Faculty Opinions recommendation of 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.9007956.9704055 ◽

2011 ◽

Author(s):

Antony Carr

Keyword(s):

Replication Stress ◽

Dna Lesions ◽

Nuclear Bodies

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Faculty Opinions recommendation of 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.9007956.9573054 ◽

2011 ◽

Author(s):

Junjie Chen

Keyword(s):

Replication Stress ◽

Dna Lesions ◽

Nuclear Bodies

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Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication Timing

International Journal of Molecular Sciences ◽

10.3390/ijms22094959 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4959

Author(s):

Lilas Courtot ◽

Elodie Bournique ◽

Chrystelle Maric ◽

Laure Guitton-Sert ◽

Miguel Madrid-Mencía ◽

...

Keyword(s):

Replication Timing ◽

Replication Stress ◽

Chromatin Accessibility ◽

Cell Type ◽

Replicative Stress ◽

Daughter Cells ◽

Origin Activation ◽

Cell Type Specific ◽

Cellular Identity ◽

Dna Replication Timing

DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains.

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A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans

The Journal of Cell Biology ◽

10.1083/jcb.200506124 ◽

2005 ◽

Vol 171 (2) ◽

pp. 267-279 ◽

Cited By ~ 156

Author(s):

Anjon Audhya ◽

Francie Hyndman ◽

Ian X. McLeod ◽

Amy S. Maddox ◽

John R. Yates ◽

...

Keyword(s):

Cell Division ◽

Caenorhabditis Elegans ◽

Rna Binding ◽

Rna Binding Protein ◽

Rna Helicase ◽

Aurora B ◽

Aurora B Kinase ◽

Daughter Cells ◽

Sm Protein ◽

Spindle Structure

Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1–depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

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Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions

Blood ◽

10.1182/blood-2017-01-764274 ◽

2017 ◽

Vol 130 (13) ◽

pp. 1523-1534 ◽

Cited By ~ 14

Author(s):

Ana Martín-Pardillos ◽

Anastasia Tsaalbi-Shtylik ◽

Si Chen ◽

Seka Lazare ◽

Ronald P. van Os ◽

...

Keyword(s):

Dna Damage ◽

Mitochondrial Dysfunction ◽

Replication Stress ◽

Precursor Cells ◽

Dna Lesions ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Functional Integrity ◽

Key Points

Key Points Tolerance of oxidative DNA lesions ensures the genomic and functional integrity of hematopoietic stem and precursor cells. Endogenous DNA damage–induced replication stress is associated with mitochondrial dysfunction.

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

Oncogenesis ◽

10.1038/s41389-020-00270-2 ◽

2020 ◽

Vol 9 (10) ◽

Cited By ~ 1

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.

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TEX14 Interacts with CEP55 To Block Cell Abscission

Molecular and Cellular Biology ◽

10.1128/mcb.01392-09 ◽

2010 ◽

Vol 30 (9) ◽

pp. 2280-2292 ◽

Cited By ~ 51

Author(s):

Tokuko Iwamori ◽

Naoki Iwamori ◽

Lang Ma ◽

Mark A. Edson ◽

Michael P. Greenbaum ◽

...

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Intercellular Bridge ◽

Male Germ Cells ◽

Physical Separation ◽

Targeted Deletion ◽

Intercellular Bridges ◽

Daughter Cells ◽

Stable Component ◽

Evolutionarily Conserved

ABSTRACT In somatic cells, abscission, the physical separation of daughter cells at the completion of cytokinesis, requires CEP55, ALIX, and TSG101. In contrast, cytokinesis is arrested prior to abscission in differentiating male germ cells that are interconnected by TEX14-positive intercellular bridges. We have previously shown that targeted deletion of TEX14 disrupts intercellular bridges in all germ cells and causes male sterility. Although these findings demonstrate that intercellular bridges are essential for spermatogenesis, it remains to be shown how TEX14 and other proteins come together to prevent abscission and form stable intercellular bridges. Using a biochemical enrichment of male germ cell intercellular bridges, we identified additional bridge proteins, including CEP55. Although CEP55 is highly expressed in testes at the RNA level, there is no report of the presence of CEP55 in germ cells. We show here that CEP55 becomes a stable component of the intercellular bridge and that an evolutionarily conserved GPPX3Y motif of TEX14 binds strongly to CEP55 to block similar GPPX3Y motifs of ALIX and TSG101 from interacting and localizing to the midbody. Thus, TEX14 prevents the completion of cytokinesis by altering the destiny of CEP55 from a nidus for abscission to an integral component of the intercellular bridge.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00260-17 ◽

2017 ◽

Vol 37 (22) ◽

Cited By ~ 2

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.

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