p53 suppresses mutagenic RAD52 and POLθ pathways by orchestrating DNA replication restart homeostasis

Roy, Sunetra; Tomaszowski, Karl-Heinz; Luzwick, Jessica W.; Park, Soyoung; Li, Jun; Murphy, Maureen E.; Schlacher, Katharina

doi:10.1101/204057

Cited by 3 publications

(3 citation statements)

References 68 publications

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“…These signatures are consistent with an enhanced reliance upon ATM for DSBR in TP53-mutant GBMs. Taken together with evidence that p53 loss or mutation enhances expression of HR and MMEJ genes and causes replication stress and defective fork restart 56,66,67 , we suggest that this contributes to enhanced ATMi response in TP53mutant GBM.…”

Section: Discussionsupporting

confidence: 66%

ATM inhibition exploits checkpoint defects and ATM-dependent double strand break repair in TP53-mutant glioblastoma

Laverty,

Gupta,

Bradshaw

et al. 2024

Nat Commun

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Determining the balance between DNA double strand break repair (DSBR) pathways is essential for understanding treatment response in cancer. We report a method for simultaneously measuring non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). Using this method, we show that patient-derived glioblastoma (GBM) samples with acquired temozolomide (TMZ) resistance display elevated HR and MMEJ activity, suggesting that these pathways contribute to treatment resistance. We screen clinically relevant small molecules for DSBR inhibition with the aim of identifying improved GBM combination therapy regimens. We identify the ATM kinase inhibitor, AZD1390, as a potent dual HR/MMEJ inhibitor that suppresses radiation-induced phosphorylation of DSBR proteins, blocks DSB end resection, and enhances the cytotoxic effects of TMZ in treatment-naïve and treatment-resistant GBMs with TP53 mutation. We further show that a combination of G2/M checkpoint deficiency and reliance upon ATM-dependent DSBR renders TP53 mutant GBMs hypersensitive to TMZ/AZD1390 and radiation/AZD1390 combinations. This report identifies ATM-dependent HR and MMEJ as targetable resistance mechanisms in TP53-mutant GBM and establishes an approach for simultaneously measuring multiple DSBR pathways in treatment selection and oncology research.

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Section: Discussionsupporting

confidence: 66%

ATM inhibition exploits checkpoint defects and ATM-dependent double strand break repair in TP53-mutant glioblastoma

Laverty,

Gupta,

Bradshaw

et al. 2024

Nat Commun

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“…Moreover, the technique is labor-intensive and requires copious amounts of starting material (~100,000,000 cells per condition). The assay system described here termed in situ protein interaction with nascent DNA replication forks (SIRF) (Roy et al, 2018a and2018b) combines EdU-biotin Click-chemistry with proximity ligation assay (PLA) to overcome these challenges.…”

mentioning

confidence: 99%

“…In this case, primary and secondary antibody staining for IF is performed prior to the PLA reaction to avoid interference of PLA secondary antibodies from crossreacting with IF primary antibodies. The SIRF assay can be used to study protein dynamics at ongoing and stalled DNA replication forks, as well as chromatin maturation at newly replicated DNA regions (Petruk et al, 2017, Roy et al, 2018aand 2018b.…”

mentioning

confidence: 99%

SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks

Roy¹,

Schlacher²

2019

BIO-PROTOCOL

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The duplication of DNA is a fundamental process that is required for the transfer of the genetic information from parent to daughter cells. Aberrant DNA replication processes are associated with diverse disease phenotypes, including developmental defects, ageing disorders, blood disorders such as Fanconi Anemia, increased inflammation and cancer. Therefore, the development of tools to study proteins associated with error-free DNA replication processes is of paramount importance. So far, methods to study proteins associated with nascent replication forks relied on conventional immunofluorescence and immunoprecipitation assays of 5′-ethylene-2′-deoxyuridine (EdU) labeled DNA (iPOND). While greatly informative and important, these methods lack specificities for nascent fork interactions (e.g., IF) or assay an average change of millions of cells without single-cell resolution (e.g., iPOND). The assay system described here combines proximity ligation assay (PLA) with EdU coupled click-iT chemistry, which we termed "in situ Protein Interaction with Nascent DNA Replication Forks (SIRF)". This method enables sensitive and quantitative analysis of protein interactions with nascent DNA replication forks with single-cell resolution, and can further be paired with conventional immunofluorescence marker analysis for added multi-parameter analysis.

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RAD52 Prevents Excessive Replication Fork Reversal and Protects from Nascent Strand Degradation

et al. 2018

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Stabilisation of stalled replication forks prevents excessive fork reversal and their pathological degradation, which can undermine genome integrity. Here we investigate a physiological role of RAD52 at stalled replication forks by using human cell models depleted of RAD52, a specific small-molecule inhibitor of the RAD52-ssDNA interaction, in vitro and singlemolecule analyses. We demonstrate that RAD52 prevents excessive degradation of reversed replication forks by MRE11. Mechanistically, RAD52 binds to the stalled replication fork, promotes its occlusion and counteracts loading of SMARCAL1 in vitro and in vivo. Loss of the RAD52 function results in a slightly-defective replication restart, persistence of underreplicated regions and chromosome instability. Moreover, the RAD52-inhibited cells rely on RAD51 for completion of replication and viability upon replication arrest. Collectively, our data suggest an unexpected gatekeeper mechanism by which RAD52 limits excessive remodelling of stalled replication forks, thus indirectly assisting RAD51 and BRCA2 in protecting forks from unscheduled degradation and preventing genome instability.

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p53 suppresses mutagenic RAD52 and POLθ pathways by orchestrating DNA replication restart homeostasis

Cited by 3 publications

References 68 publications

ATM inhibition exploits checkpoint defects and ATM-dependent double strand break repair in TP53-mutant glioblastoma

ATM inhibition exploits checkpoint defects and ATM-dependent double strand break repair in TP53-mutant glioblastoma

SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks

RAD52 Prevents Excessive Replication Fork Reversal and Protects from Nascent Strand Degradation

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