Methods for the Detection of Genome Instability Derived from Replication Stress in Primary Mouse Embryonic Fibroblasts

Luebben, Spencer W.; Shima, Naoko; Kawabata, Tsuyoshi

doi:10.1007/978-1-4939-1215-5_19

Cited by 3 publications

(2 citation statements)

References 17 publications

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“…The DNA fiber assays were performed as described ( 39 , 40 ). Cells were sequentially labeled with 100 μM IdU for 10 min and 100 μM CldU for 20 min.…”

Section: Methodsmentioning

confidence: 99%

SMARCA4deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma

Kurashima¹,

Kashiwagi²,

Shimomura

et al. 2020

NAR Cancer

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The SWI/SNF chromatin remodeling complex regulates transcription through the control of chromatin structure and is increasingly thought to play an important role in human cancer. Lung adenocarcinoma (LADC) patients frequently harbor mutations in SMARCA4, a core component of this multisubunit complex. Most of these mutations are loss-of-function mutations, which disrupt critical functions in the regulation of chromatin architecture and can cause DNA replication stress. This study reports that LADC cells deficient in SMARCA4 showed increased DNA replication stress and greater sensitivity to the ATR inhibitor (ATRi) in vitro and in vivo. Mechanistically, loss of SMARCA4 increased heterochromatin formation, resulting in stalled forks, a typical DNA replication stress. In the absence of SMARCA4, severe ATRi-induced single-stranded DNA, which caused replication catastrophe, was generated on nascent DNA near the reversed forks around heterochromatin in an Mre11-dependent manner. Thus, loss of SMARCA4 confers susceptibility to ATRi, both by increasing heterochromatin-associated replication stress and by allowing Mre11 to destabilize reversed forks. These two mechanisms synergistically increase susceptibility of SMARCA4-deficient LADC cells to ATRi. These results provide a preclinical basis for assessing SMARCA4 defects as a biomarker of ATRi efficacy.

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“…The DNA fiber assays were performed as described ( 39 , 40 ). Cells were sequentially labeled with 100 μM IdU for 10 min and 100 μM CldU for 20 min.…”

Section: Methodsmentioning

confidence: 99%

SMARCA4deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma

Kurashima¹,

Kashiwagi²,

Shimomura

et al. 2020

NAR Cancer

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“…DNA fiber assays were performed primarily as described previously (Luebben et al, 2014;Sekimoto et al, 2015). Cells were labeled with 100 μM IdU (Sigma) for 10 min and then with 100 μM CldU (Sigma) for 20 min.…”

Section: Dna Fiber Assaysmentioning

confidence: 99%

Polη, a Y-family translesion synthesis polymerase, promotes cellular tolerance of Myc-induced replication stress

Kurashima

Sekimoto

Oda

et al. 2018

Journal of Cell Science

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Growth of precancerous and cancer cells relies on their tolerance of oncogene-induced replication stress (RS). Translesion synthesis (TLS) plays an essential role in the cellular tolerance of various types of RS and bypasses replication barriers by employing specialized polymerases. However, limited information is available about the role of TLS polymerases in oncogene-induced RS. Here, we report that Polη, a Y-family TLS polymerase, promotes cellular tolerance of Myc-induced RS. Polη was recruited to Myc-induced RS sites, and Polη depletion enhanced the Myc-induced slowing and stalling of replication forks and the subsequent generation of double-strand breaks (DSBs). Overexpression of a catalytically dead Polη also promoted Myc-induced DSB formation. In the absence of Polη, Myc-induced DSB formation depended on MUS81-EME2 (the S-phase-specific endonuclease complex), and concomitant depletion of MUS81-EME2 and Polη enhanced RS and cell death in a synergistic manner. Collectively, these results indicate that Polη facilitates fork progression during Myc-induced RS, thereby helping cells tolerate the resultant deleterious effects. Additionally, the present study highlights the possibility of a synthetic sickness or lethality between Polη and MUS81-EME2 in cells experiencing Myc-induced RS.

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High-resolution genomic assays provide insight into the division of labor between TLS and HDR in mammalian replication of damaged DNA

et al. 2016

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Methods for the Detection of Genome Instability Derived from Replication Stress in Primary Mouse Embryonic Fibroblasts

Cited by 3 publications

References 17 publications

SMARCA4deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma

SMARCA4deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma

Polη, a Y-family translesion synthesis polymerase, promotes cellular tolerance of Myc-induced replication stress

High-resolution genomic assays provide insight into the division of labor between TLS and HDR in mammalian replication of damaged DNA

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