RESUMEN
Persistent DNA double-strand breaks (DSBs) are recruited to the nuclear periphery in budding yeast. Both the Nup84 pore subcomplex and Mps3, an inner nuclear membrane (INM) SUN domain protein, have been implicated in DSB binding. It was unclear what, if anything, distinguishes the two potential sites of repair. Here, we characterize and distinguish the two binding sites. First, DSB-pore interaction occurs independently of cell-cycle phase and requires neither the chromatin remodeler INO80 nor recombinase Rad51 activity. In contrast, Mps3 binding is S and G2 phase specific and requires both factors. SWR1-dependent incorporation of Htz1 (H2A.Z) is necessary for break relocation to either site in both G1- and S-phase cells. Importantly, functional assays indicate that mutations in the two sites have additive repair defects, arguing that the two perinuclear anchorage sites define distinct survival pathways.
Asunto(s)
Sitios de Unión/genética , Ensamble y Desensamble de Cromatina/fisiología , ADN de Hongos/genética , Proteínas Fúngicas/fisiología , Saccharomycetales/genética , Adenosina Trifosfatasas/fisiología , Sitios de Unión/fisiología , Ciclo Celular/genética , Ciclo Celular/fisiología , Ensamble y Desensamble de Cromatina/genética , Roturas del ADN de Doble Cadena , Histonas/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Saccharomycetales/metabolismoRESUMEN
We show here that the transformation efficiency of Saccharomyces cerevisiae is improved by altering carbon sources in media for pre-culturing cells prior to the transformation reactions. The transformation efficiency was increased up to sixfold by combination with existing transformation protocols. This method is widely applicable for yeast research since efficient transformation can be performed easily without changing any of the other procedures in the transformation.
