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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Anderson, Richard A
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Harvard University
Schools of Arts and Sciences
United States
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Kleckner, Nancy E; Chatzi, Katerina; White, Martin A et al. (2018) Coordination of Growth, Chromosome Replication/Segregation, and Cell Division in E. coli. Front Microbiol 9:1469
Gutu, Andrian; Chang, Frederick; O'Shea, Erin K (2018) Dynamical localization of a thylakoid membrane binding protein is required for acquisition of photosynthetic competency. Mol Microbiol 108:16-31
Liu, Chenli; Danilowicz, Claudia; Kleckner, Nancy et al. (2017) Single molecule identification of homology-dependent interactions between long ssRNA and dsDNA. Nucleic Acids Res 45:894-901
Gladyshev, Eugene; Kleckner, Nancy (2017) Recombination-independent recognition of DNA homology for repeat-induced point mutation. Curr Genet 63:389-400
Yoon, Sang-Wook; Lee, Min-Su; Xaver, Martin et al. (2016) Meiotic prophase roles of Rec8 in crossover recombination and chromosome structure. Nucleic Acids Res 44:9296-9314
Zheng, Hai; Ho, Po-Yi; Jiang, Meiling et al. (2016) Interrogating the Escherichia coli cell cycle by cell dimension perturbations. Proc Natl Acad Sci U S A 113:15000-15005
Kleckner, Nancy (2016) Questions and Assays. Genetics 204:1343-1349
Gladyshev, Eugene; Kleckner, Nancy (2016) Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence. PLoS Genet 12:e1006015
Yang, Darren; Boyer, Benjamin; Prévost, Chantal et al. (2015) Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure. Nucleic Acids Res 43:10251-63
Peacock-Villada, Alexandra; Coljee, Vincent; Danilowicz, Claudia et al. (2015) ssDNA Pairing Accuracy Increases When Abasic Sites Divide Nucleotides into Small Groups. PLoS One 10:e0130875

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