RESUMEN
Xeroderma pigmentosum (XP) is a genodermatosis defined by cutaneous photosensitivity with an increased risk of skin tumors because of DNA repair deficiency. The worldwide prevalence of XP is ~1 to 4 in million, with higher incidence in some countries and regions including Japan (1 in 22,000) and North Africa due to founder mutations and a high degree of consanguinity. Among XP, the complementation group F (XP-F), is a rare form (1% of worldwide XP); however, this is underdiagnosed, because the ERCC4/XPF gene is essential for fetal development and most of previously reported ERCC4/XPF pathogenic variants are hypomorphs causing relatively mild phenotypes. From the largest Japanese XP cohort study, we report 17 XP-F cases bearing two pathogenic variants, both identified in deep intronic regions of the ERCC4/XPF gene. The first variant, located in intron 1, is a Japanese founder mutation, which additionally accounts for ~10% of the entire Japanese XP cases (MAF = 0.00196), causing an aberrant pre-mRNA splicing due to a miss-binding of U1snRNA. The second mutation located in intron eight induces an alternative polyadenylation. Both mutations cause a reduction of the ERCC4/XPF gene expression, resulting in XP clinical manifestations. Most cases developed early-onset skin cancers, indicating that these variants need critical attention. We further demonstrate that antisense oligonucleotides designed for the mutations can restore the XPF protein expression and DNA repair capacity in the patients' cells. Collectively, these pathogenic variants can be potential therapeutic targets for XP.
Asunto(s)
Dermatitis , Xerodermia Pigmentosa , Humanos , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/terapia , Xerodermia Pigmentosa/metabolismo , Reparación del ADN/genética , Intrones/genética , Estudios de Cohortes , Mutación , Dermatitis/genéticaRESUMEN
BACKGROUND: It is unclear whether hip and pelvic mobility in the sagittal plane are associated with hip function in FAIS. This study aimed to determine whether hip-pelvis-lumbar mobility is associated with preoperative hip function and postoperative outcomes in FAIS. METHODS: This was a level 3 case-control study. This study included 111 patients who underwent arthroscopic FAI correction and labral preservation between 2015 and 2019. The Hip-Pelvic-Lumbar Mobility Test (HPLMT) was performed preoperatively; hip flexion with the hip adducted and internally rotated was examined in the lateral decubitus position, and a total hip flexion angle of less than 120° was diagnosed as positive. HPLMT-positive patients were classified as cases, and HPLMT-negative patients were classified as controls. Hip muscle strength was measured preoperatively using a hand-held dynamometer. The modified Harris hip score (mHHS), Nonarthritic Hip Score (NAHS), and International Hip Outcome Tool-12 score were obtained preoperatively and postoperatively. The rates of patient acceptable symptomatic state (PASS) achievement for patient-reported outcome scores (PROSs) were compared between groups. RESULTS: The preoperative PROSs for the HPLMT-positive patients were significantly lower than those for the HPLMT-negative patients. HPLMT-positive patients had significantly weaker hip strength on the affected side than HPLMT-negative patients (flexion; p < 0.001, abduction; p = 0.001). HPLMT-positive patients had significantly lower postoperative mHHS than HPLMT-negative patients. HPLMT-positive patients were significantly less likely to achieve a PASS for the mHHS (62% versus 85%) and NAHS (48% versus 71%) than HPLMT-negative patients. CONCLUSION: Hip-pelvis-lumbar mobility is associated with hip function and clinical outcomes in FAIS patients. The HPLMT is an efficient tool for assessing FAIS patients.
Asunto(s)
Pinzamiento Femoroacetabular , Humanos , Pinzamiento Femoroacetabular/cirugía , Estudios de Casos y Controles , Articulación de la Cadera/diagnóstico por imagen , Articulación de la Cadera/cirugía , Resultado del Tratamiento , Artroscopía , Actividades Cotidianas , Pelvis , Fuerza Muscular , Estudios de Seguimiento , Estudios RetrospectivosRESUMEN
PURPOSE: The purpose of this study was to assess the additional effect of anterior inferior iliac spine (AIIS) decompression on knee extensor and hip flexor strength and compare functional outcomes after arthroscopic FAI correction with and without AIIS decompression. METHODS: Sixty patients who underwent arthroscopic FAI correction surgery were divided into two groups matched for AIIS morphology: 31 patients who underwent arthroscopic FAI surgery only (without AIIS decompression) (FAI group) (AIIS Type I; n = 5, Type II; n = 26, Type III; n = 0) and 29 patients who underwent arthroscopic FAI surgery with AIIS decompression (AIIS group) (AIIS Type I; n = 5, Type II; n = 24, Type III; n = 0). Knee extensor and hip flexor strength were evaluated preoperatively and at 6 months after surgery. Patient-reported outcome (PRO) scores using the modified Harris hip score (MHHS), the nonarthritic hip score (NAHS) and iHOT-12 were obtained preoperatively and at 6 months after surgery. RESULTS: In the AIIS group, there was no significant difference between knee extensor strength pre- and postoperatively (n.s.). In the AIIS group, hip flexor strength was significantly improved postoperatively compared to preoperative measures (p < 0.05). In the FAI group, there were no significant improvements regarding muscle strength (n.s.). While there were no significant differences of preoperative and postoperative MHHS and NAHS between both groups (MHHS; n.s., NAHS; n.s.), the mean postoperative iHOT-12 in the FAI group was inferior to that in the AIIS group. (p < 0.01). The revision surgery rate for the AIIS group was significantly lower compared with that in the FAI group (p < 0.05). CONCLUSION: Anterior inferior iliac spine decompression, as a part of an arthroscopic FAI corrective procedure, had a lower revision surgery rate and did not compromise knee extensor and hip flexor strength, and it improved clinical outcomes comparable to FAI correction without AIIS decompression. AIIS decompression for FAI correction improved postoperative PRO scores without altering the muscle strength of hip flexor and knee extensor. LEVEL OF EVIDENCE: III.
Asunto(s)
Artroscopía/métodos , Descompresión Quirúrgica/métodos , Pinzamiento Femoroacetabular/cirugía , Ilion/anatomía & histología , Fuerza Muscular , Adolescente , Adulto , Variación Anatómica , Artroscopía/rehabilitación , Descompresión Quirúrgica/rehabilitación , Femenino , Pinzamiento Femoroacetabular/etiología , Articulación de la Cadera/cirugía , Humanos , Ilion/diagnóstico por imagen , Articulación de la Rodilla , Masculino , Persona de Mediana Edad , Dinamómetro de Fuerza Muscular , Medición de Resultados Informados por el Paciente , Periodo Posoperatorio , Reoperación/estadística & datos numéricos , Resultado del Tratamiento , Adulto JovenRESUMEN
Replication fork maintenance pathways preserve chromosomes, but their faulty application at nonallelic repeats could generate rearrangements causing cancer, genomic disorders and speciation. Potential causal mechanisms are homologous recombination and error-free postreplication repair (EF-PRR). Homologous recombination repairs damage-induced DNA double-strand breaks (DSBs) and single-ended DSBs within replication. To facilitate homologous recombination, the recombinase RAD51 and mediator BRCA2 form a filament on the 3' DNA strand at a break to enable annealing to the complementary sister chromatid while the RecQ helicase, BLM (Bloom syndrome mutated) suppresses crossing over to prevent recombination. Homologous recombination also stabilizes and restarts replication forks without a DSB. EF-PRR bypasses DNA incongruities that impede replication by ubiquitinating PCNA (proliferating cell nuclear antigen) using the RAD6-RAD18 and UBC13-MMS2-RAD5 ubiquitin ligase complexes. Some components are common to both homologous recombination and EF-PRR such as RAD51 and RAD18. Here we delineate two pathways that spontaneously fuse inverted repeats to generate unstable chromosomal rearrangements in wild-type mouse embryonic stem (ES) cells. Gamma-radiation induced a BLM-regulated pathway that selectively fused identical, but not mismatched, repeats. By contrast, ultraviolet light induced a RAD18-dependent pathway that efficiently fused mismatched repeats. Furthermore, TREX2 (a 3'â5' exonuclease) suppressed identical repeat fusion but enhanced mismatched repeat fusion, clearly separating these pathways. TREX2 associated with UBC13 and enhanced PCNA ubiquitination in response to ultraviolet light, consistent with it being a novel member of EF-PRR. RAD18 and TREX2 also suppressed replication fork stalling in response to nucleotide depletion. Interestingly, replication fork stalling induced fusion for identical and mismatched repeats, implicating faulty replication as a causal mechanism for both pathways.
Asunto(s)
Inestabilidad Cromosómica/genética , Cromosomas de los Mamíferos/genética , Reparación del ADN/genética , Replicación del ADN/genética , Recombinación Homóloga/genética , Secuencias Invertidas Repetidas/genética , Animales , Secuencia de Bases , Rotura Cromosómica , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Exodesoxirribonucleasas/metabolismo , Hidroxiurea/farmacología , Ratones , Nucleótidos/deficiencia , Nucleótidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Recombinasa Rad51/metabolismo , RecQ Helicasas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/efectos de la radiación , Rayos UltravioletaRESUMEN
Translesion DNA synthesis, a process orchestrated by monoubiquitinated PCNA, is critical for DNA damage tolerance. While the ubiquitin-conjugating enzyme RAD6 and ubiquitin ligase RAD18 are known to monoubiquitinate PCNA, how they are regulated by DNA damage is not fully understood. We show that NBS1 (mutated in Nijmegen breakage syndrome) binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Polη focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting domain of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting domain of RAD6. These domains of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously and are crucial for Polη-dependent UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in translesion DNA synthesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Células Cultivadas , Reparación del ADN , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Humanos , Ratones , Ratones Noqueados , Mutación , Proteínas Nucleares/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitinación , Rayos UltravioletaRESUMEN
BACKGROUNDS: The International Hip Outcome Tool 12 (iHOT12) was authorized by the Multicenter Arthroscopy of the Hip Outcomes Research Network (MAHORN). iHOT12 is increasingly being adopted in orthopedic studies to report patient outcomes. This study aimed to develop a Japanese version of the International Hip Outcome Tools "iHOT12J", and to establish its reliability, validity, and responsiveness. METHODS: To assess test-retest reliability, an identical set of patients reported outcome measures with five qualitative scoring measures including iHOT12; these were filled out by each patient twice. Reliability was explored using Cronbachss alpha and intraclass correlation coefficient. The Bland-Altman plot was used to explore the absolute agreement. To evaluate validity, we examined the relationships between SF36 and iHOT12. Responsiveness was assessed by comparing the smallest detectable change to the minimal important change by applying an anchor-based approach. RESULTS: Fifty patients (51 joints) were analyzed from March 2016 to October 2017 in Japanese four facility. The Cronbach α coefficient was 0.90 and the average value of intraclass coefficient (ICC) was 0.89. Bland-Altman plot analysis showed a solid agreement. Regarding the validity, the Spearman rank correlation coefficients were strong with PF (r = 0.69, p < 0.01), BP (r = 0.71, p < 0.01) and PCS (r = 0.69, p < 0.01). The smallest detectable change (3.19) was smaller than the minimum important change (12.40). CONCLUSIONS: We developed iHOT12J, which seems to show sufficient reliability, validity, and responsiveness. We believe that this patient reported outcome measure is beneficial in studying Japanese patients with femoroacetabular impingement.
Asunto(s)
Artroscopía , Pinzamiento Femoroacetabular/cirugía , Medición de Resultados Informados por el Paciente , Adolescente , Adulto , Femenino , Pinzamiento Femoroacetabular/fisiopatología , Humanos , Japón , Masculino , Persona de Mediana Edad , Variaciones Dependientes del Observador , Calidad de Vida , Rango del Movimiento Articular , Recuperación de la Función , Reproducibilidad de los Resultados , Encuestas y Cuestionarios , Traducciones , Adulto JovenRESUMEN
BACKGROUND: This study aimed to develop a Japanese version of the international PROMs "Vail Hip Score (Vail10)" and to establish its reliability, validity, and responsiveness with COSMIN check-list. METHODS: The study was conducted from March 2016 to October 2017 and included 46 patients totaling 47 joints. Disorders included 30 cases of FAI (55%), 13 cases of DDH (28%), and 4 others (8%). We administered an identical set of PROMs (5 measures: Japanese-version iHOT12 (pilot draft), Japanese-version Vail10, Japanese-version Oxford Hip Score, JHEQ, and SF36) twice in these subjects. We determined interclass correlation coefficients for the first and second round [ICC(1,2)], as well as the Cronbach α coefficient for patient responses to each of the 10 items in Vail10. In addition, we determined Spearman rank correlation coefficients of Vail10, OHS, JHEQ, satisfaction VAS, the 8 subscales of SF36, and the 3 QOL summary scores. RESULTS: ICC for the total score of all 10 items in Vail10 was 0.96. Cronbach α coefficient was 0.96. Bland-Altman plot analysis showed a solid agreement. Regarding the validity, Spearman rank correlation coefficients, only satisfaction VAS, and SF36 subscales of PF and BP had r > 0.45 (p < 0.01 in both administration rounds). The SDC (1.32) was smaller than the MIC (8.14). CONCLUSIONS: After developing the Japanese version of Vail10, we examined its Reliability, validity, and responsiveness by administering the measure to patients with acetabular labral tear. Correlations were strong and demonstrated the efficacy of the Japanese version of Vail10.
Asunto(s)
Lista de Verificación , Lesiones de la Cadera/fisiopatología , Articulación de la Cadera/fisiopatología , Medición de Resultados Informados por el Paciente , Rango del Movimiento Articular/fisiología , Traumatismos de los Tendones/fisiopatología , Adolescente , Adulto , Artroscopía , Femenino , Lesiones de la Cadera/cirugía , Articulación de la Cadera/cirugía , Humanos , Japón , Lenguaje , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Traumatismos de los Tendones/cirugía , Traducciones , Adulto JovenRESUMEN
In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18(-/-) mice. Moreover, primary Rad18(-/-) mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18(-/-) HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18(-/-) mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/fisiología , Células Madre Hematopoyéticas/fisiología , Animales , Células Cultivadas , Reparación del ADN , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Hematopoyesis , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Mutágenos/farmacologíaRESUMEN
Medical staffs like radiological technologists, doctors, and nurses are at an increased risk of exposure to radiation while assisting the patient in a position or monitor contrast medium injection during computed tomography (CT). However, methods to protect medical staff from radiation exposure and protocols for using radiological protection equipment have not been standardized and differ among hospitals. In this study, the distribution of scattered X-rays in a CT room was measured by placing electronic personal dosimeters in locations where medical staff stands beside the CT scanner gantry while assisting the patient and the exposure dose was measured. Moreover, we evaluated non-uniform exposure and revealed effective techniques to reduce the exposure dose to medical staff during CT. The dose of the scattered X-rays was the lowest at the gantry and at the examination table during both head and abdominal CT. The dose was the highest at the trunk of the upper body of the operator corresponding to a height of 130 cm during head CT and at the head corresponding to a height of 150 cm during abdominal CT. The maximum dose to the crystalline lens was approximately 600 µSv during head CT. We found that the use of volumetric CT scanning and X-ray protective goggles, and face direction toward the gantry reduced the exposure dose, particularly to the crystalline lens, for which lower equivalent dose during CT scan has been recently recommended in the International Commission on Radiological Protection Publication 118.
Asunto(s)
Cuerpo Médico , Exposición a la Radiación , Protección Radiológica/métodos , Tomógrafos Computarizados por Rayos X , Humanos , Protección Radiológica/instrumentaciónRESUMEN
Trans-lesion DNA synthesis (TLS) is a DNA damage-tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome xeroderma pigmentosum variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates proliferating cell nuclear antigen (PCNA), but the basis for recruitment of Rad18 to PCNA is not completely understood. Here, we show that Rad18 is targeted to PCNA by DNA polymerase eta (Polη), the XPV gene product that is mutated in XPV patients. The C-terminal domain of Polη binds to both Rad18 and PCNA and promotes PCNA monoubiquitination, a function unique to Polη among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length catalytically-inactive Polη exhibit increased recruitment of other error-prone TLS polymerases (Polκ and Polι) after UV irradiation. These results define a novel non-catalytic role for Polη in promoting PCNA monoubiquitination and provide a new potential mechanism for mutagenesis and genome instability in XPV individuals.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/fisiología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Secuencia de Consenso , Daño del ADN , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Inducción Enzimática , Inestabilidad Genómica , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas , Xerodermia Pigmentosa/enzimología , Xerodermia Pigmentosa/genéticaRESUMEN
Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.
Asunto(s)
Reparación del ADN , Replicación del ADN , ADN/biosíntesis , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitinación , Animales , Cisplatino/farmacología , ADN/efectos de los fármacos , ADN/genética , Daño del ADN , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/genética , Ratones , Mutagénesis , Antígeno Nuclear de Célula en Proliferación/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Rayos UltravioletaRESUMEN
The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G â T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1(-/-) MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk(-/-) Polh(-/-) Poli(-/-) triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Mutagénesis , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/análogos & derivados , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Aductos de ADN/genética , Daño del ADN , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/genética , Humanos , Proteínas Mad2 , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutagénesis/efectos de los fármacos , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismo , Alineación de SecuenciaRESUMEN
Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.
Asunto(s)
Aductos de ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Anemia de Fanconi/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Sustitución de Aminoácidos , Línea Celular , Aductos de ADN/genética , Proteínas de Unión al ADN/genética , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Humanos , Mutación Missense , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Recruitment of RAD18 to stalled replication forks facilitates monoubiquitination of PCNA during S-phase, promoting translesion synthesis at sites of UV irradiation-induced DNA damage. In this study, we show that RAD18 is also recruited to ionizing radiation (IR)-induced sites of DNA double-strand breaks (DSBs) forming foci which are co-localized with 53BP1, NBS1, phosphorylated ATM, BRCA1 and gamma-H2AX. RAD18 associates with 53BP1 and is recruited to DSB sites in a 53BP1-dependent manner specifically during G1-phase, RAD18 monoubiquitinates KBD domain of 53BP1 at lysine 1268 in vitro. A monoubiquitination-resistant 53BP1 mutant harboring a substitution at lysine 1268 is not retained efficiently at the chromatin in the vicinity of DSBs. In Rad18-null cells, retention of 53BP1 foci, efficiency of DSB repair and post-irradiation viability are impaired compared with wild-type cells. Taken together, these results suggest that RAD18 promotes 53BP1-directed DSB repair by enhancing retention of 53BP1, possibly through an interaction between RAD18 and 53BP1 and the modification of 53BP1.
Asunto(s)
Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Fase G1/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Epigénesis Genética , Fase G1/efectos de la radiación , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Proteína 1 de Unión al Supresor Tumoral P53 , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas , Ubiquitinación , Dedos de ZincRESUMEN
The E3 ubiquitin ligase RAD18 mono-ubiquitinates PCNA to promote bypass of replication fork-stalling DNA lesions. On the other hand, RAD18 also contributes to DNA double-strand break (DSB) repair. RAD18 is recruited to ionizing radiation (IR)-induced DSB and colocalizes with ubiquitinated chromatin proteins. RAD18 interacts with the ubiquitinated chromatin proteins via its ubiquitin-binding Zinc finger (UBZ) domain and is proposed to propagate DNA DSB signalling and recruit DNA repair proteins. We found that purified human RAD18 protein complexed with RAD6B (RAD6B-RAD18) catalyzes mono- and poly-ubiquitination of histone H2A in vitro while UBZ domain-mutated RAD18 complexed with RAD6B protein catalyzes mono- but not poly-ubiquitination of histone H2A. Human RAD18-/-cells synchronized at the G1 phase show a reduced signal of ubiquitinated protein in chromatin after IR when compared to that of wild-type control cells. The reduced signal of ubiquitinated protein in RAD18-/-cells is rescued by the introduction of RAD18 cDNA but to a lesser extent by the introduction of cDNA coding RAD18 lacking UBZ domain. Taken together, these results indicate that RAD18 mediates DSB-induced ubiquitination of chromatin protein during the G1 phase.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células Cultivadas , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/deficiencia , Humanos , Ubiquitina-Proteína Ligasas/deficiencia , UbiquitinaciónRESUMEN
Xeroderma pigmentosum complementation group D (XPD) is a UV-sensitive syndrome and a rare incurable genetic disease which is caused by the genetic mutation of the excision repair cross-complementation group 2 gene (ERCC2). Patients who harbor only XPD R683W mutant protein develop severe photosensitivity and progressive neurological symptoms. Cultured cells derived from patients with XPD (XPD R683W cells) demonstrate a reduced nucleotide excision repair (NER) ability. We hope to ameliorate clinical symptoms if we can identify candidate agents that would aid recovery of the cells' NER ability. To investigate such candidates, we created in silico methods of drug repurposing (in silico DR), a strategy that utilizes the recovery of ATP-binding in the XPD R683W protein after the induced fit. We chose 4E1RCat and aprepitant as the candidates for our in silico DR, and evaluated them by using the UV-induced unscheduled DNA synthesis (UDS) assay to verify the recovery of NER in XPD R683W cells. UDS values of the cells improved about 1.4-1.7 times after 4E1RCat treatment compared with solvent-only controls; aprepitant showed no positive effect. In this study, therefore, we succeeded in finding the candidate agent 4E1RCat for XPD R683W. We also demonstrated that our in silico DR method is a cost-effective approach for drug candidate discovery.
RESUMEN
The E3 ubiquitin ligase Rad18 promotes a damage-tolerant and error-prone mode of DNA replication termed trans-lesion synthesis that is pathologically activated in cancer. However, the impact of vertebrate Rad18 on cancer genomes is not known. To determine how Rad18 affects mutagenesis in vivo, we have developed and implemented a novel computational pipeline to analyze genomes of carcinogen (7, 12-Dimethylbenz[a]anthracene, DMBA)-induced skin tumors from Rad18+/+ and Rad18- / - mice. We show that Rad18 mediates specific mutational signatures characterized by high levels of A(T)>T(A) single nucleotide variations (SNVs). In Rad18- /- tumors, an alternative mutation pattern arises, which is characterized by increased numbers of deletions >4 bp. Comparison with annotated human mutational signatures shows that COSMIC signature 22 predominates in Rad18+/+ tumors whereas Rad18- / - tumors are characterized by increased contribution of COSMIC signature 3 (a hallmark of BRCA-mutant tumors). Analysis of The Cancer Genome Atlas shows that RAD18 expression is strongly associated with high SNV burdens, suggesting RAD18 also promotes mutagenesis in human cancers. Taken together, our results show Rad18 promotes mutagenesis in vivo, modulates DNA repair pathway choice in neoplastic cells, and mediates specific mutational signatures that are present in human tumors.
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The Japanese Orthopaedic Association Hip Disease Evaluation Questionnaire (JHEQ) was created for patient-reported outcome measures (PROMs) and to evaluate the conditions of patients with hip disease. Nevertheless, the validity of the JHEQ for patients with hip labral tears remains unclear. Therefore, we validated the JHEQ in patients with labral tears. There were 51 patients (mean age 44.5, range 18-60 years; 31 women). Thirty-two patients had right-sided tears, 29 underwent hip arthroscopy, 32 had femoroacetabular impingement and 15 had developmental hip dysplasia. Five PROMs included in the JHEQ were evaluated using test-retest methods. Statistical analysis was performed using SPSS software according to the COnsensus-based Standards for the selection of health status Measurement Instruments checklist. The intra-class correlation coefficient (1, 2) of all JHEQ scores (84 points) was 0.88 and Cronbach's α was 0.94. Bland-Altman analysis revealed good test-retest reliability for the JHEQ. The Spearman's rank test, including the SF-36 subscale, showed a high correlation with physical functioning [1, 0.67 (P < 0.01); 2, 0.65 (P < 0.01)], body pain [1, 0.54 (P < 0.01); 2, 0.53 (P < 0.01)] and physical component summary [1, 0.55 (P < 0.01); 2, 0.55 (P < 0.01)]. The value of minimal important change (22.9) was higher than that of smallest detectable change (3.21), suggesting that the JHEQ has adequate responsiveness. We demonstrated the reliability, validity and responsiveness of the JHEQ in Japanese patients with hip labral tears. JHEQ is a valid assessment tool not only for patients with hip osteoarthritis or osteonecrosis but also for those with hip labral tears.
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Checkpoint kinase 2 (human CHEK2; murine Chk2) is a critical mediator of the DNA damage response and has established roles in DNA double strand break (DSB)-induced apoptosis and cell cycle arrest. DSBs may be invoked directly by ionizing radiation but may also arise indirectly from environmental exposures such as solar ultraviolet (UV) radiation. The primary forms of DNA damage induced by UV are DNA photolesions (such as cyclobutane pyrimidine dimers CPD and 6-4 photoproducts) which interfere with DNA synthesis and lead to DNA replication fork stalling. Persistently stalled and unresolved DNA replication forks can "collapse" to generate DSBs that induce signaling via Chk2 and its upstream activator the ataxia telangiectasia-mutated (ATM) protein kinase. This review focuses on recently defined roles of Chk2 in protecting against DNA replication-associated genotoxicity. Several DNA damage response factors such as Rad18, Nbs1 and Chk1 suppress stalling and collapse of DNA replication forks. Defects in the primary responders to DNA replication fork stalling lead to generation of DSB and reveal "back-up" roles for Chk2 in S-phase progression and genomic stability. In humans, there are numerous variants of the CHEK2 gene, including CHEK2*1100delC. Individuals with the CHEK2*1100delC germline alteration have an increased risk of developing breast cancer and malignant melanoma. DNA replication fork-stalling at estrogen-DNA adducts and UV-induced photolesions are implicated in the etiology of breast cancer and melanoma, respectively. It is likely therefore that the Chk2/CHEK2-deficiency is associated with elevated risk for tumorigenesis caused by replication-associated genotoxicities that are exacerbated by environmental genotoxins and intrinsic DNA-damaging agents.
Asunto(s)
Quinasa de Punto de Control 2/genética , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Exposición a Riesgos Ambientales/efectos adversos , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Humanos , Neoplasias/genéticaRESUMEN
Post-replication DNA repair facilitates the resumption of DNA synthesis upon replication fork stalling at DNA damage sites. Despite the importance of RAD18 and polymerase eta (Poleta) for post-replication repair (PRR), the molecular mechanisms by which these factors are recruited to stalled replication forks are not well understood. We present evidence that human RAD18 complexed with RAD6B protein preferentially binds to forked and single-stranded DNA (ssDNA) structures, which are known to be localized at stalled replication forks. The SAP domain of RAD18 (residues 248-282) is crucial for binding of RAD18 complexed with RAD6B to DNA substrates. RAD18 mutated in the SAP domain fails to accumulate at DNA damage sites in vivo and does not guide DNA Poleta to stalled replication forks. The SAP domain is also required for the efficient mono-ubiquitination of PCNA. The SAP domain mutant fails to suppress the ultraviolet (UV)-sensitivity of Rad18-knockout cells. These results suggest that RAD18 complexed with RAD6B is recruited to stalled replication forks via interactions with forked DNA or long ssDNA structures, a process that is required for initiating PRR.