PROBING GUANINE STRUCTURE IN NUCLEIC ACID FOLDING
Steven E Rokita, Professor
State University New York Stony Brook Stony Brook, Ny 11794
Grant 5R01GM047531-02 from National Institute Of General Medical Sciences IRG: BMT
Abstract: Transition metal complexes have captured considerable attention in the biomedical community because of their ability to bind certain nucleic acid structures and to promote chemical modification at or near the site of binding. Such processes occur "naturally" when metalloproteins operate on DNA or RNA, when metal-based toxins or drugs select genetic material as their target, and when metal complexes are used as conformation specific probes of nucleic acids. These laboratories have recently identified a series of nickel complexes to be exceptional probes for the secondary and tertiary structure of guanine in DNA and RNA. The utility and application of these complexes will now be explored in depth using well defined models and large polynucleotide systems of current interest. An accurate of nucleic acid folding must include a wide variety of conformations that significantly depart from the canonical double helix. Great interest in studying the polymorphic nature of nucleic acids has arisen from the preeminent role secondary and tertiary structure seems to play in recognition, regulation and reactivity of genetic information. While small oligonucleotide models may be examined in great detail by physical methods, larger systems may only be characterized through their chemical and biological activity. Ideally, reagents should be made available to identify the solvent accessibility of each group or site in a polynucleotide structure. Initial analysis suggests that the nickel complexes described herein are unrivaled in their absolute specificity for detecting guanine residues held in one of a number of non- Watson-Crick base pairing arrangements. Investigations will fully define the nickel reagent s selectivity with targets containing mismatched, bulged, hairpin and pseudoknot sequences. Oligonucleotide models have been chosen for these analyses so that direct correlations can be drawn between these chemical studies and the existing structural results obtained from magnetic resonance and crystallography. Polynucleotide studies will follow to provide a new perspective on key structures such as bends, cruciforms and protein-DNA complexes. Most importantly, the nickel species will also be applied to a number of contemporary problems of enormous impact. For example, the accessibility of guanine will be determined for RNA folding patterns that (i) regulate gene translation and (ii) form RNA-based catalysts.
Keywords: guanine, metal complex, nucleic acid structure, chemical model, conformation, nickel
Project start date: 1993-08-01
Project end date: 1995-07-31
5R01GM047531-02 (1994): $159615
Sponsored Links Excellgen http://Excellgen.com
NICKEL CARCINOGENESIS--MODIFICATION OF NUCLEOSOMES
Steven E Rokita, Professor
University Of Maryland College Pk Campus 3112 Lee Building College Park, Md 207425141
Grant 5R01GM047531-08 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: Our daily exposure to a variety of common materials containing nickel such as coins, kitchen equipment and costume jewelry belies the highly carcinogenic and often toxic nature of many nickel compounds. In particular, amorphous nickel subsulfide (Ni3S2) and water soluble nickel sulfate (NiSO4) have both been linked to pulmonary and renal cancers in workers exposed from an industrial environment. The molecular basis of this carcinogenic activity has not been identified and awaits a more complete understanding of nickel s biochemical reactivity. Simple nickel(II) salts alone demonstrate little ability to modify DNA and proteins irreversibly. However, when nickel binds to strong donor ligands, the resulting complex may promote a number of biologically destructive reactions. Peptides and proteins represent the most significant class of such ligands available within a cell, and DNA-binding proteins are obvious candidates for mediating genomic damage. Preliminary data suggests that histones do indeed activate the oxidative chemistry of nickel and serve, along with bound DNA, as targets of modification. The significant expertise gained by this laboratory on the biomimetic reactions of nickel during the previous funding period will now be applied to describe the role of chromatin in nickel carcinogenesis. Efforts will focus on cross-linking reactions of DNA and histones that likely represent the most detrimental processes induced by nickel in the presence of biological oxidants. The origins of crosslinking will be identified by use of nucleosomes with homogeneous positional and rotational structures. Sequence and conformational determinants of this reaction will be examined by using natural variants in nucleosome assembly. Nickel salts also promote a variety of other harmful lesions which will be subject to concurrent investigation. Our broad characterization is designed to distinguish the range of mechanisms that are stimulated by nickel in vitro as a model of the potentially mutagenic processes in vivo.
Keywords: DNA damage, chemical carcinogenesis, chromatin, histone, metal complex, nickel, nucleosome, DNA binding protein, conformation, crosslink, intermolecular interaction, ligand, metal poisoning, molecular assembly /self assembly, oxidative stress
Project start date: 1993-08-01
Project end date: 2004-05-31
5R01GM047531-08 (2002): $151992
5R01GM047531-07 (2001): $209077
5R01GM047531-06 (2000): $203046
PROBING GUANINE STRUCTURE IN NUCLEIC ACID FOLDING
Steven E Rokita, Professor
University Of Maryland College Pk Campus 3112 Lee Building College Park, Md 207425141
Grant 5R01GM047531-04 from National Institute Of General Medical Sciences IRG: BMT
Project start date: 1993-08-01
Project end date: 1998-07-31
5R01GM047531-04 (1996): $174239
Grants awarded to Steven E Rokita
PROBING GUANINE STRUCTURE IN NUCLEIC ACID FOLDING
Steven E Rokita, Professor
State University New York Stony Brook
stony Brook, Ny 11794
Grant 1R01GM047531-01A2 from National Institute Of General Medical Sciences IRG: BMT
Abstract: Transition metal complexes have captured considerable attention in the biomedical community because of their ability to bind certain nucleic acid structures and to promote chemical modification at or near the site of binding. Such processes occur "naturally" when metalloproteins operate on DNA or RNA, when metal-based toxins or drugs select genetic material as their target, and when metal complexes are used as conformation specific probes of nucleic acids. These laboratories have recently identified a series of nickel complexes to be exceptional probes for the secondary and tertiary structure of guanine in DNA and RNA. The utility and application of these complexes will now be explored in depth using well defined models and large polynucleotide systems of current interest. An accurate of nucleic acid folding must include a wide variety of conformations that significantly depart from the canonical double helix. Great interest in studying the polymorphic nature of nucleic acids has arisen from the preeminent role secondary and tertiary structure seems to play in recognition, regulation and reactivity of genetic information. While small oligonucleotide models may be examined in great detail by physical methods, larger systems may only be characterized through their chemical and biological activity. Ideally, reagents should be made available to identify the solvent accessibility of each group or site in a polynucleotide structure. Initial analysis suggests that the nickel complexes described herein are unrivaled in their absolute specificity for detecting guanine residues held in one of a number of non- Watson-Crick base pairing arrangements. Investigations will fully define the nickel reagent´s selectivity with targets containing mismatched, bulged, hairpin and pseudoknot sequences. Oligonucleotide models have been chosen for these analyses so that direct correlations can be drawn between these chemical studies and the existing structural results obtained from magnetic resonance and crystallography. Polynucleotide studies will follow to provide a new perspective on key structures such as bends, cruciforms and protein-DNA complexes. Most importantly, the nickel species will also be applied to a number of contemporary problems of enormous impact. For example, the accessibility of guanine will be determined for RNA folding patterns that (i) regulate gene translation and (ii) form RNA-based catalysts
Keywords: guanine, metal complex, nucleic acid structure chemical model, conformation, nickel
Project start date: 1993-08-01
Project end date: 1997-07-31
1R01GM047531-01A2 (1993): $175179
TARGET PROMOTED ALKYLATION OF NUCLEIC ACIDS
Steven E Rokita, Professor
Chemistry And Biochemistryuniversity Of Maryland College Pk Campus
3112 Lee Building
college Park, Md 207425141
Grant 5R01CA081571-03 from National Cancer Institute IRG: ZRG1
Abstract: Research interest in DNA alkylation continues to be sustained by numerous issues concerning nucleic acid structure, toxicology, and pharmacology. The importance of alkylating agents in chemotherapy cannot be underestimated despite the serious complications associated with such treatment. Selective modification of DNA is generally ascribed to the intrinsic chemistry and binding properties of the modifying agent since DNA typically acts as a passive receptor and target of reaction. The potential for DNA to act as a catalyst and control its own modification has received limited attention despite substantial advances in the related field of RNA catalysis. This proposal is designed to reveal a new and perhaps general mechanism of reagent activation that should broaden our understanding of potential mutagenic, chemotherapeutic and diagnostic reactants. Previous studies have demonstrated that duplex DNA can activate silyl phenol derivatives for alkylation and cross-linking. These derivatives had originally been constructed for fluoride-dependent reaction but were found to react spontaneously after binding to target nucleotide sequences. The mechanism of this process remains to be discovered. The proposed investigations will begin by localizing the region of duplex DNA that is responsible for the activation process. The mechanism will then be defined in part by its functional group and conformational requirements. These in turn will be identified by examining the effect of nucleotide analogues incorporated into the region of activation. Finally, the silyl-based chemistry will be extended to new reagent design and application to enhance its future utility in vitro and in vivo. Accordingly, the efficiency and specificity of target modification will be examined in DNA, RNA and protein complexes as a method to help define biological structure and reactivity. The efficiency of our lead compounds will also be tested with a variety of cell lines and compared to alkylating agents that are used clinically
Keywords: alkylation, chemical structure /function, nucleic acid structure binding site, chemical binding, chemical group, conformation, crosslink, nucleotide analog, phenol cell line
Project start date: 1999-05-01
Project end date: 2003-02-28
5R01CA081571-03 (2001): $246754
5R01CA081571-02 (2000): $239644
1R01CA081571-01 (1999): $220500
NICKEL CARCINOGENESIS--MODIFICATION OF NUCLEOSOMES
Steven E Rokita, Professor
Chemistry And Biochemistryuniversity Of Maryland College Pk Campus
3112 Lee Building
college Park, Md 207425141
Grant 2R01GM047531-05A2 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: Our daily exposure to a variety of common materials containing nickel such as coins, kitchen equipment and costume jewelry belies the highly carcinogenic and often toxic nature of many nickel compounds. In particular, amorphous nickel subsulfide (Ni3S2) and water soluble nickel sulfate (NiSO4) have both been linked to pulmonary and renal cancers in workers exposed from an industrial environment. The molecular basis of this carcinogenic activity has not been identified and awaits a more complete understanding of nickel´s biochemical reactivity. Simple nickel(II) salts alone demonstrate little ability to modify DNA and proteins irreversibly. However, when nickel binds to strong donor ligands, the resulting complex may promote a number of biologically destructive reactions. Peptides and proteins represent the most significant class of such ligands available within a cell, and DNA-binding proteins are obvious candidates for mediating genomic damage. Preliminary data suggests that histones do indeed activate the oxidative chemistry of nickel and serve, along with bound DNA, as targets of modification. The significant expertise gained by this laboratory on the biomimetic reactions of nickel during the previous funding period will now be applied to describe the role of chromatin in nickel carcinogenesis. Efforts will focus on cross-linking reactions of DNA and histones that likely represent the most detrimental processes induced by nickel in the presence of biological oxidants. The origins of crosslinking will be identified by use of nucleosomes with homogeneous positional and rotational structures. Sequence and conformational determinants of this reaction will be examined by using natural variants in nucleosome assembly. Nickel salts also promote a variety of other harmful lesions which will be subject to concurrent investigation. Our broad characterization is designed to distinguish the range of mechanisms that are stimulated by nickel in vitro as a model of the potentially mutagenic processes in vivo
Keywords: DNA damage, chemical carcinogenesis, chromatin, histone, metal complex, nickel, nucleosome DNA binding protein, conformation, crosslink, intermolecular interaction, ligand, metal poisoning, molecular assembly /self assembly, oxidative stress
Project start date: 1993-08-01
Project end date: 2003-05-31
2R01GM047531-05A2 (1999): $225189
2R55GM047531-05A1 (1998): $100000
REDUCTIVE DEHALOGENATION IN MAMMALS BY IODOTYROSINE DEIODINASE
Steven E Rokita, Professor
University Of Maryland College Pk Campus, 3112 Lee Building, College Park, Md 20742-5141
Grant 5R01DK084186-02 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Organohalides are ubiquitous in the environment through natural and human activities. Most organisms detoxify and degrade these compounds by enzyme-mediated hydrolysis, elimination or oxidation. Certain microbes are also capable of promoting reductive dehalogenation, although this is primarily limited to anaerobic metabolism. Mammals provide a fascinating exception to this general observation. The essential hormone, thyroxine (3-[4-[4-hydroxy-3,5-diiodophenoxy]-3,5-diiodophenyl]alanine), is reductively deiodinated in a variety of tissues by selenocysteine-containing enzymes. Quite surprisingly, an entirely different strategy has been recruited in the thyroid to deiodinate 3-iodo- and 3,5-diiodotyrosine. In this case, a unique flavoprotein, iodotyrosine deiodinase, is responsible for reducing the iodinated amino acids in order to salvage iodide for reuse in thyroxine biosynthesis. Investigations are now proposed to identify the unprecedented chemistry and mechanism of this mammalian deiodinase. The novel properties of this enzyme will extend the known repertoire of flavin-dependent catalysis and pathways available in biology to process halogenated compounds. Our of catalysis will focus on the reduction of the C-I bond and concomitant oxidation of the reduced flavin in IYD. Spectroscopic and product analyses will be used to differentiate between one and two electron processes and, for the first time, reveal the full range of substrates that are processed by IYD. Activation of both the substrate and flavin cofactor will be described by independently measuring recognition and catalytic properties of enzyme mutants and substrate analogues. Concurrently, substrate-dependent control of the flavin chemistry will be detected by changes in its redox properties. These investigations will be enriched as well by continuing crystallographic studies of IYD. Finally, the origins of this unusual deiodination will be examined by expressing and characterizing homologous genes from organisms that are successively more distant from mammals in the "Tree of Life." Iodide is a necessary component of our diet and used to produce an iodide-containing hormone in the thyroid that is required for regulating the metabolic rate of our entire body. The process by which we recycle iodide from byproducts formed during hormone biosynthesis will be investigated and is crucial to understand the basis for certain congenital defects leading to hypothyroidism
Relevance: Iodide is a necessary component of our diet and used to produce an iodide-containing hormone in the thyroid that is required for regulating the metabolic rate of our entire body. The process by which we recycle iodide from byproducts formed during hormone biosynthesis will be investigated and is crucial to understand the basis for certain congenital defects leading to hypothyroidism
Project start date: 2009-06-15
Project end date: 2013-04-30
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
PFA/PA: PA-07-070
5R01DK084186-02 (2010): $312170
CATALYSIS AND INHIBITION OF IODOTYROSINE DEIODINASE
Steven E Rokita, Professor
State University New York Stony Brook Stony Brook, Ny 11794
Grant 1R01DK045783-01 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: BNP
Abstract: Investigator s ) The catalytic properties of iodotyrosine deiodinase will be examined to identify the source of its unique reactivity. This enzyme is one of only two types of mammalian enzymes known to effect reductive iodide elimination and thus it provides a superb model for investigating the chemical basis of metabolic diversity within organisms. The rare but physiological amino acids, mono- and diiodotyrosine, are specifically activated and transformed in the thyroid by the deiodinase to yield iodide and tyrosine. This process serves as the critical salvage pathway for iodide and thus is essential for human health. The dehalogenation of other potential substrates has not yet been investigated but may play a role in the endogenous metabolism of halogenated pollutants. The deiodinase is dependent on an enzyme-bound flavin of unprecedented activity. Consequently, the chemical elaboration of this reaction will expand the known repertoire of flavin-based transformations. The investigators of catalysis will begin with the analysis of the aromatic substitution reaction essential for deiodination. Activation of this process will be measured in part by the interaction of the diodinase with a range of substituted tyrosine derivatives. Additional enzymological study include the use of (1) reversible enzyme inhibition to define both substrate recognition patterns and catalytic transition-state (reactive intermediate) properties and (2) mechanism-based inactivation to characterize the activation of tyrosine derivatives. To focus on the pharmacologically important protein chemistry of this dehalogenation system, new methods for purifying and reconstituting the deiodinase will also be assessed. Isolation of homogeneous enzyme in large quantities, while not required for most studies proposed, will be necessary for future investigations. Similarly, methods for continuous assay of enzyme turnover will be examined in order to facilitate later kinetic analyses. The novel properties revealed for this unusual catalyst should contribute to the underlying goal of finding new targets for therapeutic control of thyroid disease.
Keywords: enzyme mechanism, flavin, iodide peroxidase, iodination, chemical substitution, enzyme activity, enzyme inhibitor, protein purification, protein sequencing
Project start date: 1992-09-30
Project end date: 1995-09-29
1R01DK045783-01 (1992): $108629
5R01DK045783-03 (1994): $118837
Sponsored Links Excellgen http://Excellgen.com
5R01DK045783-02 (1993): $111549
TARGET PROMOTED ALKYLATION OF NUCLEIC ACIDS
Steven E Rokita, Professor
University Of Maryland College Pk Campus, 3112 Lee Building, College Park, Md 20742-5141
Grant 5R01CA081571-08 from National Cancer Institute
Keywords: 1, 3 diazine; 2H-1, 3, 2-Oxazaphosphorin-2-amine, N, N-bis(2-chloroethyl)tetrahydro-, 2-oxide; 2H-1, 3, 2-oxazaphosphorin-2-amine, N, N-is(2-chloroethyl)tetrahydro-, 2-oxide; Address; Affinity; Alkylation; Base Pairing; Binding; Binding (Molecular Function); Biochemical; Biological; Biomimetics; Bizelesin; Bizelesin (U-77779); C-glycoside; CTX; CYCLO-cell; Cancers; Carloxan; Chemicals; Ciclofosfamida; Ciclofosfamide; Cicloxal; Clafen; Claphene; Complementary DNA; Complex; Cycloblastin; Cycloblastine; Cyclophospham; Cyclophosphamide; Cyclophosphamidum; Cyclophosphan; Cyclophosphane; Cyclophosphanum; Cyclostin; Cyclostine; Cytophosphan; Cytophosphane; Cytoxan; DNA; DNA Alkylation; DNA Sequence; DNA, Complementary; DNA, Single-Stranded; Deoxyribonucleic Acid; Endoxan; Endoxana; Enduxan; Evaluation; Fosfaseron; Funding; Future; Gene Products, RNA; Gene Transcription; Genes; Genetic Transcription; Genoxal; Genuxal; Glean; Goals; Guidelines; In Vitro; Ledoxina; Major Groove; Malignant Neoplasms; Malignant Tumor; Methods; Mimetics, Biological; Mitoxan; Molecular Interaction; Mustard; Mustard (food); Nature; Neosar; Nucleic Acids; Oligo; Oligonucleotides; PNA; Peptide Nucleic Acids; Process; Procytox; Proteins; Purines; Pyrimidine; Pyrimidines; RNA; RNA Expression; RNA, Non-Polyadenylated; Range; Rate; Reaction; Reagent; Ribonucleic Acid; Scheme; Sendoxan; Single-Stranded DNA; Site; Specificity; Syklofosfamid; System; System, LOINC Axis 4; Testing; Therapeutic; Thermodynamic; Thermodynamics; Transcription; Transcription, Genetic; Translations; Work; Zytoxan; adduct; analog of CC-1065; anticancer treatment; base; benzo(1, 2-b[{..}]4, 3-b`)dipyrrol-4-ol, 6, 6`-(carbonylbis(imino-1H-indole-5, 2-diylcarbonyl))bis(8-(chloromethyl)-3, 6, 7, 8-tetrahydro-1-methyl-, (S-(R*, R*)); cDNA; cellular targeting; design; designing; experience; experiment; experimental research; experimental study; gene product; in vivo; malignancy; neoplasm/cancer; nucleobase; purine; qui methide; research study
Project start date: 1999-05-01
Project end date: 2010-04-30
Budget start date: 1-MAY-2008
Budget end date: 30-APR-2010
5R01CA081571-08 (2008): $0
5R01CA081571-07 (2007): $244419
5R01CA081571-06 (2006): $251719
2R01CA081571-05A2 (2005): $284006