Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination

Abstract

Summary Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.

Talazoparib monotherapy in metastatic castration-resistant prostate cancer with DNA repair alterations (TALAPRO-1): an open-label, phase 2 trial

“…30 ATM alterations have more complex effects on HRR than BRCA and PALB2 alterations, and ATM is also implicated in replication fork stability and cell cycle progression. 31 We found that germline versus somatic origin of HRR alterations had no association with response, which is consistent with other results. 20 Our study has several limitations, including the absence of a control group and potential investigator bias due to the open-label design.…”

Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair

“…Similar to recent strategies developed to study proteins interacting with nucleic acids (Alabert et al, 2014; Aranda et al, 2019; Bao et al, 2018; Kliszczak et al, 2011; Nakamura et al, 2021; Sirbu et al, 2011), we exploited the use of nucleotide mimetics as a tool to mark the DNA, leaving a chemical trace amenable for selective isolation of p rotein o n c hromatin (or iPOC). In particular, we established the conditions for the specific labeling of DNA via full incorporation of 5-Ethynyl-2’-deoxyuridine (EdU) and subsequent capture via copper-catalyzed azide-alkyne cycloaddition (CuAAC) of biotin azide [Fig.5A, Fig.S5A, B].…”

Beyond the Double-Strand Breaks: The Role of DNA Repair Proteins in Cancer Stem-Cell Regulation

“…Both pathways are highly overlapping, as DNA damage caused by RS delays the progression of replication forks or provokes their collapse. Activation of the Ataxia telangiectasia mutated (ATM) kinase only occurs through collapsed replication forks leading to DNA double-strand breaks (DSBs) [ 36 ]. The importance of error-free DNA repair for replication-associated DNA damage is expressed by the presence of de facto all DNA repair pathways at replication forks in CSC (reviewed in [ 6 ]).…”