RESUMO
Pulse stable isotope labeling with amino acids in cell culture (pSILAC) coupled to mass spectrometric analysis is a powerful tool to study propagation of histone post-translational modifications (PTMs). We describe the combination of triple pSILAC with pulse-chase labeling of newly replicated DNA by nascent chromatin capture (NCC). This technology tracks newly synthesized and recycled old histones, from deposition to transmission to daughter cells, unveiling principles of histone-based inheritance.
Assuntos
Cromatina , Histonas , Cromatina/genética , Código das Histonas , Histonas/genética , Histonas/metabolismo , Espectrometria de Massas , Processamento de Proteína Pós-TraducionalRESUMO
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.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Replicação do DNA , DNA Topoisomerases Tipo I/genética , DNA/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Camptotecina/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Células HeLa , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Piridinas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Inibidores da Topoisomerase I/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Quinase 1 Polo-LikeRESUMO
DNA replication in a human cell involves hundreds of proteins that copy the DNA accurately and completely each cell division cycle. In addition to the core DNA copying machine (the replisome), accessory proteins work to respond to replication stress, correct errors, and repackage the DNA into appropriate chromatin structures. New proteomic tools have been invented in the past few years to facilitate the purification, identification, and quantification of the replication, chromatin maturation, and replication stress response machineries. These tools, including iPOND (isolation of proteins on nascent DNA) and NCC (nascent chromatin capture), have yielded discoveries of new proteins involved in these processes and insights into the dynamic regulatory processes ensuring genome and chromatin integrity. In this review, I will introduce these experimental approaches and examine how they have been utilized to define the replication fork proteome.