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. We will investigate three key aspects of chromosome partitioning. First, condensins are essential proteins for the condensation, organization, and segregation of chromosomes that have been conserved from bacteria to man. We will focus on the condensins from yeast and Escherichia coli so that we can do parallel biochemical, biophysical, topological, and genetic studies. We have shown that these giant proteins form a filament with DNA that, in the presence of ATP, introduces a right-handed writhe into DNA and compacts it about 10-fold. The condesin filament from bacteria has extraordinary stability and elasticity and we shall determine if eukaryotic condensins share these properties. We will study the structure of the filament, with particular emphasis on the path of DNA. We will investigate the mechanism of condensation of DNA by single molecule force-extension measurements, microscopy, DNA probing, and enzyme kinetics. We will initiate the study of a yeast protein analogous to condensin that plays a key role in DNA repair. Second, we will study the mechanism of DNA translocases involved in chromosome partitioning. Our very recent results show that the E. coli translocase moves DNA at an astonishing speed and in a nucleotide sequence-directed, unidirectional fashion to promote recombination and chromosome segregation. Using a combination of single molecule and ensemble biochemistry experiments, we hope to determine the generality of our results for other DNA translocases and how unidirectional movement and energy coupling are brought about. Third, we will examine the structure, maintenance, and roles of topological domains in chromosomes. The division of the chromosomes into topologically closed supercoiled regions limits damage to DNA and facilitates DNA packaging and unlinking. The key methods that will be used are the isolation and characterization of E. coli mutants that alter topological domain boundaries or global supercoiling and the use of 330 supercoiling sensitive genes spread around the bacterial chromosome as reporters of whether an individual domain is intact. We hope to determine the proteins that maintains the boundaries of domains, how the boundaries are formed and removed, the localization of barriers, and how the global level of supercoiling is controlled. Our work will help illuminate the factors promoting chromosome partitioning. Missegregation leading to aneuploidy is an important step in the development of many cancers. An aspect of the health relationship of this work is that DNA replication is vital to all organisms and a number of the most successful anti-cancer and antibacterial agents inhibits enzymes in this process. The front line defense against many bacteria, including Bacillus anthracis, and over half of all chemotherapeutic regimens use drugs that inhibit the topological changes in DNA during replication by a mechanism discovered in my laboratory in work supported by NIGMS.
| 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 |
| Case, Ryan B; Chang, Yun-Pei; Smith, Steven B et al. (2004) The bacterial condensin MukBEF compacts DNA into a repetitive, stable structure. Science 305:222-7 |
| Simmons, Lyle A; Breier, Adam M; Cozzarelli, Nicholas R et al. (2004) Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability. Mol Microbiol 51:349-58 |
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