RESUMEN
Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteína de Replicación A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga de MutS/genética , Proteína 3 Homóloga de MutS/metabolismo , Mutación , Mapeo de Interacción de Proteínas , Proteína de Replicación A/genética , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/genética , Rayos UltravioletaRESUMEN
The Saccharomyces cerevisiae Rad1/Rad10 complex is a multifunctional, structure-specific endonuclease that processes UV-induced DNA lesions, recombination intermediates, and inter-strand DNA crosslinks. However, we do not know how Rad1/Rad10 recognizes these structurally distinct target molecules or how it is incorporated into the protein complexes capable of incising divergent substrates. Here, we have determined the order and hierarchy of assembly of the Rad1/Rad10 complex, Saw1, Slx4, and Msh2/Msh3 complex at a 3' tailed recombination intermediate. We found that Saw1 is a structure-specific DNA binding protein with high affinity for splayed arm and 3'-flap DNAs. By physical interaction, Saw1 facilitates targeting of Rad1 at 3' tailed substrates in vivo and in vitro, and enhances 3' tail cleavage by Rad1/Rad10 in a purified system in vitro. Our results allow us to formulate a model of Rad1/Rad10/Saw1 nuclease complex assembly and 3' tail removal in recombination.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Perfilación de la Expresión Génica , Mutagénesis , Recombinación Genética/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Yeast Rad1-Rad10 (XPF-ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1-Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1-Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1-Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1-Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1-Rad10 removes ICL via NER, whereas in S/G2, Rad1-Rad10 facilitates NER-independent replication-coupled ICLR.
Asunto(s)
Daño del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Animales , Células CHO , Ciclo Celular/genética , Cricetulus , Reactivos de Enlaces Cruzados/toxicidad , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Microscopía Intravital , Mutagénesis Sitio-Dirigida , Mutación , Proteínas de Saccharomyces cerevisiae/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/genética , Rayos Ultravioleta/efectos adversosRESUMEN
In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype.