DNA replication requires not just the synthesis of a copy of the genome, but the separation of old and new chromosomes and their safe delivery into daughter cells. This proposal investigates three aspects of chromosome disentanglement and partitioning. First, an important class of proteins in the condensation and segregation of chromosomes is the SMC (Structural Maintenance of Chromosomes) proteins. These essential proteins have been conserved from bacteria to man. Dr. Cozzarelli has shown that 13S condensin, an SMC protein complex of Xenopus compacts pure DNA by introducing a global positive writhe. The mechanism of its condensation of DNA will be investigated by single molecule force-extension measurements, microscopy, DNA ring closure kinetics, and enzyme kinetics. The compaction of chromatin by condensin will also be investigated. Second, the structure of intermediates in DNA replication will be studied. This work will focus on the properties of a four-way junction that is generated at the fork of replication intermediates by positive superhelical stress. Third, the structure, maintenance and roles of topological domains in bacterial chromosomes will be examined. These oft-postulated but little studied entities are presumed to divide up the chromosome into topologically closed regions that limit damage to DNA and facilitate DNA unlinking. The key method in the analysis will be the detection simultaneously of all transcribed or replicated genes in Escherichia coli by hybridization to a microarray of all the E. coli genes on a glass slide. It is hoped to determine what maintains the boundaries of domains, the size of domains, how domains are formed and removed, and what proteins influence domain stability. As part of this study, a comprehensive analysis of supercoiling-sensitive genes in E. coli will be performed. It is hoped to determine which genes are turned on and off by negative and positive supercoiling and by relaxation. This work will help illuminate the factors promoting chromosome partitioning. Mis-segregation leading to aneuploidy is an important step in many cancers. An aspect of the health relationship of this work is that replication is vital to all organisms and a number of the most successful anti-cancer and antibacterial agents inhibit enzymes in this process.
| Ptacin, Jerod L; Nollmann, Marcelo; Becker, Eric C et al. (2008) Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis. Nat Struct Mol Biol 15:485-93 |
| Nollmann, Marcelo; Crisona, Nancy J; Arimondo, Paola B (2007) Thirty years of Escherichia coli DNA gyrase: from in vivo function to single-molecule mechanism. Biochimie 89:490-9 |
| Nollmann, Marcelo; Stone, Michael D; Bryant, Zev et al. (2007) Multiple modes of Escherichia coli DNA gyrase activity revealed by force and torque. Nat Struct Mol Biol 14:264-71 |
| Ptacin, Jerod L; Nollmann, Marcelo; Bustamante, Carlos et al. (2006) Identification of the FtsK sequence-recognition domain. Nat Struct Mol Biol 13:1023-5 |
| Stray, James E; Crisona, Nancy J; Belotserkovskii, Boris P et al. (2005) The Saccharomyces cerevisiae Smc2/4 condensin compacts DNA into (+) chiral structures without net supercoiling. J Biol Chem 280:34723-34 |
| Levy, Oren; Ptacin, Jerod L; Pease, Paul J et al. (2005) Identification of oligonucleotide sequences that direct the movement of the Escherichia coli FtsK translocase. Proc Natl Acad Sci U S A 102:17618-23 |
| Breier, Adam M; Weier, Heinz-Ulrich G; Cozzarelli, Nicholas R (2005) Independence of replisomes in Escherichia coli chromosomal replication. Proc Natl Acad Sci U S A 102:3942-7 |
| Camara, Johanna E; Breier, Adam M; Brendler, Therese et al. (2005) Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication. EMBO Rep 6:736-41 |
| Breier, Adam M; Cozzarelli, Nicholas R (2004) Linear ordering and dynamic segregation of the bacterial chromosome. Proc Natl Acad Sci U S A 101:9175-6 |
| Manna, Dipankar; Breier, Adam M; Higgins, N Patrick (2004) Microarray analysis of transposition targets in Escherichia coli: the impact of transcription. Proc Natl Acad Sci U S A 101:9780-5 |
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