Tom E. Ellenberger - US grants

[unreadable] DESCRIPTION (provided by applicant): This proposal focuses on DNA repair enzymes and signaling proteins with multiple roles in cellular responses to DNA damage. We view these multitasking proteins as potential control nodes that may integrate DNA damage-specific signals and marshal the appropriate repair process(es) to the sites of DNA damage. A frontier area in the field of DNA repair is to understand how the biochemical pathways of DNA repair are coordinated with one another, in a manner analogous to other intracellular signaling pathways. Our immediate efforts are focused on 1) defining the mechanism of substrate selection by mammalian DNA ligase III in DNA damage responses, 2) creating a "chemical genetic switch" to shut off the NER pathway in order to explore other diverse functions of the repair endonuclease ERCC1-XPF, and 3) the enzymatic regulation of Sir2, a protein deacetylase with diverse activities, including DNA repair functions. We are using a structure-based approach to develop small molecule modulators of protein- protein interactions and enzymatic activities that can ultimately be used to probe the physical interactions and functional crosstalk between DNA repair pathways in living cells. Inhibitors of DNA repair activities may ultimately be useful in treating cancers that have become resistant to DNA crosslinkers, alkylating agents, and other DNA-targeted therapies. PUBLIC HEALTH RELEVANCE: We are studying how damaged DNA is repaired. These repair processes are essential for the normal maintenance of our genetic blueprint, but they can also cause resistance to anti-cancer drugs that kill tumors by inflicting damage to DNA. A better understanding of basic DNA repair mechanisms could lead to the development of drugs used to temporarily switch off repair during cancer chemotherapy. [unreadable] [unreadable] [unreadable]

DESCRIPTION: This is an ambitious program of x-ray crystal structure analyses, aimed at understanding the mode of action of the E. coli T7 DNA polymerase. T7 is important, in that it is one of the simplest of all replicative polymerases. It is a heterodimer consisting of an 80 kD gene 5 protein and a 12 kD E. coli thioredoxin. Both polymerase activity and the 3'-5' exonuclease activity of the holoenzyme reside in the gene 5 subunit. Thioredoxin is a processivity factor stabilizing interaction of the polymerase with DNA, increasing processivity from 10-50 bases to several thousand bases before the polymerase falls off the DNA template. In contrast to T7, most other replicative polymerases are large multiprotein complexes. Although T7 catalyzes DNA synthesis from primed single-stranded DNA in vitro, replication in E. coli requires assistance of several other phage-endcoded proteins. Among them, gene 2.5 protein, a dimer of 25.5 kD subunits, binds ssDNA with high affinity and greatly accelerates annealing of homologous DNA strands. It assists lagging-strand synthesis and contributes to the coupling of leading and lagging-strand syntheses. Specific projects planned include crystal structure analyses of: (1) ternary complexes of T7 polymerase, primer/template DNA, and mononucleotide, employing each of the four mononucleotides in turn: dGTP, dATP, dCTP and dTTP, in each case with the proper DNA template; (2) the "apopolymerase" alone, without bound DNA, (3) a complex of polymerase and primer/template having the wrong mononucleotide incorporated at the 3' position, or a 3' mismatch (following a search for the most stable mismatched complexes) and (4) the complex of T7 polymerase or one of its domains with the T7 gene 2.5 protein. This entire project represents a long-term collaboration with Prof. Charles Richardson, also of Harvard.

The metalloenzyme nitric oxide synthase (NOS) regulates nitric oxide (NO) synthesis and thereby its biological activity. NO has a dual role as 1) a diffusible, biological messenger for neurotransimission, long-term potentiatltion, platelet aggregation, and blood pressure regulation and 2) a cytotoxic agent for defense against tumor cells and intracellulare parasites. NOS enzymes, found in inducible (iNOS), constitutive endothelial (eNOS), and constitutive neuronal (nNOS) isoforms acheive their important biological function by adopting an intriguing calcium-regulated mechanism and incorporating a unique assembly of five cofactors: heme, tetrahydrobiopterin, contained in an oxygenase domain, and FMN, FAD and NADPH contained in a reductase domain. We have obtained crystals of the murine iNOS oxygenase domain that diffract to 2.5 angstrom resolution with a synchrotron source at 100 K. Cysteine mutants of iNOSox, designed to facilitate derivatization, are being produced and one has already been crystallized. An atomic structure of iNOSox should lend much insight to the understanding of a unique catalytic mechanism and potentially the physiological role of NOS and NO.

DESCRIPTION: This proposal is aimed at obtaining three-dimensional structural information about the process of recombination catalyzed by the lambda integrase (Int) protein. Int catalyzes the integration of the bacteriophages lambda into the bacterial host, as well as the excision of the genome during lysis. The reaction has some similarity to that catalyzed by Type I topoisomerases, in that a covalent linkage is formed between a tyrosine residue in the protein and the phosphate backbone of DNA. Building on previous work from his laboratory, Dr. Ellenberger proposes to study by x-ray crystallography the structures of lambda integrase bound to pieces of DNA corresponding to various stages in the reaction cycle. Crystals have been obtained of a dimer of lambda integrase bound to uncleaved DNA through the tyrosine linkage. This structure will resolve issues relating to whether the tyrosine residue of one integrase protein catalyzes bond cleavage within the active site of the same molecule, or whether it does so in that of the neighboring molecule. Interest in this issue arises because of a fundamental interest in understanding how cooperativity is utilized in driving DNA cleavage and religation. Further aims of the proposal include the determination of the structure of lambda integrase complexed with a four way Holliday Junction, as well as a very large (in terms of crystallography) DNA hairpin structure stabilized by the DNA bending protein known as IHF.

DESCRIPTION (provided by applicant): Phage lambda integrase (Int) is the archetype of a large family of site-specific DNA recombinases that function in the segregation of plasmids, viral and cellular chromosomes, the regulation of gene expression, and programmed gene rearrangements. These enzymes catalyze DNA cleavage and religation without the addition of high energy cofactors. They are the only enzymes known to both create branched Holliday junction intermediates and to resolve them into recombinant DNA duplexes. Recent crystal structures of the related P 1 Cre recombinase and the yeast Flp protein complexed to Holliday junctions have provided many insights into the recombination reaction. In contrast to these simple recombinases, lambda Int functions in higher order structures comprising multiple DNA sites and accessory factors that bend the DNA into a compact shape. These additional interactions, which are essential for Int-catalyzed recombination in vivo, allosterically regulate the efficiency and fidelity of DNA cleavage and strand transfer. We have determined a crystal structure of a covalent Int-DNA complex that, in comparison to an earlier structure of unbound Int, reveals a DNA-mediated switch in the structure of the enzyme active site. We now propose crystallographic studies of the higher order Int-DNA complexes that will address the physical basis for the allosteric regulation of recombination through interactions of Int's two autonomous DNA binding domains. Diffracting crystals of several of these larger Int-Holliday junction complexes have been grown and isomorphous heavy atom derivatives have been identified. The proposed crystal structure determinations, together with site-directed mutational studies of the protein subunit interfaces in these complexes, will address how DNA cleavage and ligation activities are regulated by the physical organization of the Int-DNA complexes. A physical description of the enzymatic processing of Holliday junctions and other types of DNA recombination joints is a key to mechanistic understanding of a variety of biological processes that maintain chromosome structure or create genetic diversity. The lambda Int recombination system is a model for studying the chemistry of the trans-esterification reactions and the orchestration of pairwise DNA strand exchanges that create the Holliday junction intermediate and then resolve it into recombinant DNA products.

crystallization; macromolecule; chemical structure; biomedical resource; structural biology; synchrotrons;

The Project (Nucleotide Excision Repair and Base Excision Repair) focuses on understanding the interactions of key proteins with DNA substrates and partner proteins critical for functional DNA excision repair complexes. Our goal is to leverage structural and biochemical studies of the repair endonuclease ERCC1-XPF and of DNA ligase to illuminate the coordination of the multi-step reaction pathways that comprise Nucleotide Excision Repair (NER) and the completion of B_ase Excision Repair (NER). Defects in DNA excision repair proteins and pathways result in increased rates of mutation, chromosomal breakage, and an increased incidence of cancers. Distortions of the helical structure of DNA are specifically recognized by repair enzymes and can be read out in a way that does not depend on the chemical identity of the damage. This generalized strategy enables one enzyme to initiate the repair of a variety of lesions in DNA. Moreover, interactions with other DNA binding and repair proteins provide additional biological specificity and contribute to the efficiency of repair. Although the enzymatic activities constituting the basic NER and BER pathways are known, it remains to be determined how these activities are coordinated into a multi-step reaction pathway by the physical interactions within enzyme-DNA complexes catalyzing the excision repair of DNA damage. Structural analyses of the relevant enzyme-DNA complexes will reveal distinct conformational states of the enzymes and their DNA substrates corresponding to different steps of the repair reaction. Low-resolution structures and conformations derived from x-ray scattering in solution will complement high-resolution images of the repair complexes that can be crystallized. In this way, the dynamic assembly and disassembly of multi-protein complexes catalyzing DNA repair will be characterized. We propose to test and develop these hypotheses by investigating specific excision repair components as follows: 1) Catalytic Substrate specificity of XPF-ERCC1, and related DNA structure-specific nucleases;2) Damage senses by XPA that recruits XPFERCC1 to NER complexes;3) Catalytic selectivity of DNA ligases;Nick-sensing and DNA repair;4) Interactions of DNA Ligase I with DNA sliding clamps;and 5) Interactions of ligase I with the clamp loaders.

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We are studying the structural organization of DNA repair complexes that excise DNA damage and the functional consequences of disrupting these protein-protein interactions by mutation and small molecule inhibitors. Alternative pathways of repair are available for many types of DNA damage, and posttranslational modifications generated in response to DNA damage control the differential assembly of repair complexes, providing a mechanism for regulating pathway choice. Cancer-associated defects in DNA maintenance activities can be exploited therapeutically by targeting the remaining repair activities with mechanism-based inhibitors. Our work focuses on the mechanisms of repairing DNA single strand breaks generated by the base excision and nucleotide excision repair pathways. We are studying the physical assembly of DNA damage excision complexes in vitro and in cultured cells, and the mechanism of coupling DNA cleavage to end processing and ligation. Small angle x-ray scattering of purified DNA repair complexes reveals dynamic conformational states that we propose are important for handoffs of DNA repair intermediates to successive enzymes in a pathway. High resolution crystal structures and small molecule screening experiments are being used to predict and identify inhibitors of repair protein interactions, which are candidates for anti-tumor therapies and serve as reversible chemical probes of cellular physiology during DNA damage responses. This integrated approach takes advantage of the broad expertise of investigators in Projects 1, 2, and 6 for assays and biological materials, as well as the unique capabilities of the Expression and Molecular Biology Core and the Structural Cell Biology Core of the SBDR Program to produce proteins and structurally evaluate repair complexes.

? DESCRIPTION (provided by applicant) Molecularly-targeted cancer therapies have revolutionized the treatment of this heterogeneous and increasingly prevalent disease. Genetic instability is a hallmark of many cancers that generates mutations to support uncontrolled tumor growth and resistance to chemotherapies. The underlying DNA repair defects in these tumors can be exploited in tumor-selective therapies that block critical remaining DNA repair functions to trigger catastrophic damage and cell death. This idea is borne out by the clinical successes of inhibitors of poly(ADP-ribose) polymerase 1 (PARP1) to treat breast and ovarian cancers with mutations in BRCA1 or BRCA2. However, these BRCA-deficient tumors account for a minority of cancers so it is important to identify other physiological defects of tumors that are synthetically lethal in combination with molecularly targeted therapies. Additionally, the current PARP inhibitors suffer from dose-limiting toxicities, which may result from off-target effects on other members of the large PARP superfamily. As an alternative to PARP inhibitors, we used high-throughput screening to identify selective inhibitors of the human poly(ADP-ribose) glycohydrolase PARG. PARG is a monogenic enzyme that removes the poly(ADP-ribose) posttranslational modification of proteins modified by PARP1. A genetic knockdown of PARG sensitizes cancer cells to DNA damaging agents and radiation and phenocopies the tumor-specific killing effects of PARP1 enzymatic inhibitors in BRCA- deficient cancer cells. In this application, we propose experiments to improve the potency and selectivity of small molecule PARG inhibitors through structure-guided chemical synthesis and testing in vitro, and to advance selected compounds to preclinical trials of tumor killing activity in cultured cells and xenograft models of breast cancer. We will synthesize focused libraries of analogs that exploit unique features of the PARG active site and screen small molecule fragment library to identify new chemotypes and interactions that can be incorporated into our inhibitor design strategy. Selective inhibitors of PARG will be useful probes of cellular responses to cancer chemotherapeutics that damage DNA, and may be useful cancer therapies in their own right by exploiting the genomic instability phenotype of many tumors.