RESUMEN
The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in Förster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5' extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends.
Asunto(s)
Proteínas de Unión al ADN/genética , ADN/genética , Autoantígeno Ku/genética , Proteínas Nucleares/genética , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Proteína Quinasa Activada por ADN/genética , Transferencia Resonante de Energía de Fluorescencia/métodos , Ratones , Fosforilación/genéticaRESUMEN
One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Endonucleasas/metabolismo , Proteínas Nucleares/metabolismo , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Exodesoxirribonucleasas , Células HeLa , Humanos , Proteínas Nucleares/genéticaRESUMEN
Here, we identify a phylogenetically conserved Schizosaccharomyces pombe factor, named Rtf2, as a key requirement for efficient replication termination at the site-specific replication barrier RTS1. We show that Rtf2, a proliferating cell nuclear antigen-interacting protein, promotes termination at RTS1 by preventing replication restart; in the absence of Rtf2, we observe the establishment of "slow-moving" Srs2-dependent replication forks. Analysis of the pmt3 (SUMO) and rtf2 mutants establishes that pmt3 causes a reduction in RTS1 barrier activity, that rtf2 and pmt3 are nonadditive, and that pmt3 (SUMO) partly suppresses the rtf2-dependent replication restart. Our results are consistent with a model in which Rtf2 stabilizes the replication fork stalled at RTS1 until completion of DNA synthesis by a converging replication fork initiated at a flanking origin.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Replicación del ADN , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Genoma Fúngico , Datos de Secuencia Molecular , Mutación , Fenotipo , Plásmidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Homología de Secuencia de Aminoácido , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The Streptomyces coelicolor gene SCC88.10c encodes a protein (RNase ES) which is homologous to endoribonucleases in the RNase E/G family. We expressed S. coelicolor RNase ES as a 6 x His-tagged protein in an Escherichia coli mutant carrying a rng (which encodes RNase G) or a rne (which encodes RNase E) mutation to study whether S. coelicolor RNase ES is able to complement these mutations in host E. coli cells. The results clearly indicated that the S. coelicolor RNase ES can partially abrogate either the rng::cat or rne-1 mutation, as measured by the ability to suppress the several aberrant phenotypes resulting from the rng or rne mutation. Thus, S. coelicolor RNase ES appears to have the dual ability to supplant the functions of both RNase G and RNase E in E. coli.
