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1.
Mol Cell ; 84(4): 659-674.e7, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38266640

RESUMO

Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors.


Assuntos
Proteína BRCA1 , Neoplasias , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Dano ao DNA , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Recombinação Homóloga
2.
Annu Rev Biochem ; 82: 55-80, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23414304

RESUMO

Genetic, biochemical, and cellular studies have uncovered many of the molecular mechanisms underlying the signaling and repair of chromosomal DNA breaks. However, efficient repair of DNA damage is complicated in that genomic DNA is packaged, through histone and nonhistone proteins, into chromatin. The DNA repair machinery has to overcome this physical barrier to gain access to damaged DNA and repair DNA lesions. Posttranslational modifications of chromatin as well as ATP-dependent chromatin remodeling factors help to overcome this barrier and facilitate access to damaged DNA by altering chromatin structure at sites of DNA damage. Here we review and discuss our current knowledge of and recent advances in chromatin changes induced by chromosome breakage in mammalian cells and their implications for genome stability and human disease.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Quebra Cromossômica , Reparo do DNA/genética , Histonas/genética , Transdução de Sinais/genética , Animais , Cromatina/metabolismo , Quebras de DNA , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos
3.
Mol Cell ; 75(3): 483-497.e9, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31253574

RESUMO

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.


Assuntos
Proteína BRCA1/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Enzimas Desubiquitinantes/genética , Chaperonas de Histonas/genética , Neoplasias/genética , Sítios de Ligação/genética , Proteínas de Transporte/genética , Núcleo Celular/genética , Núcleo Celular/imunologia , Citoplasma/genética , Citoplasma/imunologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/imunologia , Enzimas Desubiquitinantes/imunologia , Células HeLa , Humanos , Imunidade Celular/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Neoplasias/imunologia , Proteínas Associadas à Matriz Nuclear/genética , Ligação Proteica/genética , Ubiquitina/genética , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética
4.
Genes Dev ; 33(11-12): 684-704, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31048545

RESUMO

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
5.
Mol Cell Proteomics ; 23(8): 100802, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38880245

RESUMO

The ATR kinase protects cells against DNA damage and replication stress and represents a promising anti-cancer drug target. The ATR inhibitors (ATRi) berzosertib and gartisertib are both in clinical trials for the treatment of advanced solid tumors as monotherapy or in combination with genotoxic agents. We carried out quantitative phospho-proteomic screening for ATR biomarkers that are highly sensitive to berzosertib and gartisertib, using an optimized mass spectrometry pipeline. Screening identified a range of novel ATR-dependent phosphorylation events, which were grouped into three broad classes: (i) targets whose phosphorylation is highly sensitive to ATRi and which could be the next generation of ATR biomarkers; (ii) proteins with known genome maintenance roles not previously known to be regulated by ATR; (iii) novel targets whose cellular roles are unclear. Class iii targets represent candidate DNA damage response proteins and, with this in mind, proteins in this class were subjected to secondary screening for recruitment to DNA damage sites. We show that one of the proteins recruited, SCAF1, interacts with RNAPII in a phospho-dependent manner and recruitment requires PARP activity and interaction with RNAPII. We also show that SCAF1 deficiency partly rescues RAD51 loading in cells lacking the BRCA1 tumor suppressor. Taken together these data reveal potential new ATR biomarkers and new genome maintenance factors.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Dano ao DNA , Proteômica , Pirazinas , Humanos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteômica/métodos , Fosforilação , Pirazinas/farmacologia , Linhagem Celular Tumoral , Biomarcadores Tumorais/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fosfoproteínas/metabolismo , Sulfonas/farmacologia , Reparo do DNA/efeitos dos fármacos , Isoxazóis
6.
Nature ; 560(7716): 117-121, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30022168

RESUMO

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.


Assuntos
Reparo do DNA , Complexos Multiproteicos/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/genética , Feminino , Genes BRCA1 , Humanos , Switching de Imunoglobulina/genética , Camundongos , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/deficiência , Complexos Multiproteicos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/metabolismo , Proteína Supressora de Tumor p53/deficiência
7.
Mol Cell ; 61(4): 547-562, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26895424

RESUMO

The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.


Assuntos
Cromatina/genética , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Células HEK293 , Humanos , Poli(ADP-Ribose) Polimerase-1
8.
Mol Cell ; 63(3): 514-25, 2016 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-27453043

RESUMO

An emerging therapeutic strategy for cancer is to induce selective lethality in a tumor by exploiting interactions between its driving mutations and specific drug targets. Here we use a multi-species approach to develop a resource of synthetic lethal interactions relevant to cancer therapy. First, we screen in yeast ∼169,000 potential interactions among orthologs of human tumor suppressor genes (TSG) and genes encoding drug targets across multiple genotoxic environments. Guided by the strongest signal, we evaluate thousands of TSG-drug combinations in HeLa cells, resulting in networks of conserved synthetic lethal interactions. Analysis of these networks reveals that interaction stability across environments and shared gene function increase the likelihood of observing an interaction in human cancer cells. Using these rules, we prioritize ∼10(5) human TSG-drug combinations for future follow-up. We validate interactions based on cell and/or patient survival, including topoisomerases with RAD17 and checkpoint kinases with BLM.


Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Genes Supressores de Tumor , Mutação , Medicina de Precisão/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Células HeLa , Humanos , Estimativa de Kaplan-Meier , Terapia de Alvo Molecular , Fenótipo , Interferência de RNA , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Mutações Sintéticas Letais , Fatores de Tempo , Transfecção , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/mortalidade
9.
Semin Cell Dev Biol ; 113: 65-74, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32962915

RESUMO

Zinc-Finger (ZnF) proteins represent one of the most abundant group of proteins in the human genome. At first characterized as DNA binding proteins, it has become increasingly clear that ZnF-proteins have the ability to bind a large variety of substrates such as RNAs, proteins and post-translational modifications, suggesting potential roles in a variety of biological processes. Indeed, several studies have implicated ZnF-proteins for instance in transcription regulation, signal transduction and cell migration. Intriguingly, more recently these proteins have emerged as important protectors of the genome, particularly by orchestrating the repair of highly deleterious DNA double-strand breaks. Here we provide a comprehensive summary of the roles of ZnF domain-containing proteins in DNA double-strand break repair and discuss how their dysfunction impacts genome stability and human disease.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Instabilidade Genômica/genética , Dedos de Zinco/genética , Humanos
10.
J Cell Sci ; 134(3)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33408245

RESUMO

DNA damage-induced SUMOylation serves as a signal for two antagonizing proteins that both stimulate repair of DNA double-strand breaks (DSBs). Here, we demonstrate that the SUMO-dependent recruitment of the deubiquitylating enzyme ataxin-3 to DSBs, unlike recruitment of the ubiquitin ligase RNF4, additionally depends on poly [ADP-ribose] polymerase 1 (PARP1)-mediated poly(ADP-ribosyl)ation (PARylation). The co-dependence of ataxin-3 recruitment on PARylation and SUMOylation temporally confines ataxin-3 to DSBs immediately after occurrence of DNA damage. We propose that this mechanism ensures that ataxin-3 prevents the premature removal of DNA repair proteins only during the early phase of the DSB response and does not interfere with the subsequent timely displacement of DNA repair proteins by RNF4. Thus, our data show that PARylation differentially regulates SUMO-dependent recruitment of ataxin-3 and RNF4 to DSBs, explaining how both proteins can play a stimulatory role at DSBs despite their opposing activities.


Assuntos
Ataxina-3 , Quebras de DNA de Cadeia Dupla , Poli ADP Ribosilação , Ataxina-3/genética , Linhagem Celular Tumoral , DNA , Dano ao DNA , Reparo do DNA/genética , Humanos , Poli(ADP-Ribose) Polimerase-1/genética
11.
Mol Cell ; 57(2): 273-89, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25533186

RESUMO

Mec1-Ddc2 (ATR-ATRIP) controls the DNA damage checkpoint and shows differential cell-cycle regulation in yeast. To find regulators of Mec1-Ddc2, we exploited a mec1 mutant that retains catalytic activity in G2 and recruitment to stalled replication forks, but which is compromised for the intra-S phase checkpoint. Two screens, one for spontaneous survivors and an E-MAP screen for synthetic growth effects, identified loss of PP4 phosphatase, pph3Δ and psy2Δ, as the strongest suppressors of mec1-100 lethality on HU. Restored Rad53 phosphorylation accounts for part, but not all, of the pph3Δ-mediated survival. Phosphoproteomic analysis confirmed that 94% of the mec1-100-compromised targets on HU are PP4 regulated, including a phosphoacceptor site within Mec1 itself, mutation of which confers damage sensitivity. Physical interaction between Pph3 and Mec1, mediated by cofactors Psy2 and Ddc2, is shown biochemically and through FRET in subnuclear repair foci. This establishes a physical and functional Mec1-PP4 unit for regulating the checkpoint response.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Pontos de Checagem do Ciclo Celular , Quinase do Ponto de Checagem 2/metabolismo , Replicação do DNA , Epistasia Genética , Regulação Fúngica da Expressão Gênica , Células HEK293 , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/citologia , Transdução de Sinais
12.
J Med Genet ; 59(5): 481-491, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-33811135

RESUMO

BACKGROUND: Rare protein-truncating variants (PTVs) in partner and localiser of BRCA2 (PALB2) confer increased risk to breast cancer, but relatively few studies have reported the prevalence in South-East Asian populations. Here, we describe the prevalence of rare variants in PALB2 in a population-based study of 7840 breast cancer cases and 7928 healthy Chinese, Malay and Indian women from Malaysia and Singapore, and describe the functional impact of germline missense variants identified in this population. METHODS: Mutation testing was performed on germline DNA (n=15 768) using targeted sequencing panels. The functional impact of missense variants was tested in mouse embryonic stem cell based functional assays. RESULTS: PTVs in PALB2 were found in 0.73% of breast cancer patients and 0.14% of healthy individuals (OR=5.44; 95% CI 2.85 to 10.39, p<0.0001). In contrast, rare missense variants in PALB2 were not associated with increased risk of breast cancer. Whereas PTVs were associated with later stage of presentation and higher-grade tumours, no significant association was observed with missense variants in PALB2. However, two novel rare missense variants (p.L1027R and p.G1043V) produced unstable proteins and resulted in a decrease in homologous recombination-mediated repair of DNA double-strand breaks. CONCLUSION: Despite genetic and lifestyle differences between Asian and other populations, the population prevalence of PALB2 PTVs and associated relative risk of breast cancer, are similar to those reported in European populations.


Assuntos
Neoplasias da Mama , Predisposição Genética para Doença , Animais , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Feminino , Mutação em Linhagem Germinativa , Humanos , Malásia/epidemiologia , Masculino , Camundongos , Singapura/epidemiologia
13.
J Cell Sci ; 133(10)2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32299836

RESUMO

Eukaryotic chromosomes are replicated in interphase and the two newly duplicated sister chromatids are held together by the cohesin complex and several cohesin auxiliary factors. Sister chromatid cohesion is essential for accurate chromosome segregation during mitosis, yet has also been implicated in other processes, including DNA damage repair, transcription and DNA replication. To assess how cohesin and associated factors functionally interconnect and coordinate with other cellular processes, we systematically mapped the genetic interactions of 17 cohesin genes centered on quantitative growth measurements of >52,000 gene pairs in the budding yeast Saccharomyces cerevisiae Integration of synthetic genetic interactions unveiled a cohesin functional map that constitutes 373 genetic interactions, revealing novel functional connections with post-replication repair, microtubule organization and protein folding. Accordingly, we show that the microtubule-associated protein Irc15 and the prefoldin complex members Gim3, Gim4 and Yke2 are new factors involved in sister chromatid cohesion. Our genetic interaction map thus provides a unique resource for further identification and functional interrogation of cohesin proteins. Since mutations in cohesin proteins have been associated with cohesinopathies and cancer, it may also help in identifying cohesin interactions relevant in disease etiology.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Coesinas
14.
EMBO Rep ; 21(1): e48460, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31782600

RESUMO

The cellular response to DNA breaks is influenced by chromatin compaction. To identify chromatin regulators involved in the DNA damage response, we screened for genes that affect recovery following DNA damage using an RNAi library of chromatin regulators. We identified genes involved in chromatin remodeling, sister chromatid cohesion, and histone acetylation not previously associated with checkpoint recovery. Among these is the PHD finger protein 6 (PHF6), a gene mutated in Börjeson-Forssman-Lehmann syndrome and leukemic cancers. We find that loss of PHF6 dramatically compromises checkpoint recovery in G2 phase cells. Moreover, PHF6 is rapidly recruited to sites of DNA lesions in a PARP-dependent manner and required for efficient DNA repair through classical non-homologous end joining. These results indicate that PHF6 is a novel DNA damage response regulator that promotes end joining-mediated repair, thereby stimulating timely recovery from the G2 checkpoint.


Assuntos
Hipogonadismo , Deficiência Intelectual Ligada ao Cromossomo X , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades , Pontos de Checagem da Fase G2 do Ciclo Celular , Transtornos do Crescimento , Humanos
15.
Nucleic Acids Res ; 48(9): 4915-4927, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32232336

RESUMO

Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.


Assuntos
Quebras de DNA de Cadeia Dupla , Histona Desmetilases/fisiologia , Proteínas de Homeodomínio/fisiologia , Reparo de DNA por Recombinação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Instabilidade Genômica , Células HeLa , Histona Desmetilases/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos
16.
EMBO J ; 36(8): 1066-1083, 2017 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-28275011

RESUMO

The SUMO-targeted ubiquitin ligase RNF4 functions at the crossroads of the SUMO and ubiquitin systems. Here, we report that the deubiquitylation enzyme (DUB) ataxin-3 counteracts RNF4 activity during the DNA double-strand break (DSB) response. We find that ataxin-3 negatively regulates ubiquitylation of the checkpoint mediator MDC1, a known RNF4 substrate. Loss of ataxin-3 markedly decreases the chromatin dwell time of MDC1 at DSBs, which can be fully reversed by co-depletion of RNF4. Ataxin-3 is recruited to DSBs in a SUMOylation-dependent fashion, and in vitro it directly interacts with and is stimulated by recombinant SUMO, defining a SUMO-dependent mechanism for DUB activity toward MDC1. Loss of ataxin-3 results in reduced DNA damage-induced ubiquitylation due to impaired MDC1-dependent recruitment of the ubiquitin ligases RNF8 and RNF168, and reduced recruitment of 53BP1 and BRCA1. Finally, ataxin-3 is required for efficient MDC1-dependent DSB repair by non-homologous end-joining and homologous recombination. Consequently, loss of ataxin-3 sensitizes cells to ionizing radiation and poly(ADP-ribose) polymerase inhibitor. We propose that the opposing activities of RNF4 and ataxin-3 consolidate robust MDC1-dependent signaling and repair of DSBs.


Assuntos
Ataxina-3/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteína SUMO-1/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Ataxina-3/genética , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Raios gama , Células HEK293 , Humanos , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteína SUMO-1/genética , Transativadores/genética , Fatores de Transcrição/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
17.
Mol Cell ; 49(2): 346-58, 2013 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-23273983

RESUMO

To protect the genome, cells have evolved a diverse set of pathways designed to sense, signal, and repair multiple types of DNA damage. To assess the degree of coordination and crosstalk among these pathways, we systematically mapped changes in the cell's genetic network across a panel of different DNA-damaging agents, resulting in ~1,800,000 differential measurements. Each agent was associated with a distinct interaction pattern, which, unlike single-mutant phenotypes or gene expression data, has high statistical power to pinpoint the specific repair mechanisms at work. The agent-specific networks revealed roles for the histone acetyltranferase Rtt109 in the mutagenic bypass of DNA lesions and the neddylation machinery in cell-cycle regulation and genome stability, while the network induced by multiple agents implicates Irc21, an uncharacterized protein, in checkpoint control and DNA repair. Our multiconditional genetic interaction map provides a unique resource that identifies agent-specific and general DNA damage response pathways.


Assuntos
Dano ao DNA , Epistasia Genética , Saccharomyces cerevisiae/genética , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Genoma Fúngico , Instabilidade Genômica , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
J Biol Chem ; 294(52): 20122-20134, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31699900

RESUMO

Histone post-translational modifications (PTMs) are critical for processes such as transcription. The more notable among these are the nonacetyl histone lysine acylation modifications such as crotonylation, butyrylation, and succinylation. However, the biological relevance of these PTMs is not fully understood because their regulation is largely unknown. Here, we set out to investigate whether the main histone acetyltransferases in budding yeast, Gcn5 and Esa1, possess crotonyltransferase activity. In vitro studies revealed that the Gcn5-Ada2-Ada3 (ADA) and Esa1-Yng2-Epl1 (Piccolo NuA4) histone acetyltransferase complexes have the capacity to crotonylate histones. Mass spectrometry analysis revealed that ADA and Piccolo NuA4 crotonylate lysines in the N-terminal tails of histone H3 and H4, respectively. Functionally, we show that crotonylation selectively affects gene transcription in vivo in a manner dependent on Gcn5 and Esa1. Thus, we identify the Gcn5- and Esa1-containing ADA and Piccolo NuA4 complexes as bona fide crotonyltransferases that promote crotonylation-dependent transcription.


Assuntos
Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Crotonatos/química , Histona Acetiltransferases/genética , Histonas/química , Lisina/química , Lisina/metabolismo , Espectrometria de Massas , Peptídeos/análise , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
19.
EMBO J ; 35(1): 6-23, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26628622

RESUMO

A timely and accurate cellular response to DNA damage requires tight regulation of the action of DNA damage response (DDR) proteins at lesions. A multitude of posttranslational modifications (PTMs) of chromatin and chromatin-associated proteins coordinates the recruitment of critical proteins that dictate the appropriate DNA repair pathway and enable the actual repair of lesions. Phosphorylation, ubiquitylation, SUMOylation, neddylation, poly(ADP-ribosyl)ation, acetylation, and methylation are among the DNA damage-induced PTMs that have taken center stage as important DDR regulators. Redundant and multivalent interactions of DDR proteins with PTMs may not only be a means to facilitate efficient relocalization, but also a feature that allows high temporal and spatial resolution of protein recruitment to, and extraction from, DNA damage sites. In this review, we will focus on the complex interplay between such PTMs, and discuss the importance of their interconnectivity in coding DNA lesions and maintaining the integrity of the genome.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Reparo do DNA , Regulação da Expressão Gênica , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Humanos , Análise Espaço-Temporal
20.
Nucleic Acids Res ; 46(21): 11251-11261, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30203048

RESUMO

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitinação , Cromatina/genética , Cromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares/genética , Ligação Proteica , Mapas de Interação de Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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