Mus81-Eme1–dependent aberrant processing of DNA replication intermediates in mitosis impairs genome integrity

Chromosome instability (CIN) underpins cancer evolution and is associated with drug resistance and poor prognosis. Understanding the mechanistic basis of CIN is thus a priority. The structure-specific endonuclease Mus81-Eme1 is known to prevent CIN. Intriguingly, however, here we show that the aberrant processing of late replication intermediates by Mus81-Eme1 is a source of CIN. Upon depletion of checkpoint kinase 1 (Chk1), Mus81-Eme1 cleaves under-replicated DNA engaged in mitotic DNA synthesis, leading to chromosome segregation defects. Supplementing cells with nucleosides allows the completion of mitotic DNA synthesis, restraining Mus81-Eme1–dependent DNA damage in mitosis and the ensuing CIN. We found no correlation between CIN arising from nucleotide shortage in mitosis and cell death, which were selectively linked to DNA damage load in mitosis and S phase, respectively. Our findings imply the possibility of optimizing Chk1-directed therapies by inducing cell death while curtailing CIN, a common side effect of chemotherapy.

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TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

The Journal of Cell Biology ◽

10.1083/jcb.201502107 ◽

2015 ◽

Vol 210 (4) ◽

pp. 565-582 ◽

Cited By ~ 52

Author(s):

Rune Troelsgaard Pedersen ◽

Thomas Kruse ◽

Jakob Nilsson ◽

Vibe H. Oestergaard ◽

Michael Lisby

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Chromosome Segregation ◽

Nuclear Bodies ◽

Genome Integrity ◽

Unscheduled Dna Synthesis ◽

Focus Formation ◽

Mitotic Entry ◽

Daughter Cells ◽

Early Mitosis

Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis.

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A mitosis-specific and R loop–driven ATR pathway promotes faithful chromosome segregation

Science ◽

10.1126/science.aan6490 ◽

2017 ◽

Vol 359 (6371) ◽

pp. 108-114 ◽

Cited By ~ 91

Author(s):

Lilian Kabeche ◽

Hai Dang Nguyen ◽

Rémi Buisson ◽

Lee Zou

Keyword(s):

Dna Damage ◽

Chromosome Segregation ◽

Stress Responses ◽

Chromosome Instability ◽

Protein A ◽

Aurora B ◽

Cyclin Dependent Kinase ◽

Ataxia Telangiectasia Mutated ◽

Atr Kinase

The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A–regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)–coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop–driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability.

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Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

10.1101/427344 ◽

2018 ◽

Author(s):

Laura E. Schultz-Rogers ◽

Maira P. Almeida ◽

Wesley A. Wierson ◽

Marcel Kool ◽

Maura McGrail

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Cell Death ◽

Neural Stem Cells ◽

Progenitor Cell ◽

Adaptor Protein ◽

Genome Integrity ◽

Neural Precursors ◽

Chromatin Remodelers

AbstractRetinoblastoma-binding protein 4 (Rbbp4) is a WDR adaptor protein for multiple chromatin remodelers implicated in human oncogenesis. Here we show Rbbp4 is overexpressed in zebrafish rb1-embryonal brain tumors and is upregulated across the spectrum of human embryonal and glial brain cancers. We demonstrate in vivo Rbbp4 is essential for zebrafish neurogenesis and has distinct roles in neural stem and progenitor cells. rbbp4 mutant neural stem cells show delayed cell cycle progression and become hypertrophic. In contrast, rbbp4 mutant neural precursors accumulate extensive DNA damage and undergo programmed cell death that is dependent on Tp53 signaling. Loss of Rbbp4 and disruption of genome integrity correlates with failure of neural precursors to initiate quiescence and transition to differentiation. rbbp4; rb1 double mutants show that survival of neural precursors after disruption of Rb1 is dependent on Rbbp4. Elevated Rbbp4 in Rb1-deficient brain tumors might drive proliferation and circumvent DNA damage and Tp53-dependent apoptosis, lending support to current interest in Rbbp4 as a potential druggable target.Author SummaryExamining the developmental mechanisms controlling neural stem and progenitor cell behavior is critical to our understanding of the processes driving brain tumor oncogenesis. Chromatin remodelers and their associated adaptor proteins are thought to be key drivers of brain development and disease through epigenetic regulation of gene expression and maintenance of genome integrity, but knowledge of their in vivo roles in vertebrate neurogenesis is limited. The chromatin remodeler adaptor protein Rbbp4 has recently been shown to function in a mouse model of neuroblastoma and in glioblastoma multiforme cell resistance to the chemotherapeutic temozolomide. However, an in vivo requirement for Rbbp4 in neurogenesis has only just been shown by isolation of a recessive lethal mutation in zebrafish rbbp4. Here we provide conclusive genetic evidence that zebrafish rbbp4 is essential in neural stem and progenitor cell function during development. Our data reveal for the first time in vivo that Rbbp4 prevents DNA damage and activation of Tp53 signaling pathway that leads to programmed cell death. Importantly, neural progenitors that are mutant for the tumor suppressor Rb1 also depend on Rbbp4 for survival. Finally, we show that neural stem cells that have lost Rbbp4 cease dividing, and may enter a senescent like state. Together, these observations provide novel evidence that elevated expression of Rbbp4 in rb1-mutant tumors may contribute to cancer cell survival by blocking senescence and/or DNA damage-induced cell death.

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Polo-Like Kinase 1 Depletion Induces DNA Damage in Early S Prior to Caspase Activation

Molecular and Cellular Biology ◽

10.1128/mcb.01277-08 ◽

2009 ◽

Vol 29 (10) ◽

pp. 2609-2621 ◽

Cited By ~ 44

Author(s):

Hyungshin Yim ◽

Raymond L. Erikson

Keyword(s):

Dna Damage ◽

Cell Death ◽

Dna Synthesis ◽

Cancer Cells ◽

S Phase ◽

Mitotic Catastrophe ◽

Serum Starvation ◽

Dna Integrity ◽

Normal Cells ◽

Sequence Of Events

ABSTRACT Polo-like kinase 1 (Plk1) plays several roles in mitosis, and it has been suggested to have a role in tumorigenesis. We have previously reported that Plk1 depletion results in cell death in cancer cells, whereas normal cells survive similar depletion. However, Plk1 depletion together with p53 depletion induces cell death in normal cells as well. This communication presents evidence on the sequence of events that leads to cell death in cancer cells. DNA damage is detected at the first S phase following Plk1 depletion and is more severe in Plk1-depleted p53-null cancer cells. As a consequence of Plk1 depletion using lentivirus-based small interfering RNA techniques, prereplicative complex (pre-RC) formation is disrupted at the G1/S transition, and DNA synthesis is reduced during S phase of the first cycle after depletion. The levels of geminin, an inhibitor of DNA pre-RC, and Emi1, an inhibitor of anaphase-promoting complex/cyclosome, are elevated in Plk1-depleted cells. The rate of cell cycling is slower in Plk1-depleted cells than in control cells when synchronized by serum starvation. Plk1 depletion results in disrupted DNA pre-RC formation, reduced DNA synthesis, and DNA damage before cells display severe mitotic catastrophe or apoptosis. Our data suggest that Plk1 is required for cell cycle progression not only in mitosis but also for DNA synthesis, maintenance of DNA integrity, and prevention of cell death.

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