RESUMEN
In most sexually reproducing organisms, meiotic recombination is initiated by DNA double-strand breaks (DSBs) formed by the Spo11 protein. In budding yeast, nine other proteins are also required for DSB formation, but the roles of these proteins and the interactions among them are poorly understood. We report further studies of the behaviors of these proteins. Consistent with other studies, we find that Mei4 and Rec114 bind to chromosomes from leptonema through early pachynema. Both proteins showed only limited colocalization with the meiotic cohesin subunit Rec8, suggesting that Mei4 and Rec114 associated preferentially with chromatin loops. Rec114 localization was independent of other DSB factors, but Mei4 localization was strongly dependent on Rec114 and Mer2. Systematic deletion analysis identified protein regions important for a previously described two-hybrid interaction between Mei4 and Rec114. We also report functional characterization of a previously misannotated 5' coding exon of REC102. Sequences encoded in this exon are essential for DSB formation and for Rec102 interaction with Rec104, Spo11, Rec114, and Mei4. Finally, we also examined genetic requirements for a set of previously described two-hybrid interactions that can be detected only when the reporter strain is induced to enter meiosis. This analysis reveals new functional dependencies for interactions among the DSB proteins. Taken together, these studies support the view that Mei4, Rec114, and Mer2 make up a functional subgroup that is distinct from other subgroups of the DSB proteins: Spo11-Ski8, Rec102-Rec104, and Mre11-Rad50-Xrs2. These studies also suggest that an essential function of Rec102 and Rec104 is to connect Mei4 and Rec114 to Spo11.
Asunto(s)
Roturas del ADN de Doble Cadena , Meiosis/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Complejo Sinaptonémico/genética , Rotura Cromosómica , ADN de Hongos , Endodesoxirribonucleasas , Esterasas , Meiosis/fisiología , Proteínas Nucleares , Fase Paquiteno , Recombinasas , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Sinaptonémico/metabolismoRESUMEN
Meiotic double-strand breaks (DSBs) are formed by Spo11 in conjunction with at least nine other proteins whose roles are not well understood. We find that two of these proteins, Rec102 and Rec104, interact physically, are mutually dependent for proper subcellular localization, and share a requirement for Spo11 and Ski8 for their recruitment to meiotic chromosomes, suggesting that they work together as a functional unit. Rec102 associated extensively with chromatin loops during leptotene and zygotene and showed preferential binding in the vicinity at least of most DSB sites, consistent with a direct role in DSB formation. However, Rec102 was associated with both DSB-hot and DSB-cold regions, ruling out a simple model in which sites of DSB formation are dictated by where Rec102/104 complexes load. Both proteins persisted on chromatin until pachytene before abruptly disappearing, indicating that they remain on chromosomes well after DSB formation. These studies reveal unexpected behaviors for Rec102 and Rec104, and point to distinct roles and subcomplexes among the DSB proteins.
Asunto(s)
Cromosomas Fúngicos/metabolismo , Proteínas de Unión al ADN/metabolismo , Meiosis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Inmunoprecipitación de Cromatina , Cromosomas Fúngicos/genética , Proteínas de Unión al ADN/genética , Unión Proteica , Transporte de Proteínas , Recombinasas , Recombinación Genética/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Meiotic recombination initiates with double-strand breaks (DSBs) catalyzed by Spo11 in conjunction with accessory proteins whose roles are not understood. Two-hybrid analysis reveals a network of interactions connecting the yeast DSB proteins to one another. Of these proteins, Ski8 was known to function in cytoplasmic RNA metabolism, suggesting that its role in recombination might be indirect. However, obligate partners of Ski8 in RNA metabolism are dispensable for recombination and Ski8 relocalizes to the nucleus and associates with chromosomes specifically during meiosis. Interaction of Ski8 with Spo11 is essential for DSB formation and Ski8 relocalization. Thus, Ski8 plays distinct roles in RNA metabolism and, as a direct partner of Spo11, in DSB formation. Ski8 works with Spo11 to recruit other DSB proteins to meiotic chromosomes, implicating Ski8 as a scaffold protein mediating assembly of a multiprotein complex essential for DSB formation.
Asunto(s)
Daño del ADN , Esterasas/metabolismo , Meiosis/genética , ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Cromosomas Fúngicos/metabolismo , Citoplasma/metabolismo , Endodesoxirribonucleasas , Esterasas/genética , Técnica del Anticuerpo Fluorescente Indirecta , Modelos Biológicos , Modelos Moleculares , Recombinación Genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Pancreatic endocrine tumors (PETs) arise from neuroendocrine cells in and around the pancreas. As loss of heterozygosity (LOH) of chromosome 3 has been reported in sporadic PETs, we examined 16 sporadic PETs for LOH of 10 polymorphic DNA markers spanning both arms of chromosome 3. LOH was demonstrated in 4 of 8 (50%) sporadic PETs with hepatic metastasis, but in none of 8 sporadic PETs without hepatic involvement. The smallest common-deleted region (SCDR) mapped to 3q27-qter. Analysis of this data with the status of markers on chromosomes 1, 11, and MEN1 mutations in these 16 sporadic PETs revealed that chromosome 3q loss may be a late event in sporadic PET tumorigenesis. These data, combined with reports from other investigators, indicate that chromosome 3q27-qter may contain a tumor suppressor gene that's important in the tumorigenesis of sporadic PETs.