RESUMEN
MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/química , Fase G2 , Reparación del ADN por Recombinación , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Rayos gamma/efectos adversos , Humanos , Proteína Homóloga de MRE11 , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismoRESUMEN
Essential genome transactions, such as homologous recombination, are achieved by concerted and dynamic interactions of multiple protein components with DNA. Which proteins do what and how, will be reflected in their relative arrangements. However, obtaining high-resolution structural information on the variable arrangements of these complex assemblies is a challenge. Here we demonstrate the versatility of a combined total internal reflection fluorescence and scanning force microscope (TIRF-SFM) to pinpoint fluorescently labeled human homologous recombination protein RAD54 interacting with presynaptic (ssDNA) and postsynaptic (dsDNA) human recombinase RAD51 nucleoprotein filaments. Labeled proteins were localized by superresolution imaging on complex structures in the SFM image with high spatial accuracy. We observed some RAD54 at RAD51 filament ends, as expected. More commonly, RAD54 interspersed along RAD51-DNA filaments. RAD54 promotes RAD51-mediated DNA strand exchange and has been described to both stabilize and destabilize RAD51-DNA filaments. The different architectural arrangements we observe for RAD54 with RAD51-DNA filaments may reflect the diverse roles of this protein in homologous recombination.
Asunto(s)
ADN/metabolismo , Microscopía/métodos , Proteínas Nucleares/metabolismo , Recombinasa Rad51/metabolismo , Sinapsis/metabolismo , ADN/química , ADN Helicasas , Proteínas de Unión al ADN , Colorantes Fluorescentes/química , Humanos , Proteínas Nucleares/química , Recombinasa Rad51/químicaRESUMEN
The Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structural and in vivo functional studies to uncover key properties of Mre11-W243R, a mutation identified in two pediatric cancer patients with enhanced ataxia telangiectasia-like disorder. Purified human Mre11-W243R retains nuclease and DNA binding activities in vitro. X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 three-dimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions. The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure. W248R differs from other ataxia telangiectasia-like disorder analog alleles by the reduced stability of its interaction with Rad50 in cell lysates. Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions.
Asunto(s)
Ataxia Telangiectasia/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/genética , Exodesoxirribonucleasas/genética , Mutación Missense , Proteínas de Schizosaccharomyces pombe/genética , Secuencia de Aminoácidos , Antígenos Nucleares/genética , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Humanos , Autoantígeno Ku , Proteína Homóloga de MRE11 , Meiosis , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Pyrococcus furiosus/enzimología , Proteína Recombinante y Reparadora de ADN Rad52/análisis , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homeostasis del Telómero , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The MRE11-RAD50-NBS1 (MRN) complex has several distinct functions in DNA repair including important roles in both non-homologous end-joining (NHEJ) and homologous recombination (HR). The biochemical activities of MR(N) have been well characterized implying specific functional roles for the components. The arrangement of proteins in the complex implies interdependence of their biochemical activities making it difficult to separate specific functions. We obtained purified human RAD50 and observed that it binds ATP, undergoes ATP-dependent conformational changes as well as having ATPase activity. Scanning force microscopy analysis clearly showed that RAD50 binds DNA although not as oligomers. RAD50 alone was not functional in tethering DNA molecules. ATP increased formation of RAD50 multimers which were however globular lacking extended coiled coils, in contrast to the MR complex where ATP induced oligomers have obvious coiled coils protruding from a central domain. These results suggest that MRE11 is important in maintaining the structural arrangement of RAD50 in the protein complex and perhaps has a role in reinforcing proper alignment of the coiled coils in the ATP-bound state.