RNA Ligase Function and Use in DNA Synthesis

University Of California Berkeley, Berkeley CA

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): 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.