RESUMEN
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.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de Ciclo Celular/genética , Replicación del ADN , ADN-Topoisomerasas de Tipo I/genética , ADN/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Reparación del ADN por Recombinación , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Camptotecina/farmacología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina/química , Cromatina/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN-Topoisomerasas de Tipo I/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Regulación de la Expresión Génica , Células HeLa , Humanos , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas/metabolismo , Piridinas/farmacología , Quinolinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Inhibidores de Topoisomerasa I/farmacología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacos , Quinasa Tipo Polo 1RESUMEN
Operons are transcriptional modules that allow bacteria to adapt to environmental changes by coordinately expressing the relevant set of genes. In humans, biological pathways and their regulation are more complex. If and how human cells coordinate the expression of entire biological processes is unclear. Here, we capture 31 higher-order co-regulation modules, which we term progulons, by help of supervised machine-learning on proteomics data. Progulons consist of dozens to hundreds of proteins that together mediate core cellular functions. They are not restricted to physical interactions or co-localisation. Progulon abundance changes are primarily controlled at the level of protein synthesis and degradation. Implemented as a web app at www.proteomehd.net/progulonFinder, our approach enables the targeted search for progulons of specific cellular processes. We use it to identify a DNA replication progulon and reveal multiple new replication factors, validated by extensive phenotyping of siRNA-induced knockdowns. Progulons provide a new entry point into the molecular understanding of biological processes.
Asunto(s)
Proteoma , Humanos , Proteoma/genética , Proteoma/metabolismoRESUMEN
During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.
Asunto(s)
Replicación del ADN , Histonas/química , Componente 2 del Complejo de Mantenimiento de Minicromosoma/química , Modelos Moleculares , Chaperonas Moleculares/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Western Blotting , Línea Celular Tumoral , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Componente 2 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Multimerización de Proteína , Interferencia de ARN , Homología de Secuencia de AminoácidoRESUMEN
Histone H3 proteins play fundamental roles in DNA packaging, gene transcription, and the transmission of epigenetic states. In addition to posttranslational modifications of their N termini, the use of H3 variants contributes to their regulatory repertoire. Canonical histone H3.2 is expressed during S phase and differs by four amino acid residues from the variant histone H3.3, which is synthesized in a cell-cycle-independent manner. Because H3.3 is enriched within actively transcribed loci, and because di- and trimethylation of H3 lysine 4 are hallmarks of chromatin at such sites in the genome, the H3.3K4 residue is considered to serve as the major regulatory determinant for the transcriptional state of a gene. Here we use genetic approaches in Drosophila to replace all 46 gene copies of His3.2 with mutant derivatives and thereby demonstrate that canonical and variant H3 can functionally replace each other. Cells are able to divide and differentiate when H3.2 is entirely absent but replaced by S phase-expressed H3.3. Moreover, although slowed down in their proliferative capacity, cells that code for a nonmethylatable residue instead of K4 in all canonical and variant H3 genes are competent to respond to major developmental signaling pathways by activating target gene expression. Hence, the presence of different H3 protein species is not essential in Drosophila and transcriptional regulation can occur in the complete absence of H3K4 methylation.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Animales , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Transcripción GenéticaRESUMEN
Di- and trimethylation of histone H3 lysine 4 (H3K4me2 and H3K4me3) are hallmarks of chromatin at active genes. The major fraction of K4-methylated histone H3 is the variant H3 (termed H3.3 in Drosophila), which replaces canonical H3 (H3.2) in transcribed genes. Here, we genetically address the in vivo significance of such K4 methylation by replacing wild-type H3.3 with a mutant form (H3.3K4A) that cannot be methylated. We monitored the transcription that occurs in response to multiple well-described signaling pathways. Surprisingly, the transcriptional outputs of these pathways remain intact in H3.3K4A mutant cells. Even the complete absence of both H3.3 genes does not noticeably affect viability or function of cells: double mutant animals are viable but sterile. Fertility can be rescued by K4-containing versions of H3.3, but not with mutant H3.3 (H3.3K4A) or with canonical H3.2. Together, these data suggest that in Drosophila, presence of H3.3K4me in the chromatin of active genes is dispensable for successful transcription in most cells and only plays an important role in reproductive tissues.