RESUMEN
Propagation of chromatin states through DNA replication is central to epigenetic regulation and can involve recruitment of chromatin proteins to replicating chromatin through interactions with replication fork components. Here we show using a fully reconstituted T7 bacteriophage system that eukaryotic proteins are not required to tether the Polycomb complex PRC1 to templates during DNA replication. Instead, DNA binding by PRC1 can withstand passage of a simple replication fork.
Asunto(s)
Replicación del ADN , ADN Viral/química , Proteínas del Grupo Polycomb/química , Bacteriófago T7/genética , Unión Competitiva , ADN Helicasas/química , ADN Polimerasa Dirigida por ADN/química , Plásmidos/química , Unión Proteica , Proteínas Virales/químicaRESUMEN
Epigenetic regulation may involve heritable chromatin states, but how chromatin features can be inherited through DNA replication is incompletely understood. We address this question using cell-free replication of chromatin. Previously, we showed that a Polycomb group complex, PRC1, remains continuously associated with chromatin through DNA replication. Here we investigate the mechanism of persistence. We find that a single PRC1 subunit, Posterior sex combs (PSC), can reconstitute persistence through DNA replication. PSC binds nucleosomes and self-interacts, bridging nucleosomes into a stable, oligomeric structure. Within these structures, individual PSC-chromatin contacts are dynamic. Stable association of PSC with chromatin, including through DNA replication, depends on PSC-PSC interactions. Our data suggest that labile individual PSC-chromatin contacts allow passage of the DNA replication machinery while PSC-PSC interactions prevent PSC from dissociating, allowing it to rebind to replicated chromatin. This mechanism may allow inheritance of chromatin proteins including PRC1 through DNA replication to maintain chromatin states.
Asunto(s)
Replicación del ADN , ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Represoras/metabolismo , Animales , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Drosophila/metabolismo , Humanos , Nucleosomas/metabolismo , Proteínas del Grupo Polycomb , Proteínas Represoras/químicaRESUMEN
Mre11/Rad50 complexes in all organisms function in the repair of DNA double-strand breaks. In budding yeast, genetic evidence suggests that the Sae2 protein is essential for the processing of hairpin DNA intermediates and meiotic double-strand breaks by Mre11/Rad50 complexes, but the biochemical basis of this functional relationship is not known. Here we demonstrate that recombinant Sae2 binds DNA and exhibits endonuclease activity on single-stranded DNA independently of Mre11/Rad50 complexes, but hairpin DNA structures are cleaved cooperatively in the presence of Mre11/Rad50 or Mre11/Rad50/Xrs2. Hairpin structures are not processed at the tip by Sae2 but rather at single-stranded DNA regions adjacent to the hairpin. Truncation and missense mutants of Sae2 inactivate this endonuclease activity in vitro and fail to complement Deltasae2 strains in vivo for meiosis and recombination involving hairpin intermediates, suggesting that the catalytic activities of Sae2 are important for its biological functions.
Asunto(s)
ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Conformación de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ADN de Hongos/química , ADN de Cadena Simple/metabolismo , Endonucleasas/metabolismo , Proteínas Mutantes/metabolismo , Fenotipo , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
Mre11 and Rad50 are the catalytic components of a highly conserved DNA repair complex that functions in many aspects of DNA metabolism involving double-strand breaks. The ATPase domains in Rad50 are related to the ABC transporter family of ATPases, previously shown to share structural similarities with adenylate kinases. Here we demonstrate that Mre11/Rad50 complexes from three organisms catalyze the reversible adenylate kinase reaction in vitro. Mutation of the conserved signature motif reduces the adenylate kinase activity of Rad50 but does not reduce ATP hydrolysis. This mutant resembles a rad50 null strain with respect to meiosis and telomere maintenance in S. cerevisiae, correlating adenylate kinase activity with in vivo functions. An adenylate kinase inhibitor blocks Mre11/Rad50-dependent DNA tethering in vitro and in cell-free extracts, indicating that adenylate kinase activity by Mre11/Rad50 promotes DNA-DNA associations. We propose a model for Rad50 that incorporates both ATPase and adenylate kinase reactions as critical activities that regulate Rad50 functions.
Asunto(s)
Adenilato Quinasa/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Ácido Anhídrido Hidrolasas , Adenina/metabolismo , Adenosina Trifosfato/metabolismo , Adenilato Quinasa/antagonistas & inhibidores , Secuencias de Aminoácidos , Animales , Proteínas Arqueales/metabolismo , Catálisis/efectos de los fármacos , Enzimas Reparadoras del ADN/metabolismo , Fosfatos de Dinucleósidos/metabolismo , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasas/metabolismo , Humanos , Hidrólisis/efectos de los fármacos , Proteína Homóloga de MRE11 , Proteínas Mutantes/metabolismo , Mutación/genética , Pyrococcus furiosus/efectos de los fármacos , Pyrococcus furiosus/enzimología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , XenopusRESUMEN
The repair of double-strand breaks in DNA is an essential process in all organisms, and requires the coordinated activities of evolutionarily conserved protein assemblies. One of the most critical of these is the Mre11/Rad50 (M/R) complex, which is present in all three biological kingdoms, but is not well-understood at the biochemical level. Previous structural analysis of a Rad50 homolog from archaebacteria illuminated the catalytic core of the enzyme, an ATP-binding domain related to the ABC transporter family of ATPases. Here, we present the crystallographic structure of the Rad50 mutant S793R. This missense signature motif mutation changes the key serine residue in the signature motif that is conserved among Rad50 homologs and ABC ATPases. The S793R mutation is analogous to the mutation S549R in the cystic fibrosis transmembrane conductance regulator (CFTR) that results in cystic fibrosis. We show here that the serine to arginine change in the Rad50 protein prevents ATP binding and disrupts the communication among the other ATP-binding loops. This structural change, in turn, alters the communication between Rad50 monomers and thus prevents Rad50 dimerization. The equivalent mutation was made in the human Rad50 gene, and the resulting mutant protein did form a complex with Mre11 and Nbs1, but was specifically deficient in all ATP-dependent enzymatic activities. This signature motif structure-function homology extends to yeast, because the same mutation introduced into the Saccharomyces cerevisiae RAD50 gene generated an allele that failed to complement a rad50 deletion strain in DNA repair assays in vivo. These structural and biochemical results extend our understanding of the Rad50 catalytic domain and validate the use of the signature motif mutant to test the role of Rad50 ATP binding in diverse organisms.