RESUMO
The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.
Assuntos
Genes Essenciais , Teorema de Bayes , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Biblioteca Gênica , Humanos , MutaçãoRESUMO
Viral infection often trigger an ATM serine/threonine kinase (ATM)-dependent DNA damage response in host cells that suppresses viral replication. Viruses evolved different strategies to counteract this antiviral surveillance system. Here, we report that human herpesvirus 6B (HHV-6B) infection causes genomic instability by suppressing ATM signaling in host cells. Expression of immediate-early protein 1 (IE1) phenocopies this phenotype and blocks homology-directed double-strand break repair. Mechanistically, IE1 interacts with NBS1, and inhibits ATM signaling through two distinct domains. HHV-6B seems to efficiently inhibit ATM signaling as further depletion of either NBS1 or ATM do not significantly boost viral replication in infected cells. Interestingly, viral integration of HHV-6B into the host's telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair. Given that spontaneous IE1 expression has been detected in cells of subjects with inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases.
Assuntos
Herpesvirus Humano 6 , Proteínas Imediatamente Precoces , Infecções por Roseolovirus , Humanos , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/metabolismo , Infecções por Roseolovirus/genética , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Integração Viral , Instabilidade Genômica , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here we identified MBTD1 as a stable subunit of the complex, and we reveal that, via a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double-strand breaks by homologous recombination. It was previously suggested that TIP60-dependent acetylation of H4 regulates binding of the non-homologous end joining factor 53BP1, which engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub. We find that the TIP60 complex regulates association of 53BP1 partly by competing for H4K20me2 and by regulating H2AK15ub. Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex to regulate 53BP1-dependent repair through competitive bivalent binding and modification of chromatin.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sítios de Ligação , Ligação Competitiva , Sistemas CRISPR-Cas , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histona Acetiltransferases/genética , Histonas/genética , Humanos , Células K562 , Lisina Acetiltransferase 5 , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , UbiquitinaçãoRESUMO
The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.
Assuntos
Proteínas Nucleares , Ubiquitina , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.
Assuntos
Microscopia Crioeletrônica , Histonas/química , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Metilação , Modelos Moleculares , Nucleossomos/química , Nucleossomos/genética , Maleabilidade , Multimerização Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell's DNA replication and repair machineries to replicate their own genomes. How this host-pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.
Assuntos
Quebras de DNA de Cadeia Dupla , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Feminino , Instabilidade Genômica , Recombinação Homóloga , Interações Hospedeiro-Patógeno , Humanos , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Neoplasias do Colo do Útero/virologiaRESUMO
DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.
Assuntos
Proteína BRCA1/genética , Proteínas de Transporte/genética , Ciclo Celular/genética , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Proteínas de Ligação a Telômeros/genética , Proteína BRCA1/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Reparo do DNA por Junção de Extremidades/genética , Endodesoxirribonucleases , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fase S , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
The BMRF1 protein of Epstein-Barr virus (EBV) has multiple roles in viral lytic infection, including serving as the DNA polymerase processivity factor, activating transcription from several EBV promoters and inhibiting the host DNA damage response to double-stranded DNA breaks (DSBs). Using affinity purification coupled to mass spectrometry, we identified the nucleosome remodeling and deacetylation (NuRD) complex as the top interactor of BMRF1. We further found that NuRD components localize with BMRF1 at viral replication compartments and that this interaction occurs through the BMRF1 C-terminal region previously shown to mediate transcriptional activation. We identified an RBBP4 binding motif within this region that can interact with both RBBP4 and MTA2 components of the NuRD complex and showed that point mutation of this motif abrogates NuRD binding as well as the ability of BMRF1 to activate transcription from the BDLF3 and BLLF1 EBV promoters. In addition to its role in transcriptional regulation, NuRD has been shown to contribute to DSB signaling in enabling recruitment of RNF168 ubiquitin ligase and subsequent ubiquitylation at the break. We showed that BMRF1 inhibited RNF168 recruitment and ubiquitylation at DSBs and that this inhibition was at least partly relieved by loss of the NuRD interaction. The results reveal a mechanism by which BMRF1 activates transcription and inhibits DSB signaling and a novel role for NuRD in transcriptional activation in EBV.IMPORTANCE The Epstein-Barr virus (EBV) BMRF1 protein is critical for EBV infection, playing key roles in viral genome replication, activation of EBV genes, and inhibition of host DNA damage responses (DDRs). Here we show that BMRF1 targets the cellular nucleosome remodeling and deacetylation (NuRD) complex, using a motif in the BMRF1 transcriptional activation sequence. Mutation of this motif disrupts the ability of BMRF1 to activate transcription and interfere with DDRs, showing the importance of the NuRD interaction for BMRF1 functions. BMRF1 was shown to act at the same step in the DDR as NuRD, suggesting that it interferes with NuRD function.
Assuntos
Antígenos Virais/metabolismo , Dano ao DNA , Herpesvirus Humano 4/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Antígenos Virais/genética , Linhagem Celular Tumoral , Replicação do DNA , DNA Viral/genética , Proteínas de Ligação a DNA/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Células HEK293 , Células HeLa , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Glicoproteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Transativadores/metabolismo , Ativação Transcricional , Proteínas Virais/metabolismo , Replicação ViralRESUMO
The response to DNA double-strand breaks (DSBs) entails the hierarchical recruitment of proteins orchestrated by ATM-dependent phosphorylation and RNF8-mediated chromatin ubiquitylation. As in most ubiquitin-dependent processes, the ordered accumulation of DNA repair factors at the break site relies on ubiquitin-binding domains (UBDs). However, how UBDs select their ligands is poorly understood, and therefore we sought to uncover the basis for selectivity in the ubiquitin-dependent DSB response. We show that RNF168, its paralog RNF169, RAD18, and the BRCA1-interacting RAP80 protein accumulate at DSB sites through the use of bipartite modules composed of UBDs juxtaposed to peptide motifs that provide specificity. These sequences, named LR motifs (LRMs), are transferable, and we show that the RNF169 LRM2 binds to nucleosomes, the substrates of RNF168. The LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the DNA damage response.
Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HCT116 , Células HEK293 , Chaperonas de Histonas , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação/fisiologia , Ubiquitina/fisiologia , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/fisiologiaRESUMO
Interpretation of genetic variation is needed for deciphering genotype-phenotype associations, mechanisms of inherited disease, and cancer driver mutations. Millions of single nucleotide variants (SNVs) in human genomes are known and thousands are associated with disease. An estimated 21% of disease-associated amino acid substitutions corresponding to missense SNVs are located in protein sites of post-translational modifications (PTMs), chemical modifications of amino acids that extend protein function. ActiveDriverDB is a comprehensive human proteo-genomics database that annotates disease mutations and population variants through the lens of PTMs. We integrated >385,000 published PTM sites with â¼3.6 million substitutions from The Cancer Genome Atlas (TCGA), the ClinVar database of disease genes, and human genome sequencing projects. The database includes site-specific interaction networks of proteins, upstream enzymes such as kinases, and drugs targeting these enzymes. We also predicted network-rewiring impact of mutations by analyzing gains and losses of kinase-bound sequence motifs. ActiveDriverDB provides detailed visualization, filtering, browsing and searching options for studying PTM-associated mutations. Users can upload mutation datasets interactively and use our application programming interface in pipelines. Integrative analysis of mutations and PTMs may help decipher molecular mechanisms of phenotypes and disease, as exemplified by case studies of TP53, BRCA2 and VHL. The open-source database is available at https://www.ActiveDriverDB.org.
Assuntos
Bases de Dados Genéticas , Bases de Dados de Proteínas , Doença/genética , Mutação , Processamento de Proteína Pós-Traducional/genética , Substituição de Aminoácidos , Mineração de Dados/métodos , Conjuntos de Dados como Assunto , Estudos de Associação Genética , Variação Genética , Genoma Humano , Genômica , Humanos , Anotação de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Proteínas Quinases/genética , Proteômica , Software , Interface Usuário-ComputadorRESUMO
To replicate and persist in human cells, linear double-stranded DNA (dsDNA) viruses, such as Epstein-Barr virus (EBV), must overcome the host DNA damage response (DDR) that is triggered by the viral genomes. Since this response is necessary to maintain cellular genome integrity, its inhibition by EBV is likely an important factor in the development of cancers associated with EBV infection, including gastric carcinoma. Here we present the first extensive screen of EBV proteins that inhibit dsDNA break signaling. We identify the BKRF4 tegument protein as a DDR inhibitor that interferes with histone ubiquitylation at dsDNA breaks and recruitment of the RNF168 histone ubiquitin ligase. We further show that BKRF4 binds directly to histones through an acidic domain that targets BKRF4 to cellular chromatin and is sufficient to inhibit dsDNA break signaling. BKRF4 transcripts were detected in EBV-positive gastric carcinoma cells (AGS-EBV), and these increased in lytic infection. Silencing of BKRF4 in both latent and lytic AGS-EBV cells (but not in EBV-negative AGS cells) resulted in increased dsDNA break signaling, confirming a role for BKRF4 in DDR inhibition in the context of EBV infection and suggesting that BKRF4 is expressed in latent cells. BKRF4 was also found to be consistently expressed in EBV-positive gastric tumors in the absence of a full lytic infection. The results suggest that BKRF4 plays a role in inhibiting the cellular DDR in latent and lytic EBV infection and that the resulting accumulation of DNA damage might contribute to development of gastric carcinoma.IMPORTANCE Epstein-Barr virus (EBV) infects most people worldwide and is causatively associated with several types of cancer, including â¼10% of gastric carcinomas. EBV encodes â¼80 proteins, many of which are believed to manipulate cellular regulatory pathways but are poorly characterized. The DNA damage response (DDR) is one such pathway that is critical for maintaining genome integrity and preventing cancer-associated mutations. In this study, a screen for EBV proteins that inhibit the DDR identified BKRF4 as a DDR inhibitor that binds histones and blocks their ubiquitylation at the DNA damage sites. We also present evidence that BKRF4 is expressed in both latent and lytic forms of EBV infection, where it downregulates the DDR, as well as in EBV-positive gastric tumors. The results suggest that BKRF4 could contribute to the development of gastric carcinoma through its ability to inhibit the DDR.
Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiologia , Histonas/metabolismo , Neoplasias Gástricas/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Infecções por Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , Biblioteca Gênica , Células HEK293 , Humanos , Domínios Proteicos , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Virais/química , Replicação ViralRESUMO
53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone 'code' produced by DSB signalling.
Assuntos
Dano ao DNA , Histonas/química , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Defective signaling or repair of DNA double-strand breaks has been associated with developmental defects and human diseases. The E3 ligase RING finger 168 (RNF168), mutated in the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome, was shown to ubiquitylate H2A-type histones, and this ubiquitylation was proposed to facilitate the recruitment of p53-binding protein 1 (53BP1) to the sites of DNA double-strand breaks. In contrast to more upstream proteins signaling DNA double-strand breaks (e.g., RNF8), deficiency of RNF168 fully prevents both the initial recruitment to and retention of 53BP1 at sites of DNA damage; however, the mechanism for this difference has remained unclear. Here, we identify mechanisms that regulate 53BP1 recruitment to the sites of DNA double-strand breaks and provide evidence that RNF168 plays a central role in the regulation of 53BP1 functions. RNF168 mediates K63-linked ubiquitylation of 53BP1 which is required for the initial recruitment of 53BP1 to sites of DNA double-strand breaks and for its function in DNA damage repair, checkpoint activation, and genomic integrity. Our findings highlight the multistep roles of RNF168 in signaling DNA damage.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Reparo do DNA/genética , Fibroblastos , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
The human NuA4/TIP60 co-activator complex, a fusion of the yeast SWR1 and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4/H2A/H2A.Z to regulate gene expression and maintain genome stability. Our cryo-electron microscopy studies show that, within the NuA4/TIP60 complex, the EP400 subunit serves as a scaffold holding the different functional modules in specific positions, creating a unique arrangement of the ARP module. EP400 interacts with the TRRAP subunit using a footprint that overlaps with that of the SAGA acetyltransferase complex, preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.
RESUMO
The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin in vivo. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1- and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target.
Assuntos
Replicação do DNA , Regulação para Baixo , Papillomavirus Humano 31/genética , Infecções por Papillomavirus/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 31/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Peptídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Spinal muscular atrophy is the leading genetic cause of infant mortality and results from depleted levels of functional survival of motor neuron (SMN) protein by either deletion or mutation of the SMN1 gene. SMN is characterized by a central TUDOR domain, which mediates the association of SMN with arginine methylated (Rme) partners, such as coilin, fibrillarin, and RNA pol II (RNA polymerase II). Herein, we biochemically demonstrate that SMN also associates with histone H3 monomethylated on lysine 79 (H3K79me1), defining SMN as not only the first protein known to associate with the H3K79me1 histone modification but also the first histone mark reader to recognize both methylated arginine and lysine residues. Mutational analyzes provide evidence that SMNTUDOR associates with H3 via an aromatic cage. Importantly, most SMNTUDOR mutants found in spinal muscular atrophy patients fail to associate with H3K79me1.
Assuntos
Código das Histonas , Atrofia Muscular Espinal , Proteína 1 de Sobrevivência do Neurônio Motor , Humanos , Lactente , Arginina , Lisina , Atrofia Muscular Espinal/genética , RNA Polimerase II , Fatores de Transcrição , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Fanconi Anemia (FA) is a rare, genome instability-associated disease characterized by a deficiency in repairing DNA crosslinks, which are known to perturb several cellular processes, including DNA transcription, replication, and repair. Formaldehyde, a by-product of metabolism, is thought to drive FA by generating DNA interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs). However, the impact of formaldehyde on global cellular pathways has not been investigated thoroughly. Herein, using a pangenomic CRISPR-Cas9 screen, we identify EXO1 as a critical regulator of formaldehyde-induced DNA lesions. We show that EXO1 knockout cell lines exhibit formaldehyde sensitivity leading to the accumulation of replicative stress, DNA double-strand breaks, and quadriradial chromosomes, a typical feature of FA. After formaldehyde exposure, EXO1 is recruited to chromatin, protects DNA replication forks from degradation, and functions in parallel with the FA pathway to promote cell survival. In vitro, EXO1-mediated exonuclease activity is proficient in removing DPCs. Collectively, we show that EXO1 limits replication stress and DNA damage to counteract formaldehyde-induced genome instability.
Assuntos
Sistemas CRISPR-Cas , Tolerância a Medicamentos , Exodesoxirribonucleases , Anemia de Fanconi , Formaldeído , Humanos , DNA , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Anemia de Fanconi/induzido quimicamente , Anemia de Fanconi/genética , Formaldeído/toxicidade , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Tolerância a Medicamentos/genéticaRESUMO
Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.
Assuntos
Reparo do DNA , Rad51 Recombinase , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas/genética , DNA/genética , MitoseRESUMO
Human acetyltransferases MOZ and MORF are implicated in chromosomal translocations associated with aggressive leukemias. Oncogenic translocations involve the far amino terminus of MOZ/MORF, the function of which remains unclear. Here, we identified and characterized two structured winged helix (WH) domains, WH1 and WH2, in MORF and MOZ. WHs bind DNA in a cooperative manner, with WH1 specifically recognizing unmethylated CpG sequences. Structural and genomic analyses show that the DNA binding function of WHs targets MORF/MOZ to gene promoters, stimulating transcription and H3K23 acetylation, and WH1 recruits oncogenic fusions to HOXA genes that trigger leukemogenesis. Cryo-EM, NMR, mass spectrometry and mutagenesis studies provide mechanistic insight into the DNA-binding mechanism, which includes the association of WH1 with the CpG-containing linker DNA and binding of WH2 to the dyad of the nucleosome. The discovery of WHs in MORF and MOZ and their DNA binding functions could open an avenue in developing therapeutics to treat diseases associated with aberrant MOZ/MORF acetyltransferase activities.
Assuntos
Acetiltransferases , Histona Acetiltransferases , Leucemia , Humanos , Acetilação , Acetiltransferases/metabolismo , Ilhas de CpG/genética , Histona Acetiltransferases/metabolismo , Leucemia/genética , Translocação GenéticaRESUMO
Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability.