Arthur Kornberg - US grants

This program entitled "DNA Transactions and Aging" consists of an interrelated set of six projects directed by Drs. Paul Berg, Ronald W. Davis, David S. Hogness, Dale Kaiser, Arthur Kornberg and Robert Lehman. The program focuses on DNA transactions that we think are relevant to aging because they either cause or prevent alterations in a cell's repertoire of genetic functions. The rate at which such genetic errors accumulate in cell populations is clearly important to any consideration of the aging problem and, of course, that rate is determined by the interplay between these causative and preventive transactions. The program encompasses two areas of concentration within this central theme. One of these concerns the transposable DNA elements that move about the genome and generate mutational rearrangements in the chromosomal DNA. The other concerns those transactions that repair damage to the chromosomal DNA and regulate the fidelity of DNA replication.

We intend to characterize the numerous enzymes and proteins which we have identified and isolated as participants in the initiation of a cycle of replication of the E. coli chromosome at its origin (oriC) and the initiation of nascent chains at replications forks. These are some of our specific objectives: (i) Described in molecular detail how the origin of the E. coli chromosome is recognized by dnaA protein and so configured to create a bubble for the priming of replication. (ii) Discover how the dnaB helicase, with the aid of dnaC protein, is positioned to create bidirectional forks of replication. (iii) Determine the mechanisms and varieties of transcription activation of initiation at oriC. (iv) Examine the fate and cycling of the proteins that participate in initiation, the influence of membranes, and of ATP and energy charge. (v) Explore existence of factors (positive or negative) which may serve as signals from the increase in cell mass that commit the cell to initiate a new cycle of replication. (vi) Determine the genetic loci and prepare mutants of the primosomal proteins (n, n', and n") to evaluate their physiological importance and their operation at the replication forks of the E. coli chromosome. These biochemical studies are essential for revealing the facts and patterns of replication basic to understanding cell growth and cell division in eukaryotic as well as prokaryotic organisms. With a grasp of the biochemical nature of the switch that operates the initiation of replication, will come opportunities to discover the factors that regulate orderly growth and those responsible for uncontrolled proliferation (e.g. cancer) or its premature cessation.

We intend to characterize the numerous enzymes and proteins which we have identified and isolated as participants in the initiation of a cycle of replication of the E. coli chromosome at its origin (oriC) and the initiation of nascent chains at replications forks. These are some of our specific objectives: (i) Described in molecular detail how the origin of the E. coli chromosome is recognized by dnaA protein and so configured to create a bubble for the priming of replication. (ii) Discover how the dnaB helicase, with the aid of dnaC protein, is positioned to create bidirectional forks of replication. (iii) Determine the mechanisms and varieties of transcription activation of initiation at oriC. (iv) Examine the fate and cycling of the proteins that participate in initiation, the influence of membranes, and of ATP and energy charge. (v) Explore existence of factors (positive or negative) which may serve as signals from the increase in cell mass that commit the cell to initiate a new cycle of replication. (vi) Determine the genetic loci and prepare mutants of the primosomal proteins (n, n', and n") to evaluate their physiological importance and their operation at the replication forks of the E. coli chromosome. These biochemical studies are essential for revealing the facts and patterns of replication basic to understanding cell growth and cell division in eukaryotic as well as prokaryotic organisms. With a grasp of the biochemical nature of the switch that operates the initiation of replication, will come opportunities to discover the factors that regulate orderly growth and those responsible for uncontrolled proliferation (e.g. cancer) or its premature cessation.

DESCRIPTION: Inorganic polyphosphate is a linear polymer of hundreds of phosphate residues linked by the high-energy phosphoanhydride bonds found in ATP. In pre-biotic evolution, PolyP likely served as a precursor and catalyst in the synthesis of nucleic acids and proteins. Now poly P is found in every cell in Nature-bacteria, fungi, plants and animals. In human brain for example, the poly P chains are about 1000 residues long and concentrated in the nuclei of several cell types. In neuron-like PC-12 cells, poly P is dynamic with a turnover time of about one-hour. The purpose of this study is to gain an understanding of the biochemistry of poly P, the genetic determinants of its synthesis and utilization, and its physiological functions, especially with regard to cellular responses to nutritional deficiencies, environmental stresses and other factors in the aging process. Special emphasis will be given to the role of poly P in E. coli and other micro-organisms in which biochemical and genetic data are already in hand and the role of poly P in regulatory networks shown to be essential for survival in the stationary phase of growth. The proposed research will focus on three areas: (1) advances in methodology for high-throughput assays of poly P and related enzymes suitable for screening a thousand samples a day; (2) intensive study of the structure and function of the biosynthetic enzyme poly P kinase, and the principal exopolyphosphatases; and (3) genetic studies of networking of poly P accumulation and utilization with the regulatory proteins PhoB, Re1 A, Ntrc, and unidentified salt-stress proteins. Notable about poly P are its conservation, ubiquity, substitution for ATP, chelation, and storage of phosphate and pathogenesis of Neisseria meningitides, Helicobacter pylori, Pseudomonas aeruginosa. In view of these many possible functions, it seems likely that poly P will prove to have one or more roles in human metabolism, differentiation, and aberrations of growth and aging.

DESCRIPTION (provided by applicant): Inorganic polyphosphate (poly P), a polymer of hundreds of phosphate residues linked by high-energy bonds, is found in every cell in Nature-bacteria, fungi, plants and animals. Among poiy P functions are: kinase donor to glucose and nucleoside diphosphates, phosphate (P1) reservoir, divalent metal (Ca2+, Mg2+, Mn2+) chelator and component of a membrane complex in bacterial transformation. Our recent studies have disclosed three major roles: 1) "alarmone" in response to stresses and deficiencies, 2) adaptations for survival in the stationary phase and 3) virulence in some pathogens. Thus, poly P has a variety of functions depending on the cell and circumstances. The multiplicity of functions and the plasticity of microbial organisms has complicated the search for the biochemical mechanisms responsible for each action. The proposed research will focus on mechanisms of metabolic regulation. Based on promising preliminary studies, we intend to pursue two major lines: 1) Regulation of protein synthesis and turnover by the supply of amino acids by biosynthesis and protein turnover, and 2) Operations of phosphate uptake and efflux and concomitant divalent metal ion transport. In addition, we will be mindful of control of in vitro transcription of genes known to be activated by poly P in vivo and a possible role for poly P as a donor for protein kinases. The widespread conservation of the enzyme, poly P kinase (PPK), responsible for poiy P synthesis in bacteria, including many of the major pathogens, has led us to knockout ppk in several of these pathogens. In Pseudomonas aeruginosa poly P is essential for motility, quorum sensing, bioflim formation and virulence in mice. We expect that the proposed biochemical studies will reveal mechanisms that will assist in the current search for antimicrobial drugs aimed at PPK as a target. The research may also provide clues to the biosynthesis and metabolism of poly P in eukaryotes about which little is known. In view of the ubiquity of poly P in animal cells and subcellular organelles (particularly nuclei), it seems likely that poly P serves multiple functions in human metabolism that affect growth, differentiation and disease.

DESCRIPTION (provided by applicant): Inorganic polyphosphate (poly P) is a polymer of hundreds of phosphate residues linked by high-energy phosphoanhydride bonds found in ATP. In prebiotic evolution, poly P likely served as a precursor and catalyst in the synthesis of nucleic acids and proteins. Now, poly P is found in every cell in nature-bacteria, fungi, plants and animals. In human brain, for example, the poly P chains are about 1000 residues long and concentrated in the nuclei of several cell types. In neuron-like PC- 12 cells, poly P is dynamic with a turnover time of one hour.The purpose of this study is to gain an understanding of the biochemistry of poly P, the genetic determinants of its synthesis and utilization, and its physiologic functions, especially with regard to cellular responses to nutritional deficiencies, environmental stresses and virulence in pathogenic bacteria.The proposed research will focus on three areas: 1) Novel pathways of poly P synthesis in addition to that of poly P kinase (PPK 1) already characterized in Escherichia coli and other bacteria. A new enzyme (PPK2) discovered in Pseudomonas aeruginosa will be isolated, characterized and its gene cloned, deleted and overexpressed. The physiological roles, distribution in pro- and eukaryotic species will be explored. 2) Endopoly Pase (PPN) will be purified in its proteolytically activated state and its gene cloned, transferred and overexpressed. The basis for requirement of PPN for survival in yeast will be studied as will the use of PPN as a unique reagent in vivo and in vitro. 3) Poly P in animal cells and tissues, about which so little is now known will be investigated as we have done for bacteria.The extraordinary ubiquity and conservation of poly P and its proven regulatory roles in bacteria suggest that poly P may serve a variety of functions in human cells in relation to growth, development, aging phenomena, responses to environmental stresses and aberrations of disease.