RESUMEN
Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loops with near base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.
Asunto(s)
Reparación del ADN/fisiología , ADN de Cadena Simple/química , Imagen Individual de Molécula , Sulfitos , Citosina/química , Técnicas de Amplificación de Ácido Nucleico , Uracilo/químicaRESUMEN
Displacement loops (D-loops) are critical intermediates formed during homologous recombination. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54/Tid1 is also present in somatic cells where its function is less understood. While Rdh54/Tid1 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54. Here, we show that Rdh54/Tid1 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast. We propose that Rdh54/Tid1 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54/Tid1 acts as a physical roadblock to Rad54 translocation, limiting D-loop formation and D-loop length.
Asunto(s)
ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN/fisiología , ADN-Topoisomerasas/metabolismo , Recombinasa Rad51/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , ADN-Topoisomerasas/genética , ADN de Hongos/química , Regulación Fúngica de la Expresión Génica/fisiología , Mutación , Recombinasa Rad51/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Displacement loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 h of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps. The majority of nascent D-loops are disrupted by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a layer of quality control of HR relying on nascent D-loop dynamics.
Asunto(s)
Daño del ADN , ADN de Hongos/genética , Reparación del ADN por Recombinación , Saccharomyces cerevisiae/genética , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN-Topoisomerasas/genética , ADN-Topoisomerasas/metabolismo , ADN de Hongos/química , ADN de Hongos/metabolismo , Cinética , Conformación de Ácido Nucleico , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-ActividadRESUMEN
Homologous recombination enables the cell to access and copy intact DNA sequence information in trans, particularly to repair DNA damage affecting both strands of the double helix. Here, we discuss the DNA transactions and enzymatic activities required for this elegantly orchestrated process in the context of the repair of DNA double-strand breaks in somatic cells. This includes homology search, DNA strand invasion, repair DNA synthesis, and restoration of intact chromosomes. Aspects of DNA topology affecting individual steps are highlighted. Overall, recombination is a dynamic pathway with multiple metastable and reversible intermediates designed to achieve DNA repair with high fidelity.