RESUMEN
The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in Saccharomyces cerevisiae and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1-Top3-Rmi1-Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70-Ku80 becomes a suitable substrate for long-range 5'-3' resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , RecQ Helicasas/metabolismo , Proteína de Replicación A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , ADN Helicasas/genética , Reparación del ADN , Proteínas de Unión al ADN/genética , Exodesoxirribonucleasas/genética , Recombinación Homóloga , RecQ Helicasas/genética , Proteína de Replicación A/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica , Proteína 1 de Unión al Supresor Tumoral P53/genética , Animales , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Regulación de la Expresión Génica/genética , Humanos , Ratones , Reparación del ADN por Recombinación/genética , Proteína 1 de Unión al Supresor Tumoral P53/antagonistas & inhibidoresRESUMEN
The budding yeast Mre11-Rad50-Xrs2 (MRX) complex and Sae2 function together in DNA end resection during homologous recombination. Here we show that the Ku complex shields DNA ends from exonucleolytic digestion but facilitates endonucleolytic scission by MRX with a dependence on ATP and Sae2. The incision site is enlarged into a DNA gap via the exonuclease activity of MRX, which is stimulated by Sae2 without ATP being present. RPA renders a partially resected or palindromic DNA structure susceptible to MRX-Sae2, and internal protein blocks also trigger DNA cleavage. We present models for how MRX-Sae2 creates entry sites for the long-range resection machinery.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Reparación del ADN/fisiología , Endonucleasas/metabolismo , Exonucleasas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , División del ADN , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Activación Enzimática/genética , Exodesoxirribonucleasas/metabolismo , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
DNA double-strand breaks can be eliminated via non-homologous end joining or homologous recombination. Non-homologous end joining is initiated by the association of Ku with DNA ends. In contrast, homologous recombination entails nucleolytic resection of the 5'-strands, forming 3'-ssDNA tails that become coated with replication protein A (RPA). Ku restricts end access by the resection nuclease Exo1. It is unclear how partial resection might affect Ku engagement and Exo1 restriction. Here, we addressed these questions in a reconstituted system with yeast proteins. With blunt-ended DNA, Ku protected against Exo1 in a manner that required its DNA end-binding activity. Despite binding poorly to ssDNA, Ku could nonetheless engage a 5'-recessed DNA end with a 40-nucleotide (nt) ssDNA overhang, where it localized to the ssDNA-dsDNA junction and efficiently blocked resection by Exo1. Interestingly, RPA could exclude Ku from a partially resected structure with a 22-nt ssDNA tail and thus restored processing by Exo1. However, at a 40-nt tail, Ku remained stably associated at the ssDNA-dsDNA junction, and RPA simultaneously engaged the ssDNA region. We discuss a model in which the dynamic equilibrium between Ku and RPA binding to a partially resected DNA end influences the timing and efficiency of the resection process.