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

Abstract Aims Glioblastoma (GBM) is a treatment refractory cancer of extreme unmet need which exhibits treatment resistance due to a subpopulation of GBM cancer stem cells which have constitutive DNA damage response activation driven by elevated replication stress (RS). RS response inhibition is potently cytotoxic to GSC, however mechanistic understanding will be key to biomarker discovery and successful clinical translation. We investigated response to combined ATR and PARP inhibition (CAiPi) to gain mechanistic insight and inform biomarker development. Method A panel of patient-derived GBM cell lines were cultured as stem enriched (GSCs) or stem depleted (bulk), to characterise response to combined ATR inhibition (VE821 5μM) and PARP inhibition (Olaparib 1μM), by CellTiter-Glo viability assay. Mechanistic investigations included immunofluorescence of 53BP1 nuclear bodies and DNA fibre analysis. Studies into the importance of PARP trapping included another PARPi Veliparib (1μM), and investigations into inhibition of origin firing used the CDK inhibitor Roscovitine. Results Responses to CAiPi in a panel of primary paired GBM GSCs vs differentiated progeny were heterogenous. CAiPi is selectively GSC cytotoxic in a subpopulation of tumours. DNA fibre analysis identified increased new origin firing with PARPi, which was correlated with increased PARP trapping. Inhibition of origin firing by exposure to roscovitine rescued the CAiPi cytotoxic phenotype, suggesting origin firing has an important role in selective GSC cytotoxicity. A population of treatment-sensitive GSCs with increased numbers of 53BP1 nuclear bodies in G1 phase with CAiPi were identified, indicative of under-replication of DNA in S phase. Conclusion Selective GSC cytotoxicity is induced by CAiPi via dysregulation of replication, by both DNA under-replication resulting in DNA lesions, and the novel finding of increased new origin firing in GSC due to PARPi.

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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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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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Author response: The RIF1-long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress

10.7554/elife.58020.sa2 ◽

2020 ◽

Author(s):

Lotte P Watts ◽

Toyoaki Natsume ◽

Yuichiro Saito ◽

Javier Garzon ◽

Qianqian Dong ◽

...

Keyword(s):

Splice Variant ◽

Replication Stress ◽

Nuclear Bodies ◽

Author Response ◽

G1 Phase

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The Mechanism of Replication Stalling and Recovery within Repetitive DNA

10.1101/2021.06.02.446729 ◽

2021 ◽

Author(s):

Corella S Casas-Delucchi ◽

Manuel Daza-Martin ◽

Sophie L Williams ◽

Gideon Coster

Keyword(s):

Cellular Response ◽

Repetitive Sequences ◽

Replication Stress ◽

Underlying Mechanism ◽

Dna Lesions ◽

Chromosomal Dna ◽

Leading Strand ◽

Fork Stalling ◽

Dna Template

SUMMARYAccurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstituted replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We found that structure-prone repeats are sufficient to impair replication. Whilst template unwinding was unaffected, leading strand synthesis was inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats relied on replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats required an extrinsic accessory helicase. DNA-induced fork stalling was mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress also applies to stalling induced by repetitive sequences.

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