Norbert O. Reich - US grants

This proposal addresses a fundamental problem in biochemistry-the nature of specific protein-DNA interactions. The general goal is to elucidate how the EcoRI methylase catalyzes transfer of methyl group from S-adenosylmethionine (AdoMet) to the second adenine in the double stranded sequence: 5' GAATTC3'. Specific goals are to characterize: (1) the kinetic mechanism, (2) the chemical mechanism, (3) the contributions toward specificity deriving from individual enzyme-substrate interactions, (4) the structure of the methylase-DNA complex. These goals will be addressed with steady and presteady state kinetics, pH analyses, and modified DNA substrates. Specific chenical modification and genetic substitution of amino acids will be used with the kinetic studies to implicate specific residues in catalysis and substrate binding. The applicability of DNA-footprinting assays to determine the methylase-DNA complex topology and binding affinity will be tested. Collaborative crystallographic and 1H N.M.R. analyses of the methylase-DNA complex are ongoing. DNA methylases are essential for the in vitro manipulation of DNA and play a critical role in the regulation of gene expression in eukaryotes. Yet no structural and little function information is available for any DNA methylase. The proposed work is aimed at obtaining this information for the EcoRI methylase, a member of this important class of enzymes.

The PI propose to test if the prokaryotic EcoRI DNA methyltransferase (MTase) catalyzes methylation at N6 of adenine via a covalent intermediate involving cysteine 223 and the C6 position of adenine. This will be tested by constructing and testing mutant enzymes containing serine, glycine or alanine in place of cysteine 223. The existence of a covalent intermediate will be further tested with a mechanism-based inhibitor designed to activate the enzyme by forming a covalent linkage between cysteine 223 and the inhibitor. In addition to elucidating the chemical mechanism of this enzyme, these results may provide a new class of highly specific inhibitors as well as providing insights into the design of (bio)catalysts with novel substrate specificities and mechanisms. using both random and nonrandom in vitro mutagenesis methods, the substrate specificity of the enzyme will be offered. Specificity mutants will be identified through molecular selection and gene amplification using polymerase chain reaction techniques. Understanding which amino acids are critical for DNA recognition will aid in understanding of sequence- specific DNA modification mechanisms and may aid the design of agents with novel specificities.

DNA methylation is one of numerous mechanisms whereby mammalian gene expression is regulated, and changes in DNA methylation are implicated in mammalian cellular transformation. Methylation patterns are established during gametogenesis and early embryogenesis through the action of DNA (cytosine-5-)-methyltransferase (DNA Mtase); these patterns are maintained by DNA Mtase, enabling the clonal propagation of patterns of gene expression during differentiation. Little is known about what determines the methylation patterns, and in particular, the involvement of DNA Mtase in this process. The recent availability of homogeneous Mtase preparations and the protein sequence provide the opportunity for detailed biochemical investigation of this important enzyme. Using homogeneous DNA Mtase isolated from Friend murine erythroleukemia cells and synthetic DNA substrates we propose to elucidate various aspects of Mtase-DNA interactions. Based on our experience with EcoRI DNA Mtase, we propose to develop a sequence-specific DNA binding assay. This will be used to map the enzyme-substrate interface with DNA- footprinting methods, thus providing detailed information about the size of the interface and whether major and/or minor grooves are contacted. Further, this will allow investigation of what role(s) the large (1000 amino acids) N-terminal domain plays in DNA binding and discrimination of hemi-methylated substrates. Regions of the enzyme involved in DNA and AdoMet recognition will be identified using several cross-linking strategies in combination with mass spectrometric analysis. The binding assay will be used to quantitate enzyme interactions with native, hemi-methylated and single stranded substrates. Comparison of these binding affinities with the corresponding specificity constants (k(cat)/K(m)) should aid our understanding of the enzyme's well known preference for hemi-methylated substrates. We will determine if the recently reported substrate inhibition occurs as a result of complex enzyme-enzyme interactions or through multiple DNA substrates binding the same enzyme molecule. The reported inhibition of enzyme activity deriving from "nonsubstrate nucleic acids" will be mechanistically investigated because of the possible regulatory importance. Results from the proposed experiments will be used in future studies of sequence- specificity and isolation of cellular factors which modulate Mtase activity.

We propose to purchase an electrospray HPLC mass spectrometer (single quadrupole) to carry out four diverse structure-function studies, to provide training in an emerging analytical method for graduate students and post doctoral fellows, and to provide other researchers within the Chemistry Department and UCSB campus access to a contemporary molecular mass determination method, a technique presently unavailable on the campus. The first major project focuses on both bacterial and mammalian DNA methyltransferases. The target bacterial enzyme is the EcoRI DNA methyltransferase. Ongoing work on this NSF supported project has used various LC-MS methods, and the proposed work extends this effort. Characterization of critical histidines within the enzyme is proposed; a novel electrospray LC-MS method is under development and application to the methyltransferase should identify the essential histidine(s). The proposed LC-MS method is a significant improvement over presently used spectrophotometric methods and may be generally applicable. An LC-MS based strategy is proposed to determine which elements of the mammalian cytosine DNA methyltransferase are in contact with its substrate. A related strategy is proposed to identify portions of the DNA substrate contacted by the enzyme. The extent and site of post- translational modification of the mammalian enzyme are also proposed to be investigated by LC-MS methods. The characterization of the mammalian enzyme is important for understanding what role this protein plays in gene regulation, cancer, and genetic imprinting. The second major project focuses on the acetylcholine transporter of synaptic vesicles. Identification of the acetylcholine binding site within the protein is proposed using radiolabeled photoaffinity analogs and LC-MS methods. Characterization of various site directed mutants of the transporter is proposed, including the extent to which these mutants are modified in their post-trans lational modification. A related proposed set of experiments is designed to assess how different growth conditions impact on the post-translational processing of the transporter. The proposed structure-function analysis of the transporter should help elucidate how the neurotransmitter acetylcholine functions. The third project proposes to use LC-MS methods to elucidate metal ion binding selectivities of siderophores produced by open ocean marine bacteria. The metal binding affinity and selectivity of the recently identified siderophore Alterobactin A, the first structurally characterized siderophore from an open ocean organism, will be determined. Thus the speciation of the siderophore in seawater can be determined. A novel LC-MS method is proposed to screen a large number of potential metals; application to other marine siderophores is proposed. The fourth project focuses on the use of high field N.M.R. to investigate protein- nucleic acid and protein-protein interactions. LC-MS is proposed as an additional means of characterizing the target biomolecules. The purchase of the proposed LC-MS instrument would enable the training of numerous graduate students and research fellows involved in these projects, as well as undergraduates and other individuals associated with intended minor users. This proposed training targets the increasing need in the biotechnology industry for protein chemists trained in contemporary analytical methods.

9412078 Reich The information content of DNA is expanded in almost all organisms by methylation of adenine (N6) or cytosine (C5 and N4). DNA methylation is involved in the control of tissue specific and developmentally regulated gene expression; the protection of host (bacterial) DNA against endonuclease cleavage; DNA repair; aging; and genetic imprinting. The study of two bacterial DNA methyltransferases is proposed: EcoRI N6 adenine methyltransferase, which methylates the second adenine in GAATTC, and BamHI N4 cytosine methyltransferase, which methylates the first cytosine in GGATCC. The ready availability of the genes and large quantities of homogeneous protein provides the basis for the structure-function analyses to be performed. The general goal is to understand the molecular details of sequence-specific DNA methylation sufficiently to design novel biocatalysts, enzyme "mimics", and/or modify the sequence-specificity of the target enzyme. Two specific aims are detailed. The first is to identify structural features of the EcoRI methyltransferase or its substrate, and to a lesser extent the BamHI methyltransferase, that are essential for catalysis. The second aim is to determine the three-dimensional structure of the EcoRI or BamHI methyltransferase and their corresponding protein-cofactor as well as protein DNA-cofactor complexes using X-ray crystallographic analyses of the wild type proteins as well as point and deletion mutants. This structural information will aid our understanding of past structure-function studies with the EcoRI methyltransferase as well as provide the basis for future studies aimed at elucidating sequence-specific DNA modification. %%% These experiments are designed to characterize bacterial DNA methyltransferases. These enzymes modify unique DNA sequences by methylating adenine and cytosine. Structural characterization using X-ray crystallography and nuclear magnetic resonance is proposed. Also portions of the enzymes which contact the DNA will be identified using laser cross linking and mass spectrometry. Functional characterization will largely be through protein engineering and protein modification experiments. The goal of this research is to obtain a molecular understanding of how these enzymes modify unique sites within DNA. Sequence-specific DNA modification is a universal biological process which has practical applications in the field of molecular biology. ***

DNA methylation is one of numerous mechanisms whereby mammalian gene expression is regulated, and changes in DNA methylation are implicated in mammalian cellular transformation. Methylation patterns are established during gametogenesis and early embryogenesis through the action of DNA (cytosine-5-)-methyltransferase (DNA Mtase); these patterns are maintained by DNA Mtase, enabling the clonal propagation of patterns of gene expression during differentiation. Little is known about what determines the methylation patterns, and in particular, the involvement of DNA Mtase in this process. The recent availability of homogeneous Mtase preparations and the protein sequence provide the opportunity for detailed biochemical investigation of this important enzyme. Using homogeneous DNA Mtase isolated from Friend murine erythroleukemia cells and synthetic DNA substrates we propose to elucidate various aspects of Mtase-DNA interactions. Based on our experience with EcoRI DNA Mtase, we propose to develop a sequence-specific DNA binding assay. This will be used to map the enzyme-substrate interface with DNA- footprinting methods, thus providing detailed information about the size of the interface and whether major and/or minor grooves are contacted. Further, this will allow investigation of what role(s) the large (1000 amino acids) N-terminal domain plays in DNA binding and discrimination of hemi-methylated substrates. Regions of the enzyme involved in DNA and AdoMet recognition will be identified using several cross-linking strategies in combination with mass spectrometric analysis. The binding assay will be used to quantitate enzyme interactions with native, hemi-methylated and single stranded substrates. Comparison of these binding affinities with the corresponding specificity constants (k(cat)/K(m)) should aid our understanding of the enzyme's well known preference for hemi-methylated substrates. We will determine if the recently reported substrate inhibition occurs as a result of complex enzyme-enzyme interactions or through multiple DNA substrates binding the same enzyme molecule. The reported inhibition of enzyme activity deriving from "nonsubstrate nucleic acids" will be mechanistically investigated because of the possible regulatory importance. Results from the proposed experiments will be used in future studies of sequence- specificity and isolation of cellular factors which modulate Mtase activity.

9318142 Christoffersen The goal of this proposal is to purchase a modem computer- controlled fermentor that will be used to express a wide variety of proteins in microbial cells. The various projects that will utilize this facility reflect the breadth of interests present on the UCSB campus. The areas represented include proteins involved in neural growth factor receptor, DNA methylation, interferon regulation of cellular activities, DNA binding domains, plant hormone biosynthesis, plant secondary metabolism, the cellular cytoskeleton, egg sperm receptor and others. The common need to produce large amounts of protein for further biochemical or biophysical studies brings together these diverse investigators on this single proposal. The need for this instrumentation is due to advances in a variety of disciplines but most importantly is the development highly efficient expression vectors for E. coli or yeast cells. These simple, easy to growth organisms are extremely versatile in their ability or produce a variety of proteins under the right experimental conditions. Optimal expression for large scale experiments requires careful control of temperature, dissolved oxygen and agitation. The proposed fermentor would allow complete control of culture conditions and thus facilitate the recovery of milligram amounts of these various proteins.

This project is purchasing a network of six graphics workstations that, together with existing Macintosh and DEC computers, serves as the basis for new course developments in the area of computational chemistry within the undergraduate curriculum in the chemistry department and the Pharmacology Program of the biological sciences department. The new computers are being used in courses in Freshman chemistry, organic and advanced organic chemistry, biochemistry, polymer chemistry, computational chemistry, and upper-division pharmacology courses and are being used to support undergraduate research projects in the academic year and the summers. The capabilities of this new instrumentation makes it possible to communicate to students the reality and excitement of modern developments in chemistry and to involve these students in exercises and projects that will give them "hands-on" access to these computational techniques as an integral part of our undergraduate curriculum. Thus far, experimental courses in this area have been very enthusiastically accepted by students and give students a set of skills that serve them in graduate and professional schools and careers in the bulk chemical and pharmaceutical industries.

DNA methylation is essential for normal mammalian development, and inhibition of the enzyme responsible for DNA methylation [DNA cytosine C(5) methyltransferase, DCMTase] aids in alleviating oncogenesis. The long-term goal of the proposed characterization of the mammalian DCMTase is the development of novel inhibitors with potential applications as anticancer drugs. 1) The first specific aim is to determine the mechanism of cytosine methylation by the mammalian DCMTase and the structurally characterized bacterial DCMTase, M.Hhal. The goal is to provide a detailed kinetic description of the events starting with substrate addition, and ending with the methyl transfer step. This information is essential for the evaluation of inhibitor potency and for a mechanistic understanding of inhibitor action. This kinetic analysis will also form the basis for a quantitative assignment of the effects of designated DCMTase mutants. This specific aim will be addressed using pre-steady state kinetic methods, partition analysis, steady-and pre- steady state fluorescence spectroscopy, and mutagenesis of active site residues. 2) The second specific aim is to characterize the mechanism of mammalian DCMTase inhibition observed with single-stranded DNA. The DNA structural features that are essential for this potent inhibition will be identified. The hypothesis that the large, N-terminal domain of the DCMTase, is involved in binding the inhibitor will be tested. 3) The third specific aim is to determine if the mammalian DCMTse can catalyze the deamination of 5-methylcytosine to generate thymine. Others have proposed that this mutagenic reaction may account for many human genetic diseases including cancer. However, the reaction has only been demonstrated with bacterial DCMTases.

9983125
Reich
Enzymes that alter the structure of DNA perform functions that are essential for life. DNA methyltransferases expand the information content of DNA by modifying specific adenines and cytosines. DNA methylation is the most common form of epigenetic control of gene expression and occurs in organisms ranging from bacteria to man. This is an essential modification whose mechanism is not completely understood. This project addresses this topic using bacterial enzymes in an attempt to understand the molecular basis of sequence-specific DNA modification. DNA methylases bend the DNA and flip out their target bases prior to catalysis. This project builds on recent discoveries funded by NSF and conducted in this PI's laboratory, that allow the tracking of both DNA bending and base flipping in real time. This research area, the study of conformational mechanisms of DNA modifying enzymes, is only feasible through the use of several kinetic and spectroscopic methods pioneered by the PI and in conjunction with collaborators. Understanding the underlying mechanisms of such conformational changes is critical if we are to appreciate how such enzymes discriminate among DNA sequences, and carry out their particular chemistries. Most importantly, other methods such as x-ray crystallography do not provide the necessary quantitative and functional insights since they cannot provide dynamic information. This information is clearly important to gain an understanding of conformational transitions to enzyme specificity. The insights provided by this research should have implications for understanding the mechanism of action of other enzymes such as those involved in DNA repair since many of them also appear to use similar conformational mechanisms, as revealed by other investigators using methods similar to those pioneered here.

DESCRIPTION (provided by the applicant): The principal investigator and his collaborators propose to investigate the importance of protein dynamics on the catalytic rate enhancement of enzymatic methyl transfer. While much attention in the field of enzymatic catalysis is given to transition state stabilization or to entropic arguments, evidence in support of the importance of protein dynamics to catalysis is scant. Proteins are certainly known to undergo various types of conformational changes; for example, evidence in support of correlated motions contributing to ligand binding is available. Similar correlated motions have been identified for several enzyme-catalyzed reactions using molecular dynamic simulations. The investigators propose to apply a combination of x-ray crystallography, MD simulations, and functional analyses to provide an experimental and theoretical basis for relating protein dynamics and enzyme reaction rates. They propose to investigate M.HhaI, a bacterial S-adenosyl methionine-dependent DNA cytosine C5 methyltransferase. S-adenosyl methionine-dependent enzymes are widespread in biology and modify nucleic acids, proteins, lipids, carbohydrates, as well as drugs. Mammalian and bacterial DNA methyltransferases are important drug targets for anticancer and antibiotic drugs, respectively. M.HhaI is the best understood S-adenosyl methionine-dependent methyltransferase of any type, since numerous high-resolution cocrystal structures are available and both the kinetic and chemical mechanisms are well known. The principal investigator and his collaborators identified structural elements that appear to be important for protein dynamics through inspection of both the M.HhaI-DNA cocrystal structure and MD analysis of the same structure. Several mutants have been prepared and are undergoing crystallographic, multiple conformer (MCA), and MD analyses. They propose to compare this structural and dynamic information with measurements of the methyl transfer rate. Data analysis will focus on correlations between methylation and 1) protein flexibility, 2) interatomic distances, 3) correlated motions, 4) frequency of near attack conformations (NACs), and 5) distribution if conformers. Multiple conformer analysis of the WT M.HhaI-DNA and mutant cocrystal structures will be used to design additional mutants to test the relationship between methylation and correlated motions. An extract-based screen will follow random mutagenesis of segments implicated in correlated motions; candidate mutants will be submitted to the structural and functional analyses. The combination of structural, molecular dynamic, and functional analyses should provide unique insights with potentially broad impact, into how correlated conformational changes contribute to catalysis. M.HhaI represents a class of enzymes with significant medical applications, and a better mechanistic property.

The Chemistry of Life Processes Program in the Division of Chemistry is supporting Professor Norbert Reich of University of California Santa Barbara to investigate how cellular proteins locate specific positions on DNA. It is well known that DNA stores genetic information that is transcribed and translated within cells in other biological molecules and that underscores the life of every cell. The "reading and interpretation" of the DNA information require and are modulated by interactions with other molecules in the cell, particularly proteins. The search by proteins of specific locations on DNA is challenging because DNA appears very uniform. This proposal focuses on how the proteins identify specific locations on DNA to bind to and then how they translocate on the DNA to which they are bonded. This work will have a broader impact on scientists' ability to determine how the information stored in DNA is productively interpreted, which is a process common to all cells. It is having further impact on the education of the next generation of scientists, both undergraduate and graduate students, capable of recognizing and addressing scientific issues relevant for human health. Furthermore, Professor Reich and his students actively engage in activities aimed at explaining to broad audiences how science works and what benefits science brings to society.

Proteins that act on DNA are responsible for the vast majority of the functions of DNA. The overarching goal of this proposal is to provide a deeper understanding of one such protein, the bacterial DNA adenine methyltransferase (Dam, modifies 5'-GATC-3'). The ability of Dam and related enzymes to carry out multiple cycles of catalysis (processive catalysis) is critical to their biological roles. Yet, current models of processive catalysis are based largely on enzymes faced with entirely different biological challenges and do not account for the characteristics of these enzymes. For example, DNA methyltransferases efficiently modify multiple recognition sites with highly variable intersite distances, and this processivity is modulated by interacting proteins and DNA sequences that flank the target sites. The aims of the research are to investigate the mechanism, regulation, and in vivo importance of processive catalysis by Dam. Several strategies will be pursued to address broadly relevant and unanswered questions of how Dam and other proteins act processively with DNA substrates over intersite distances beyond the ones predicted by conventional models, how some proteins, such as Dam, rely on intra-segment transfer to efficiently move large distances, and what is the mechanism whereby the monomeric Dam modifies both strands of DNA without leaving the DNA.

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Norbert Reich from the University of California at Santa Barbara to understand how proteins scan DNA in order to find specific sites for action. This process is essential to all known cellular systems. A new idea about how this happens is that enhanced motion over large distances requires DNA looping. This highly efficient approach is investigated and modeled using a newly developed theoretical approach. The implications of this work are quite broad as this means of movement is likely to be used by various proteins. This project also supports the development of a university course focused on teaching STEM students how to design experiments through extensive lab work as well as lectures and discussions. Furthermore, the expansion of a large outreach program is proposed (SciTrek) which reaches thousands of K-12 students by bringing trained university students into the classroom for several weeks to guide self-directed investigations on diverse topics.

In studying the bacterial DNA adenine methyltransferase (Dam, modifies 5'-GATC-3'), it was noted that the ability to rapidly modify two or more sites is enhanced when the sites are separated up to 500 bp. Furthermore, current models of processive and distributive DNA modification fail to fully account for the efficient search processes by Dam and other proteins. A new understanding of the underlying mechanism is needed. Aim 1 attempts to answer the question: Do proteins relying on different mechanisms display distinct trajectories away from the DNA? Sliding, hopping, intersegmental transfer and intersegmental hopping mechanisms predict distinct protein movements away from the DNA and anovel experimental approach to address this is proposed. The second aim attempts to answer the question: What is the mechanism of intrasite processivity? How widespread is this activity? Dam as well as several other DNA modifying enzymes modifies both strands of DNA within the same recognition site, without dissociating (intrasite processivity). The third aim attempts to answer the question: Is efficient multisite DNA modification important in vivo? The fourth aim develops and applies quantitative theoretical/numerical tools for analyzing activity-based kinetic assays of DNA modifying enzymes. Particular emphasis is placed on elucidating the behavior of Dam, but additional systems (CcrM, uracil DNA glycosylase) are also studied to test and validate the approach more generally.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.