RESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteína Quinasa Activada por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Cromatina/genética , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Roturas del ADN de Doble Cadena , Inestabilidad Genómica , Células HEK293 , Humanos , Poli(ADP-Ribosa) Polimerasa-1RESUMEN
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.
Asunto(s)
Hipogonadismo , Discapacidad Intelectual Ligada al Cromosoma X , Proteínas Represoras/genética , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , Puntos de Control de la Fase G2 del Ciclo Celular , Trastornos del Crecimiento , HumanosRESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Histona Demetilasas/fisiología , Proteínas de Homeodominio/fisiología , Reparación del ADN por Recombinación , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Línea Celular , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Regulación de la Expresión Génica , Inestabilidad Genómica , Células HeLa , Histona Demetilasas/metabolismo , Proteínas de Homeodominio/metabolismo , HumanosRESUMEN
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.
Asunto(s)
Ataxina-3/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Proteína SUMO-1/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Ataxina-3/genética , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Rayos gamma , Células HEK293 , Humanos , Proteínas Nucleares/genética , Proteínas Represoras/genética , Proteína SUMO-1/genética , Transactivadores/genética , Factores de Transcripción/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5'-3' degradation of DSB ends that yields the 3' single-stranded DNA required for the loading of checkpoint and recombination proteins. In yeast, the Mre11-Rad50-Xrs2 complex (Xrs2 is known as NBN or NBS1 in humans) and Sae2 (known as RBBP8 or CTIP in humans) initiate end resection, whereas long-range resection depends on the exonuclease Exo1, or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 together with the endonuclease Dna2 (refs 1-6). DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood. Here we show that the yeast Saccharomyces cerevisiae Fun30 protein and its human counterpart SMARCAD1 (ref. 8), two poorly characterized ATP-dependent chromatin remodellers of the Snf2 ATPase family, are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates the repair of camptothecin-induced DNA lesions, although it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs, and the kinetics of recruitment is similar to that of EXO1. The loss of SMARCAD1 impairs end resection and recombinational DNA repair, and renders cells hypersensitive to DNA damage resulting from camptothecin or poly(ADP-ribose) polymerase inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodellers in controlling end resection, homologous recombination and genome stability in the context of chromatin.
Asunto(s)
Ensamble y Desensamble de Cromatina , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Reparación del ADN , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Camptotecina/farmacología , Línea Celular , Supervivencia Celular , ADN/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , ADN Helicasas/deficiencia , ADN Helicasas/genética , Reparación del ADN/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Inestabilidad Genómica/genética , Histonas/metabolismo , Recombinación Homóloga/genética , Humanos , Mutación , Nucleosomas/genética , Nucleosomas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , RecQ Helicasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
The faithful repair of DNA double-strand breaks (DSBs) is essential to safeguard genome stability. DSBs elicit a signaling cascade involving the E3 ubiquitin ligases RNF8/RNF168 and the ubiquitin-dependent assembly of the BRCA1-Abraxas-RAP80-MERIT40 complex. The association of BRCA1 with ubiquitin conjugates through RAP80 is known to be inhibitory to DSB repair by homologous recombination (HR). However, the precise regulation of this mechanism remains poorly understood. Through genetic screens we identified USP26 and USP37 as key de-ubiquitylating enzymes (DUBs) that limit the repressive impact of RNF8/RNF168 on HR. Both DUBs are recruited to DSBs where they actively remove RNF168-induced ubiquitin conjugates. Depletion of USP26 or USP37 disrupts the execution of HR and this effect is alleviated by the simultaneous depletion of RAP80. We demonstrate that USP26 and USP37 prevent excessive spreading of RAP80-BRCA1 from DSBs. On the other hand, we also found that USP26 and USP37 promote the efficient association of BRCA1 with PALB2. This suggests that these DUBs limit the ubiquitin-dependent sequestration of BRCA1 via the BRCA1-Abraxas-RAP80-MERIT40 complex, while promoting complex formation and cooperation of BRCA1 with PALB2-BRCA2-RAD51 during HR. These findings reveal a novel ubiquitin-dependent mechanism that regulates distinct BRCA1-containing complexes for efficient repair of DSBs by HR.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Reparación del ADN por Recombinación , Proteína BRCA1/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Chaperonas de Histonas , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Recombinasa Rad51/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
XRCC2 genetic variants have been associated with breast cancer susceptibility. However, association studies have been complicated because XRCC2 variants are extremely rare and consist mainly of amino acid substitutions whose grouping is sensitive to misclassification by the predictive algorithms. We therefore functionally characterized variants in XRCC2 by testing their ability to restore XRCC2-DNA repair deficient phenotypes using a cDNA-based complementation approach. While the protein-truncating variants p.Leu117fs, p.Arg215*, and p.Cys217* were unable to restore XRCC2 deficiency, 19 out of 23 missense variants showed no or just a minor (<25%) reduction in XRCC2 function. The remaining four (p.Cys120Tyr, p.Arg91Trp, p.Leu133Pro, and p.Ile95Leu) had a moderate effect. Overall, measured functional effects correlated poorly with those predicted by in silico analysis. After regrouping variants from published case-control studies based on the functional effect found in this study and reanalysis of the prevalence data, there was no longer evidence for an association with breast cancer. This suggests that if breast cancer susceptibility alleles of XRCC2 exist, they are likely restricted to protein-truncating variants and a minority of missense changes. Our study emphasizes the use of functional analyses of missense variants to support variant classification in association studies.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Mutación Missense , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Reparación del ADN , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Células HEK293 , HumanosRESUMEN
The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks. Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Autoantígenos/metabolismo , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Cricetinae , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of γH2AX into damaged chromatin, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA damage. In support of this, we show that SMARCA5 and RNF168 interact in a DNA damage- and PARP-dependent manner. RNF168 became poly(ADP-ribosyl)ated after DNA damage, while RNF168 and poly(ADP-ribose) chains were required for SMARCA5 binding in vivo, explaining how SMARCA5 is linked to the RNF168 ubiquitin cascade. Moreover, SMARCA5 was found to regulate the ubiquitin response by promoting RNF168 accumulation at DSBs, which subsequently facilitates efficient ubiquitin conjugation and BRCA1 assembly. Underlining the importance of these findings, we show that SMARCA5 depletion renders cells sensitive to IR and results in DSB repair defects. Our study unveils a functional link between DNA damage-induced poly(ADP-ribosyl)ation, SMARCA5-mediated chromatin remodeling and RNF168-dependent signaling and repair of DSBs.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfatasas/genética , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Daño del ADN/genética , Reparación del ADN/genética , Reparación del ADN/fisiología , Células HeLa , Humanos , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The protein predicted to be defective in individuals with Fanconi anemia complementation group J (FA-J), FANCJ, is a missing component in the Fanconi anemia pathway of genome maintenance. Here we identify pathogenic mutations in eight individuals with FA-J in the gene encoding the DEAH-box DNA helicase BRIP1, also called FANCJ. This finding is compelling evidence that the Fanconi anemia pathway functions through a direct physical interaction with DNA.
Asunto(s)
Cromosomas Humanos Par 17 , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Anemia de Fanconi/genética , Mutación/genética , ARN Helicasas/deficiencia , ARN Helicasas/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Prueba de Complementación Genética , Humanos , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Eliminación de SecuenciaRESUMEN
During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Proteína del Grupo de Complementación L de la Anemia de Fanconi , Recombinación Homóloga , Enzimas Ubiquitina-Conjugadoras , Humanos , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación L de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación L de la Anemia de Fanconi/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Sistemas CRISPR-Cas , Ubiquitinación , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Células HEK293 , Reparación del ADN por Recombinación , Reparación del ADN , Reparación del ADN por Unión de Extremidades , ADN HelicasasRESUMEN
Radiosensitive T-B- severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-PKcs missense mutation (L3062R). The causative mutation did not affect the kinase activity or DNA end-binding capacity of DNA-PKcs itself; rather, the presence of long P-nucleotide stretches in the immunoglobulin coding joints indicated that it caused insufficient Artemis activation, something that is dependent on Artemis interaction with autophosphorylated DNA-PKcs. Moreover, overall end-joining activity was hampered, suggesting that Artemis-independent DNA-PKcs functions were also inhibited. This study demonstrates that the presence of DNA-PKcs kinase activity is not sufficient to rule out a defect in this gene during diagnosis and treatment of RS-SCID patients. Further, the data suggest that residual DNA-PKcs activity is indispensable in humans.
Asunto(s)
Proteína Quinasa Activada por ADN/genética , Mutación Missense , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Tolerancia a Radiación , Recombinación Genética , Inmunodeficiencia Combinada Grave/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Preescolar , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Ligasas/metabolismo , Análisis Mutacional de ADN , Reparación del ADN , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN , Endonucleasas , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Fibroblastos/efectos de la radiación , Genotipo , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Linaje , Alineación de Secuencia , Inmunodeficiencia Combinada Grave/diagnósticoRESUMEN
Heterozygous carriers of germline loss-of-function variants in the tumor suppressor gene checkpoint kinase 2 (CHEK2) are at an increased risk for developing breast and other cancers. While truncating variants in CHEK2 are known to be pathogenic, the interpretation of missense variants of uncertain significance (VUS) is challenging. Consequently, many VUS remain unclassified both functionally and clinically. Here we describe a mouse embryonic stem (mES) cell-based system to quantitatively determine the functional impact of 50 missense VUS in human CHEK2. By assessing the activity of human CHK2 to phosphorylate one of its main targets, Kap1, in Chek2 knockout mES cells, 31 missense VUS in CHEK2 were found to impair protein function to a similar extent as truncating variants, while 9 CHEK2 missense VUS resulted in intermediate functional defects. Mechanistically, most VUS impaired CHK2 kinase function by causing protein instability or by impairing activation through (auto)phosphorylation. Quantitative results showed that the degree of CHK2 kinase dysfunction correlates with an increased risk for breast cancer. Both damaging CHEK2 variants as a group [OR 2.23; 95% confidence interval (CI), 1.62-3.07; P < 0.0001] and intermediate variants (OR 1.63; 95% CI, 1.21-2.20; P = 0.0014) were associated with an increased breast cancer risk, while functional variants did not show this association (OR 1.13; 95% CI, 0.87-1.46; P = 0.378). Finally, a damaging VUS in CHEK2, c.486A>G/p.D162G, was also identified, which cosegregated with familial prostate cancer. Altogether, these functional assays efficiently and reliably identified VUS in CHEK2 that associate with cancer. SIGNIFICANCE: Quantitative assessment of the functional consequences of CHEK2 variants of uncertain significance identifies damaging variants associated with increased cancer risk, which may aid in the clinical management of patients and carriers.
Asunto(s)
Quinasa de Punto de Control 2/genética , Predisposición Genética a la Enfermedad/genética , Mutación Missense , Neoplasias/genética , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Células Cultivadas , Quinasa de Punto de Control 2/metabolismo , Femenino , Humanos , Masculino , Ratones de la Cepa 129 , Ratones Noqueados , Neoplasias/enzimología , Linaje , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Factores de RiesgoRESUMEN
DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a 'Ku-adaptor' that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance.
Asunto(s)
Roturas del ADN de Doble Cadena , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , ADN/metabolismo , Humanos , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction.
Asunto(s)
Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Riñón/patología , Riñón/fisiopatología , Mutación/genética , Alelos , Sustitución de Aminoácidos , Secuencia de Bases , Línea Celular , Citoplasma/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Endonucleasas/deficiencia , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Luz , Hígado/patología , Hígado/fisiopatología , Proteínas Mutantes/metabolismo , Mutación Missense/genética , Estabilidad Proteica , HermanosRESUMEN
Chromatin structure is dynamically reorganized at multiple levels in response to DNA double-strand breaks (DSBs). Yet, how the different steps of chromatin reorganization are coordinated in space and time to differentially regulate DNA repair pathways is insufficiently understood. Here, we identify the Chromodomain Helicase DNA Binding Protein 7 (CHD7), which is frequently mutated in CHARGE syndrome, as an integral component of the non-homologous end-joining (NHEJ) DSB repair pathway. Upon recruitment via PARP1-triggered chromatin remodeling, CHD7 stimulates further chromatin relaxation around DNA break sites and brings in HDAC1/2 for localized chromatin de-acetylation. This counteracts the CHD7-induced chromatin expansion, thereby ensuring temporally and spatially controlled 'chromatin breathing' upon DNA damage, which we demonstrate fosters efficient and accurate DSB repair by controlling Ku and LIG4/XRCC4 activities. Loss of CHD7-HDAC1/2-dependent cNHEJ reinforces 53BP1 assembly at the damaged chromatin and shifts DSB repair to mutagenic NHEJ, revealing a backup function of 53BP1 when cNHEJ fails.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP)/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Histona Desacetilasa 1/metabolismo , Humanos , Autoantígeno Ku/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
V(D)J recombination of Ig and TCR loci is a stepwise process during which site-specific DNA double-strand breaks (DSBs) are made by RAG1/RAG2, followed by DSB repair by nonhomologous end joining. Defects in V(D)J recombination result in SCID characterized by absence of mature B and T cells. A subset of T-B-NK+ SCID patients is sensitive to ionizing radiation, and the majority of these patients have mutations in Artemis. We present a patient with a new type of radiosensitive T-B-NK+ SCID with a defect in DNA ligase IV (LIG4). To date, LIG4 mutations have only been described in a radiosensitive leukemia patient and in 4 patients with a designated LIG4 syndrome, which is associated with chromosomal instability, pancytopenia, and developmental and growth delay. The patient described here shows that a LIG4 mutation can also cause T-B-NK+ SCID without developmental defects. The LIG4-deficient SCID patient had an incomplete but severe block in precursor B cell differentiation, resulting in extremely low levels of blood B cells. The residual D(H)-J(H) junctions showed extensive nucleotide deletions, apparently caused by prolonged exonuclease activity during the delayed D(H)-J(H) ligation process. In conclusion, different LIG4 mutations can result in either a developmental defect with minor immunological abnormalities or a SCID picture with normal development.
Asunto(s)
Linfocitos B/inmunología , ADN Ligasas/genética , Células Asesinas Naturales/inmunología , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Linfocitos T/inmunología , Animales , ADN Ligasa (ATP) , Humanos , Ratones , Ratones SCID , Mutación , Valores de ReferenciaRESUMEN
Heterozygous carriers of germ-line loss-of-function variants in the DNA repair gene PALB2 are at a highly increased lifetime risk for developing breast cancer. While truncating variants in PALB2 are known to increase cancer risk, the interpretation of missense variants of uncertain significance (VUS) is in its infancy. Here we describe the development of a relatively fast and easy cDNA-based system for the semi high-throughput functional analysis of 48 VUS in human PALB2. By assessing the ability of PALB2 VUS to rescue the DNA repair and checkpoint defects in Palb2 knockout mouse embryonic stem (mES) cells, we identify various VUS in PALB2 that impair its function. Three VUS in the coiled-coil domain of PALB2 abrogate the interaction with BRCA1, whereas several VUS in the WD40 domain dramatically reduce protein stability. Thus, our functional assays identify damaging VUS in PALB2 that may increase cancer risk.