RESUMEN
SUMOylation (small ubiquitin-like modifier) in the DNA double-strand break (DSB) response regulates recruitment, activity, and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and nonhomologous end joining (NHEJ) through the investigation of the deSUMOylase SENP2. We found that regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast, we show that HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 focus retention and increases NHEJ and radioresistance. Collectively, our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Recombinación Homóloga/genética , Sumoilación/genética , Proteínas Adaptadoras Transductoras de Señales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Cisteína Endopeptidasas/genética , Roturas del ADN de Doble Cadena , Células HEK293 , Células HeLa , Humanos , Rayos Infrarrojos , Proteínas Nucleares/metabolismo , Tolerancia a Radiación/genética , Transducción de Señal/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteína que Contiene Valosina/metabolismoRESUMEN
Monoclonal antibodies (MAb) to members of the Small Ubiquitin-like modifier (SUMO) family are essential tools in the study of cellular SUMOylation. However, many anti-SUMO MAbs are poorly validated, and antibody matching to detection format is without an evidence base. Here we test the specificity and sensitivity of twenty-four anti-SUMO MAbs towards monomeric and polymeric SUMO1-4 in dot-blots, immunoblots, immunofluorescence and immunoprecipitation. We find substantial variability between SUMO MAbs for different conjugation states, for detecting increased SUMOylation in response to thirteen different stress agents, and as enrichment reagents for SUMOylated RanGAP1 or KAP1. All four anti-SUMO4 monoclonal antibodies tested cross-reacted wit SUMO2/3, and several SUMO2/3 monoclonal antibodies cross-reacted with SUMO4. These data characterize the specificity of twenty-four anti-SUMO antibodies across commonly used assays, creating an enabling resource for the SUMO research community.