RESUMEN
X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large multiprotein DNA repair complexes as well as facilitate the recruitment of DNA repair proteins to sites of DNA damage. Moreover, XRCC1 is present in constitutive DNA repair complexes, some of which associate with the replication machinery. Because of the critical role of XRCC1 in DNA repair, its common variants Arg194Trp, Arg280His and Arg399Gln have been extensively studied. However, the prevalence of these variants varies strongly in different populations, and their functional influence on DNA repair and disease remains elusive. Here we present the current knowledge about the role of XRCC1 and its variants in BER and human disease/cancer.
Asunto(s)
Roturas del ADN de Cadena Simple , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Animales , Proteínas de Unión al ADN/genética , Humanos , Proteínas de Neoplasias/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
XRCC1 is a scaffold protein involved in base excision repair and single strand break repair. It is a phosphoprotein that contains more than 45 phosphorylation sites, however only a few of these have been characterized and connected to specific kinases and functions. Mitogen activated protein kinases (MAPK) are mediators of cellular stress responses, and here we demonstrate that p38 MAPK signaling is involved in phosphorylation of XRCC1 and regulation of recruitment to oxidative stress. Inhibition of p38 MAPK caused a marked pI shift of XRCC1 towards a less phosphorylated state. Inhibition of p38 also increased the immediate accumulation of XRCC1 at site of DNA damage in a poly(ADP)-ribose (PAR) dependent manner. These results suggest a link between PARylation, p38 signaling and XRCC1 recruitment to DNA damage. Additionally, we characterized two phosphorylation sites, T358 and T367, located within, or close to, the phosphate-binding pocket of XRCC1, which is important for interaction with PAR. Mutation of these sites impairs recruitment of XRCC1 to DNA damage and binding to PARP1/PAR. Collectively, our data suggest that phosphorylation of T358 and T367 and p38 signaling are important for proper regulation of XRCC1 recruitment to DNA damage and thereby avoidance of potential toxic and mutagenic BER-intermediates.
Asunto(s)
Poli Adenosina Difosfato Ribosa/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/química , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Sitios de Unión , Línea Celular , Daño del ADN , Células HeLa , Humanos , Mutación , Estrés Oxidativo , Fosforilación , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Unión Proteica , Dominios Proteicos , Transducción de Señal , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genéticaRESUMEN
Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as targets for replacement- or adjuvant therapies.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Biomarcadores de Tumor/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/genética , Resistencia a Antineoplásicos/genética , Melfalán/farmacología , Mieloma Múltiple/genética , 8-Hidroxi-2'-Desoxicoguanosina , Apoptosis , Western Blotting , Ciclo Celular/genética , Proliferación Celular , Ensayo Cometa , Reparación del ADN/efectos de los fármacos , Replicación del ADN/genética , Proteína Quinasa Activada por ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células Tumorales CultivadasRESUMEN
XRCC1 functions as a non-enzymatic, scaffold protein in single strand break repair (SSBR) and base excision repair (BER). Here, we examine different regions of XRCC1 for their contribution to the scaffolding functions of the protein. We found that the central BRCT1 domain is essential for recruitment of XRCC1 to sites of DNA damage and DNA replication. Also, we found that ectopic expression of the region from residue 166-436 partially rescued the methyl methanesulfonate (MMS) hypersensitivity of XRCC1-deficient EM9 cells, suggesting a key role for this region in mediating DNA repair. The three most common amino acid variants of XRCC1, Arg194Trp, Arg280His and Arg399Gln, are located within the region comprising the NLS and BRCT1 domains, and these variants may be associated with increased incidence of specific types of cancer. While we could not detect differences in the intra-nuclear localization or the ability to support recruitment of POLß or PNKP to micro-irradiated sites for these variants relative to the conservative protein, we did observe lower foci intensity after micro-irradiation and a reduced stability of the foci with the Arg280His and Arg399Gln variants, respectively. Furthermore, when challenged with MMS or hydrogen peroxide, we detected small but consistent differences in the repair profiles of cells expressing these two variants in comparison to the conservative protein.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Polimorfismo de Nucleótido Simple , Animales , Células CHO , Cricetinae , Cricetulus , Daño del ADN , ADN Polimerasa beta/metabolismo , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Enzimas Reparadoras del ADN/metabolismo , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Replicación del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Humanos , Metilmetanosulfonato/farmacología , Señales de Localización Nuclear , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/efectos de la radiación , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POLß and PNK colocalize with XRCC1 in replication foci and that POLß and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLß and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLß to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes.
Asunto(s)
Roturas del ADN de Cadena Simple , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Animales , Western Blotting , Células CHO , Técnicas de Cultivo de Célula , Cricetinae , Cricetulus , Roturas del ADN de Cadena Simple/efectos de la radiación , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta en la Radiación , Células HeLa , Humanos , Inmunoprecipitación , Rayos Láser , Microscopía Confocal , Modelos Biológicos , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/genética , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
Uracil-DNA glycosylase, UNG2, interacts with PCNA and initiates post-replicative base excision repair (BER) of uracil in DNA. The DNA repair protein XRCC1 also co-localizes and physically interacts with PCNA. However, little is known about whether UNG2 and XRCC1 directly interact and participate in a same complex for repair of uracil in replication foci. Here, we examine localization pattern of these proteins in live and fixed cells and show that UNG2 and XRCC1 are likely in a common complex in replication foci. Using pull-down experiments we demonstrate that UNG2 directly interacts with the nuclear localization signal-region (NLS) of XRCC1. Western blot and functional analysis of immunoprecipitates from whole cell extracts prepared from S-phase enriched cells demonstrate the presence of XRCC1 complexes that contain UNG2 in addition to separate XRCC1 and UNG2 associated complexes with distinct repair features. XRCC1 complexes performed complete repair of uracil with higher efficacy than UNG2 complexes. Based on these results, we propose a model for a functional role of XRCC1 in replication associated BER of uracil.
Asunto(s)
ADN Glicosilasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Uracilo/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , ADN/genética , ADN/metabolismo , ADN Glicosilasas/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
SR-BI mediates exchange of cholesterol between HDL and cells, and is a crucial factor in the transport of excessive cellular cholesterol from extrahepatic tissues to the liver ("reverse cholesterol transport") and, therefore, also for cholesterol homeostasis. Hepatic SR-BI mediates transfer of HDL-cholesterol to the hepatocytes where cholesterol may be metabolised to bile acids. LXR and SREBP are key factors in the regulation of cholesterol metabolism. The purpose of the present study was to determine whether these transcription factors are involved in the regulation of SR-BI. Here we show that LXRalpha/RXR and LXRbeta/RXR induce SR-BI transcription in human and murine hepatoma cell lines, and in 3T3-L1 preadipocytes independently of SREBP-1. The LXR/RXR response was mapped within -1,200 to -937 of the promoter region. Gel mobility shift analysis confirmed that the putative LXR response element bound LXRalpha/RXR and LXRbeta/RXR heterodimers.