This application addresses chromosome behavior, organization and function in bacteria and yeast. The importance of the physico-mechanical properties of chromosomes is emphasized. I. Mechanism of Tn10 Transposition. (A) Transpososome assembly and pre- and post-assembly conformations will be investigated in vitro by analytical and crosslinking methods. (B) Structural analysis of transposase DNA complexes will be attempted, in collaboration with Dr. G. Van Duyne. II. E.coli DNA replication, cell division and chromosome organization. (A) Synchronous cell populations generated by a new baby cell method will be analyzed, by FACS, FISH, immunocytology and DNA assays, in wild type and mutant conditions. Roles of newly identified negative regulators and basic features of a new general model will be assessed. (B) Chromosome organization and physical properties will be analyzed by our new crosslinking method. (C) cis and trans coupling between/among different chromosomes and chromosomal regions will be probed by new methods. III. Eukaryotic chromosome organization and function. We will investigate basic processes in the context of chromosome breathing and concomitant chromosomal stress and stress relief. (A) Chromosome status will be analyzed using our newly developed crosslinking assay in wild type and genetically altered situations. Issues of interest include: R-band/G-band differences, chromosome expansion and relaxation, origin status and intersister relationships. (B, C) Coordinated studies in Sordaria and yeast will investigate Spo76/Pds5, proposed to be a transducer of expansional stress, and bulk chromatin proteins that could directly modulate chromatinlchromosome expansion (Bdf1/2, H1, HMG6AB and histone H3). (D) Studies of yeast DNA replication and cell cycle progression will investigate the role of newly discovered inter-origin elements (IOEs) in regulation of S-phase progression via Mecl. We will begin to investigate the hypothesis that cell cycle progression is governed by SLS's (sensors of local stress) and that Mec1/Esr1 is an integral component of this machinery and is a stress-dependent kinase.
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