Nicholas R. Cozzarelli - US grants

The major focus of the work will be on the application of topological and genetic approaches to the study of several key enzymes in DNA metabolism. This will involve developing new techniques for determining DNA structure and expanding the theory of DNA folding. In addition, we hope to isolate mutants of E. coli topoisomerase III to determine its function in vivo. We will also investigate the enzymes in E. coli that metabolize DNA knots and catenases and measure the functional level of DNA supercoiling in this organism and perhaps in yeast. Using a rigorous topological method, the mechanism of chromosome segregation in several organisms will be tested. Analogous methods will be brought to bear on the mechanism of topoisomerases. We will continue our studies of transcription by RNA polymerase III and its accessory factors. This will involve purification of the factors and determination of their role, analysis of the formation of transcription complexes, measurement of the DNA binding sites, and exploration of the striking increase in a polymerase III transcript after neoplastic transformation.

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.

The major focus of the work will be on the application of topological and genetic approaches to the study of several key enzymes in DNA metabolism. This will involve developing new techniques for determining DNA structure and expanding the theory of DNA folding. In addition, we hope to isolate mutants of E. coli topoisomerase III to determine its function in vivo. We will also investigate the enzymes in E. coli that metabolize DNA knots and catenases and measure the functional level of DNA supercoiling in this organism and perhaps in yeast. Using a rigorous topological method, the mechanism of chromosome segregation in several organisms will be tested. Analogous methods will be brought to bear on the mechanism of topoisomerases. We will continue our studies of transcription by RNA polymerase III and its accessory factors. This will involve purification of the factors and determination of their role, analysis of the formation of transcription complexes, measurement of the DNA binding sites, and exploration of the striking increase in a polymerase III transcript after neoplastic transformation.

The major focus of the work will be on the application of topological and genetic approaches to the study of several key enzymes in DNA metabolism. This will involve developing new techniques for determining DNA structure and expanding the theory of DNA folding. In addition, we hope to isolate mutants of E. coli topoisomerase III to determine its function in vivo. We will also investigate the enzymes in E. coli that metabolize DNA knots and catenases and measure the functional level of DNA supercoiling in this organism and perhaps in yeast. Using a rigorous topological method, the mechanism of chromosome segregation in several organisms will be tested. Analogous methods will be brought to bear on the mechanism of topoisomerases. We will continue our studies of transcription by RNA polymerase III and its accessory factors. This will involve purification of the factors and determination of their role, analysis of the formation of transcription complexes, measurement of the DNA binding sites, and exploration of the striking increase in a polymerase III transcript after neoplastic transformation.

The long-term objectives of this grant are a molecular understanding of two related aspects of mutation rate and its control in E. coli: (1) mechanisms that provide for the fidelity of normal DNA replication; (2) basis for the enhanced mutation rate and replicational repair induced by the SOS response to DNA damage. The major specific aims for this grant period are to understand: (1) the relative contribution and mechanism of base selection and editing for the fidelity of DNA polymerase III holoenzyme; (2) the biochemistry of the multi-protein SOS-induced pathway for point mutations (UmuCD pathway); (3) the relationship between the point-mutation pathway and other SOS pathways involving nonmutagenic replicational repair and duplication mutations. The goal in all three areas is to define the contribution of each individual protein to the overall pathway and to correlate the biochemical experiments using pure proteins with the results of genetic and physiological analysis in vivo. An understanding of mutation rate and the mutagenic effects of DNA lesions has relevance to carcinogenesis in humans. Oncogenes can clearly be activated by mutation, and there is a correlation between the mutagenic and carcinogenic effect of agents that damage DNA. Experiments on base selection will involve: (i) a more complete study of the specificity of nucleotide insertion; (ii) a more refined kinetic analysis of the reaction pathway for incorporation of correct and incorrect nucleotides. Work on editing will address: (i) the intrinsic exonuclease specificity of the editing subunit of polymerase III; (ii) contribution of the polymerase subunit to editing specificity, especially by kinetics of chain elongation from a mispaired base. For the SOS work, the experiments will seek to reconstitute the mutagenic pathway with purified proteins and to explore the mechanism of the nonmutagenic pathway termed replication-restart. Experiments will fall into four groups: (i) use of a simple replication system with currently available proteins; (ii) study of possible additional protein components; (iii) analysis of protein-protein interactions; (iv) investigation of more complex double strand replication systems.

electron microscopy; biomedical facility; bioimaging /biomedical imaging;

DNA repair; DNA replication; gene mutation; oxidative stress; enzyme activity; ultraviolet radiation; carcinogenesis; conformation; genetic recombination; iron; endonuclease; light adverse effect; DNA directed DNA polymerase; nucleic acid structure;

[unreadable] DESCRIPTION (provided by applicant): This project focuses on three giant motor proteins, toposiomerases, helicases, and FtsK that move DNA through large distances utilizing the energy of NTP hydrolysis and mechanical strain on DNA. We hope to understand how these proteins perform these vital roles in DNA replication and chromosomal segregation. We will use single DNA molecule enzymology complemented with bulk measures. The action of a single enzyme acting on DNA is measured by the resultant changes in DNA force, torque, and extension. The single DNA molecules can be supercoiled or braided at will to generate substrates for the enzymes. We will measure the rates of enzyme action, processivity, stall force, and chirality in interaction with superhelical DNA. These results will then be compared with bulk measures and measures in vivo. The clear medical relevance stems primarily from two sources. First, topoisomerases are the favored targets of antibiotics such as ciprofloxacin, and anticancer agents, such as etoposide and adriamyin. The understanding of their unusual dominant poisoning of their targets has greatly aided the development of more potent drugs. Second, interference in proper segregation of chromosomes by mutations that affect motor proteins accompanies and exacerbates human diseases, including cancer and premature aging.