RESUMEN
Saccharomyces cerevisiae Srs2, in addition to its well-documented antirecombination activity, has been proposed to play a role in promoting synthesis-dependent strand annealing (SDSA). Here we report the identification and characterization of an SRS2 mutant with a single amino acid substitution (srs2-F891A) that specifically affects the Srs2 pro-SDSA function. This residue is located within the Srs2-Rad51 interaction domain and embedded within a protein sequence resembling a BRC repeat motif. The srs2-F891A mutation leads to a complete loss of interaction with Rad51 as measured through yeast two-hybrid analysis and a partial loss of interaction as determined through protein pull-down assays with purified Srs2, Srs2-F891A, and Rad51 proteins. Even though previous work has shown that internal deletions of the Srs2-Rad51 interaction domain block Srs2 antirecombination activity in vitro, the Srs2-F891A mutant protein, despite its weakened interaction with Rad51, exhibits no measurable defect in antirecombination activity in vitro or in vivo Surprisingly, srs2-F891A shows a robust shift from noncrossover to crossover repair products in a plasmid-based gap repair assay, but not in an ectopic physical recombination assay. Our findings suggest that the Srs2 C-terminal Rad51 interaction domain is more complex than previously thought, containing multiple interaction sites with unique effects on Srs2 activity.
Asunto(s)
Intercambio Genético , ADN Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , ADN Helicasas/química , ADN Helicasas/genética , Unión Proteica , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Synthesis-dependent strand annealing (SDSA) is the preferred mode of homologous recombination in somatic cells leading to an obligatory non-crossover outcome, thus avoiding the potential for chromosomal rearrangements and loss of heterozygosity. Genetic analysis identified the Srs2 helicase as a prime candidate to promote SDSA. Here, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion and with a distinct polarity. Specifically, we partly reconstitute the SDSA pathway using Rad51, Rad54, RPA, RFC, DNA Polymerase δ with different forms of PCNA. Consistent with genetic data showing the requirement for SUMO and PCNA binding for the SDSA role of Srs2, Srs2 displays a slight but significant preference to disrupt extending D-loops over unextended D-loops when SUMOylated PCNA is present, compared to unmodified PCNA or monoubiquitinated PCNA. Our data establish a biochemical mechanism for the role of Srs2 in crossover suppression by promoting SDSA through disruption of extended D-loops.
Asunto(s)
Emparejamiento Base , ADN Helicasas/metabolismo , ADN Polimerasa III/metabolismo , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologíaRESUMEN
The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.