Scott W Morrical
University Of Vermont & St Agric College
Project start date: 1993-08-01
Project end date: 2013-01-31
Sponsored Links Excellgen http://Excellgen.com
ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES
Scott W Morrical, Dr.
University Of Vermont & St Agric College, 85 South Prospect Street, Burlington, Vt 05405
Grant 5R01GM048847-15 from National Institute Of General Medical Sciences
Abstract: The objective of this research program is to determine the mechanisms by which recombinase and helicase enzymes are assembled onto single-stranded DNA (ssDNA) in the bacteriophage T4 DNA replication/recombination system. We will study the assembly of the T4 UvsX recombinase into presynaptic filaments, and we will study the assembly of two different DNA helicases, Gp41 and Dda, onto ssDNA at replication forks and in recombination intermediates. All three enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound Gp32, the T4 ssDNA-binding protein. UvsX and Gp41 both require the activity of a specific mediator protein, UvsY or Gp59, respectively, for proper assembly onto Gp32-ssDNA complexes, whereas Dda achieves the same effect through direct protein-protein interactions with Gp32. We will explore all three enzyme loading mechanisms using classical biochemical methods (kinetics, thermodynamics, fluorescence, sedimentation, crosslinking), singlemolecule approaches (fluorescence imaging, force spectroscopy), and mutagenesis. Our SPECIFIC AIMS are (1) Determine the kinetic mechanism of UvsX-ssDNA presynaptic filament assembly and collapse. We will test a model in which UvsY protein selectively enhances filament nucleation, UvsX actively displaces gp32 from ssDNA, and filaments exhibit dynamic instability linked to ATP hydrolysis. (2) Determine how interactions of T4 Gp59 protein with replication fork DNA control helicase assembly and polymerase blockage. We will test a model in which cooperative binding of Gp32 to lagging-strand ssDNA converts Gp59 from a polymerase-blocking to a helicase-loading conformation that recruits Gp41 helicase to the replication fork. (3) Determine how interactions with Gp32 modulate the DNA helicase functions of T4 Dda protein. We will test a model in which Dda-Gp32 protein-protein interactions promote the oligomerization of Dda and enhance its DNA unwinding properties in both replication and recombination transactions. Understanding how helicase and recombinase enzymes are correctly assembled onto ssDNA is fundamental to understanding DNA replication, recombination, and repair mechanisms that are conserved in all organisms. There are clear links between errors in DNA replication/recombination/repair machineries and human disease states including cancer. Understanding how recombinase- and helicasessDNA complexes are correctly assembled and activated may therefore aid in the prevention, diagnosis, and treatment of cancer. PUBLIC HEALTH REVELANCE Proper assembly of enzyme-ssDNA complexes is critical for genome replication and maintenance in all organisms. Defects in enzyme-ssDNA assembly processes are implicated in cancer and other human disease states. Enzyme-ssDNA assembly pathways are also potential targets for new classes of antibiotic and antitumor drugs. Our studies of enzyme-ssDNA assembly mechanisms may therefore shed light on how tumors develop and how infectious diseases progress, and may also suggest new treatment strategies
Keywords: ATP Hydrolysis; Antibiotic Agents; Antibiotic Drugs; Antibiotics; Bacteriophage T4; Binding; Binding (Molecular Function); Biochemical; Biochemistry; Cancer Treatment; Cancers; Cells; Chemistry, Biological; Coliphage T4; Communicable Diseases; Complex; DNA; DNA Helicases; DNA Recombination; DNA Replication; DNA Synthesis; DNA Unwinding Proteins; DNA biosynthesis; DNA recombination (naturally occurring); DNA replication fork; DNA unwinding enzyme; DNA, Single-Stranded; Defect; Deoxyribonucleic Acid; Diagnosis; Enterobacteria phage T4; Enzyme Activation; Enzymes; Exhibits; Filament; Fluorescence; Genetic Recombination; Genetics-Mutagenesis; Genome; Homologous Recombinational Repair; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases and Manifestations; Infectious Disorder; Kinetic; Kinetics; Light; Link; Maintenance; Maintenances; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; Malignant Neoplasms; Malignant Tumor; Mediator; Mediator of Activation; Mediator of activation protein; Methods; Miscellaneous Antibiotic; Modeling; Molecular Biology, Mutagenesis; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Mutagenesis; Organism; Pathway interactions; Photoradiation; Polymerase; Prevention; Process; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Proteins; Recombination; Recombination Repair; Recombination, Genetic; Recruitment Activity; Research; SS DNA BP; Sedimentation process; Single-Stranded DNA; Single-Stranded DNA-Binding Protein; Spectroscopy; Spectrum Analyses; Spectrum Analysis; System; System, LOINC Axis 4; T4 Phage; Testing; Thermodynamic; Thermodynamics; anticancer therapy; antitumor drug; cancer therapy; conformation; conformational state; cross-link; crosslink; fluorescence imaging; gene product; helicase; human disease; living system; malignancy; neoplasm/cancer; pathway; presynaptic; programs; protein protein interaction; recombinase; recombinational repair; recruit; sedimentation; single molecule; treatment strategy; tumor
Relevance: Proper assembly of enzyme-ssDNA complexes is critical for genome replication and maintenance in all organisms. Defects in enzyme-ssDNA assembly processes are implicated in cancer and other human disease states. Enzyme-ssDNA assembly pathways are also potential targets for new classes of antibiotic and antitumor drugs. Our studies of enzyme-ssDNA assembly mechanisms may therefore shed light on how tumors develop and how infectious diseases progress, and may also suggest new treatment strategies
Project start date: 1993-08-01
Project end date: 2013-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
5R01GM048847-15 (2010): $324053
5R01GM048847-13 (2005): $302243
5R01GM048847-12 (2004): $318150
5R01GM048847-11 (2003): $331820
5R01GM048847-05 (1997): $218769
5R01GM048847-03 (1995): $170784
5R01GM048847-02 (1994): $163565
5R01GM048847-09 (2001): $270052
5R01GM048847-08 (2000): $262256
5R01GM048847-07 (1999): $272794
Sponsored Links Excellgen http://Excellgen.com
5R01GM048847-04 (1996): $177664
Grants awarded to Scott W Morrical
Assembly And Activation Of Enzyme-ssDNA Complexes
Scott W Morrical, Associate Professor
Biochemistryuniversity Of Vermont & St Agric College
Grant 2R01GM048847-14A2 from National Institute Of General Medical Sciences IRG: MGA
Abstract: The objective of this research program is to determine the mechanisms by which recombinase and helicase enzymes are assembled onto single-stranded DNA (ssDNA) in the bacteriophage T4 DNA replication/recombination system. We will study the assembly of the T4 UvsX recombinase into presynaptic filaments, and we will study the assembly of two different DNA helicases, Gp41 and Dda, onto ssDNA at replication forks and in recombination intermediates. All three enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound Gp32, the T4 ssDNA-binding protein. UvsX and Gp41 both require the activity of a specific mediator protein, UvsY or Gp59, respectively, for proper assembly onto Gp32-ssDNA complexes, whereas Dda achieves the same effect through direct protein-protein interactions with Gp32. We will explore all three enzyme loading mechanisms using classical biochemical methods (kinetics, thermodynamics, fluorescence, sedimentation, crosslinking), singlemolecule approaches (fluorescence imaging, force spectroscopy), and mutagenesis. Our SPECIFIC AIMS are (1) Determine the kinetic mechanism of UvsX-ssDNA presynaptic filament assembly and collapse. We will test a model in which UvsY protein selectively enhances filament nucleation, UvsX actively displaces gp32 from ssDNA, and filaments exhibit dynamic instability linked to ATP hydrolysis. (2) Determine how interactions of T4 Gp59 protein with replication fork DNA control helicase assembly and polymerase blockage. We will test a model in which cooperative binding of Gp32 to lagging-strand ssDNA converts Gp59 from a polymerase-blocking to a helicase-loading conformation that recruits Gp41 helicase to the replication fork. (3) Determine how interactions with Gp32 modulate the DNA helicase functions of T4 Dda protein. We will test a model in which Dda-Gp32 protein-protein interactions promote the oligomerization of Dda and enhance its DNA unwinding properties in both replication and recombination transactions. Understanding how helicase and recombinase enzymes are correctly assembled onto ssDNA is fundamental to understanding DNA replication, recombination, and repair mechanisms that are conserved in all organisms. There are clear links between errors in DNA replication/recombination/repair machineries and human disease states including cancer. Understanding how recombinase- and helicasessDNA complexes are correctly assembled and activated may therefore aid in the prevention, diagnosis, and treatment of cancer. PUBLIC HEALTH REVELANCE Proper assembly of enzyme-ssDNA complexes is critical for genome replication and maintenance in all organisms. Defects in enzyme-ssDNA assembly processes are implicated in cancer and other human disease states. Enzyme-ssDNA assembly pathways are also potential targets for new classes of antibiotic and antitumor drugs. Our studies of enzyme-ssDNA assembly mechanisms may therefore shed light on how tumors develop and how infectious diseases progress, and may also suggest new treatment strategies
Project start date: 1993-08-01
Project end date: 2013-01-31
1R01GM048847-01A1 (1993): $162061
Scott W Morrical
University Of Vermont & St Agric College, 85 South Prospect Street, Burlington, Vt 05405
Abstract: Rad51 protein is the eukaryotic representative of the RecA/Rad51 fannlly of DNA strand transferase enzymes. Homologous DNA strand exchanges catalyzed by RadSI are critical for Homology-Dlrected DNA Repair (HDR) and therefore for genome stability. To promote HDR, RadSI must first assemble onto single stranded DNA In the form of a presynaptic filament. Filament assembly allosterically activates RadSI to catalyze ATP hydrolysis, to search for homology in a sister chromosome, and to perform DNA strand exchange reactions. There Is compelling evidence that defects in the assembly and activity of RadSI presynaptic filaments are linked to human cancer. The overall goal of Project 3 Is to understand how specific changes in the structure, function, and molecular interactions of RadSI can lead to genomic instability and cancer. The SPECIFIC AIMS of Project 3 are (1) To test the hypothesis that key amino acid residues at the filament interface and in the ATPase active site of RadSI control the allosteric transitions that couple the ATPase catalytic cycle to DNA strand exchange. Using yeast RadSI as a model, the catalytic and allosteric mechanisms of RadSI will be probed using a combination of site-directed mutagenesis, biochemical and biophysical analyses, and structural biology methods. (2) To test the hypothesis that tumor-derived variants of human hRADSI protein have altered biochemical and/or regulatory properties. hRADSI variants identified in Project 1 will be characterized biochemically alongside wild-type hRADSI to identify any changes in DNA binding or catalytic properties, or in key protein-protein interactions. (3) To test the hypothesis that interactions between hRADSI and DNA polymerase beta (Pol-beta) help recruit hRADSI onto ssDNA generated as a result of abortive base excision repair (BER). hRADSIPol-beta interactions will be characterized biochemically and disrupted by mutagenesis to assess their importance for DNA repair functions. Interesting mutants from Aims 2-3 will be exported to Projects 1 and 4 for in vivo and chromatin studies. This project will provide rigorous models for the structure, function, and assembly of RadSI presynaptic filaments, and for potential cross-talk between HDR and BER pathways, in normal vs, tumor cells, which will be useful for predicting cancer susceptibility and for developing new cancer treatments
Keywords: ATP Hydrolysis; ATP phosphohydrolase; ATPase; Active Sites; Adenosine Triphosphatase; Adenosinetriphosphatase; Amino Acids; Assay; Base Excision Repairs; Binding; Binding (Molecular Function); Bioassay; Biochemical; Biologic Assays; Biological Assay; Biological Function; Biological Process; Cancer Treatment; Cancers; Cells; Chromatin; Chromosomes; DNA; DNA Base Excision Repair; DNA Binding; DNA Binding Interaction; DNA Damage Repair; DNA Polymerase IV; DNA Polymerase beta; DNA Polymerases; DNA Repair; DNA Repair Pathway; DNA-Dependent DNA Polymerases; DNA-Directed DNA Polymerase; Defect; Deoxynucleoside-triphosphate[{..}]DNA deoxynucleotidyltransferase (DNA-directed); Deoxyribonucleic Acid; Diagnosis; EC 2; EC 2.7.7.7; Enzymes; Excision Repair; Family; Filament; Funding; Genetic Alteration; Genetic Change; Genetic defect; Genetics-Mutagenesis; Genome Instability; Genome Stability; Genomic Instability; Germ Lines; Glean; Goals; Homologous Recombinational Repair; Human; Human, General; Knowledge; Lead; Link; Malignant Cell; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Methods; Modeling; Molecular; Molecular Biology, Mutagenesis; Molecular Interaction; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Ortholog; Orthologous Gene; Pathway interactions; Pb element; Phenotype; Play; Polymerase; Predisposition; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Proteins; RAD51 protein; Rad51 recombinase; Radiation, X-Rays; Radiation, X-Rays, Gamma-Rays; Reaction; Recombination Repair; Recruitment Activity; Repairs, Base Excision; Roentgen Rays; Role; SIS; Single-Stranded DNA; Sister; Site; Site-Directed Mutagenesis; Site-Specific Mutagenesis; Stability, Genomic; Structure; Susceptibility; Targeted DNA Modification; Targeted Modification; Testing; Transferase; Tumor Cell; Tumor-Derived; Unscheduled DNA Synthesis; Variant; Variation; X-Radiation; X-Rays; Xrays; Yeasts; aminoacid; anticancer therapy; base; cancer cell; cancer therapy; cell transformation; design; designing; gene product; genome mutation; heavy metal Pb; heavy metal lead; in vivo; interest; malignancy; mutant; neoplasm/cancer; neoplastic cell; novel; pathway; presynaptic; programs; protein protein interaction; recombinational repair; recruit; repair; repaired; social role; structural biology; transformed cells; treatment strategy; tumor
Relevance: The human RAD51 protein plays critical roles in DNA repair pathways that protect cells from mutations and from cancer. In this project we will isolate variant forms of RADSI that are found in human cancer cells, and we will study how these RAD51 variants differ from normal RAD51, Information from this project will help us to design better diagnosis and treatment strategies for cancer
Project start date: 2010-07-01
Project end date: 2015-06-30
Budget start date: 1-JUL-2010
Budget end date: 31-AUG-2011
PFA/PA: PAR-09-025
2P01CA098993-06A1_5475 (2010): $202463
STRUCTURE AND FUNCTION OF HOMOLOGOUS RECOMBINATION ENZYMES
Scott W Morrical, Associate Professor
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5P01CA098993-040003 from National Cancer Institute IRG: NCI
Abstract: Double-strand break repair (DSBR) by homologous recombination repairs double strand breaks produced by ionizing radiation and this DNA repair process is conserved from bacteriophage to humans. In all organisms, homologous DSBR requires the DNA strand exchange activity of an ortholog of the E. coli RecA protein. The RecA family is the most highly sequence-conserved family of DNA repair proteins, which exhibits strong conservation of global structure and function in promoting homologous recombination and DSBR transactions. Nevertheless, RecAs for which biochemical data are available exhibit surprising diversity in DNA binding properties, catalysis, and allosteric mechanisms, while others appear to be specialized recombination mediators. Does this diversity/specialization reflect different evolutionary paths towards RecA function? Also, to what extent does RecA biochemical diversity/specialization reflect the environmental demands placed on the DNA recombination and repair systems of a host organism? The work proposed in this Project intends to address these questions by studying the functional and structural diversity of RecA enzymes. In SPECIFIC AIM 1 we will compare biochemical and structural properties of divergent members of the RecA family. Target enzymes identified by computational methods will be cloned, expressed and purified. Using high throughput methods, DNA-binding and catalytic properties of each RecA ortholog will be determined. Variations in biochemical properties will be correlated with Phylogenetic and/or predicted structural variations within the RecA enzyme family. RecA orthologs representing distinct Phylogenetic and/or biochemical classes will be crystallized in the presence/absence of bound nucleotide, polynucleotide, and mediator protein ligands, and their high resolution X-ray structures will be determined. In SPECIFIC AIM 2 we will study the allosteric mechanisms of divergent RecA enzymes. The roles of key amino acid residues in the induction of the high-affinity ssDNA-binding state will be determined for selected RecA orthologs. The ability of cognate recombination mediator proteins (RMPs) to induce high-affinity RecA-ssDNA binding will also be examined in targeted systems.
Keywords: DNA repair, N glycosidase, enzyme structure, gene targeting, ionizing radiation, DNA binding protein, DNA damage, binding site, biochemistry, catalyst, crystallization, genetic recombination, genetic screening, recombinant protein, affinity chromatography, fluorescence resonance energy transfer, high throughput technology
ASSEMBLY AND ACTIVATION OF ENZYME/SSDNA COMPLEXES
Scott W Morrical, Associate Professor
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 3R01GM048847-06S1 from National Institute Of General Medical Sciences IRG: MBC
Abstract: adapted from investigator s ) The objective of Dr. Morrical s research is to characterize the protein-ssDNA and protein-protein interactions required for the assembly and activation of enzyme complexes on single-stranded DNA. Two different representative enzyme-ssDNA assembly problems in the bacteriophage T4 DNA recombination and replication systems will be examined (1) Assembly of presynaptic filaments containing the T4 uvsX recombinase bound to ssDNA; and (2) Assembly of gp41, the essential DNA helicase component of the phage primosome, onto ssDNA. A common feature is that both enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound gp32, the T4 ssDNA-binding protein. Another common feature is that both enzymes require a second "assembly factor" (uvsY or gp59, respectively) for assembly onto gp32-ssDNA complexes. This project focuses on the detailed biochemical mechanisms used by uvsY to load uvsX, and by gp59 to load gp41, onto gp32-covered ssDNA molecules. The SPECIFIC AIMS are the following AIM 1 Characterize structural and biochemical aspects of uvsY protein and uvsY-ssDNA complexes. The following specific hypotheses will be tested that ssDNA binds to a hexameric form of uvsY and causes a significant structural rearrangement of the uvsY hexamer; and that uvsY binding causes extensive unstacking of ssDNA bases consistent with wrapping or other structural distortions of the ssDNA. AIM 2 Characterize interactions of uvsY protein with gp32-2ssDNA and uvsX-ssDNA complexes. The following specific hypotheses will be tested that uvsY retains its hexameric form in complexes with gp32 and uvsX proteins; and that uvsY binding destabilizes gp32-ssDNA interactions while stabilizing uvsX-ssDNA interactions. AIM 3 Characterize interactions of gp59 protein with gp32-ssDNA complexes. The following specific hypotheses will be tested that a critical cluster of gp59 molecules bound to gp32-ssDNA forms the minimal assembly site for the gp41 helicase; and that gp59 destabilizes gp32-ssDNA complexes through protein-protein interactions with ssDNA-bound gp32 molecules. AIM # 4 Characterize interactions of gp59 protein with the gp41 DNA helicase. The specific hypothesis that gp59 promotes the hexamerization of gp41 will be tested.
Keywords: DNA binding protein, enzyme complex, enzyme induction /repression, protein biosynthesis, DNA replication, genetic recombination, helicase, intermolecular interaction, protein structure /function, recombinase, stoichiometry, virus protein, bacteriophage T4, circular dichroism, crosslink, fluorescence spectrometry, high performance liquid chromatography, sedimentation, ultraviolet radiation
Project start date: 1993-08-01
Project end date: 2002-07-31
3R01GM048847-06S1 (1999): $23305
ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES
Scott W Morrical, Associate Professor
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 3R01GM048847-06S2 from National Institute Of General Medical Sciences IRG: MBC
Abstract: adapted from investigator s ) The objective of Dr. Morrical s research is to characterize the protein-ssDNA and protein-protein interactions required for the assembly and activation of enzyme complexes on single-stranded DNA. Two different representative enzyme-ssDNA assembly problems in the bacteriophage T4 DNA recombination and replication systems will be examined (1) Assembly of presynaptic filaments containing the T4 uvsX recombinase bound to ssDNA; and (2) Assembly of gp41, the essential DNA helicase component of the phage primosome, onto ssDNA. A common feature is that both enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound gp32, the T4 ssDNA-binding protein. Another common feature is that both enzymes require a second "assembly factor" (uvsY or gp59, respectively) for assembly onto gp32-ssDNA complexes. This project focuses on the detailed biochemical mechanisms used by uvsY to load uvsX, and by gp59 to load gp41, onto gp32-covered ssDNA molecules. The SPECIFIC AIMS are the following AIM 1 Characterize structural and biochemical aspects of uvsY protein and uvsY-ssDNA complexes. The following specific hypotheses will be tested that ssDNA binds to a hexameric form of uvsY and causes a significant structural rearrangement of the uvsY hexamer; and that uvsY binding causes extensive unstacking of ssDNA bases consistent with wrapping or other structural distortions of the ssDNA. AIM 2 Characterize interactions of uvsY protein with gp32-2ssDNA and uvsX-ssDNA complexes. The following specific hypotheses will be tested that uvsY retains its hexameric form in complexes with gp32 and uvsX proteins; and that uvsY binding destabilizes gp32-ssDNA interactions while stabilizing uvsX-ssDNA interactions. AIM 3 Characterize interactions of gp59 protein with gp32-ssDNA complexes. The following specific hypotheses will be tested that a critical cluster of gp59 molecules bound to gp32-ssDNA forms the minimal assembly site for the gp41 helicase; and that gp59 destabilizes gp32-ssDNA complexes through protein-protein interactions with ssDNA-bound gp32 molecules. AIM # 4 Characterize interactions of gp59 protein with the gp41 DNA helicase. The specific hypothesis that gp59 promotes the hexamerization of gp41 will be tested.
Keywords: DNA binding protein, enzyme complex, enzyme induction /repression, protein biosynthesis, DNA replication, genetic recombination, helicase, intermolecular interaction, protein structure /function, recombinase, stoichiometry, virus protein, bacteriophage T4, circular dichroism, crosslink, fluorescence spectrometry, high performance liquid chromatography, sedimentation, ultraviolet radiation
Project start date: 1993-08-01
Project end date: 2002-07-31
3R01GM048847-06S2 (1999): $24486
2R56GM048847-14A1 (2007): $325021
Sponsored Links Excellgen http://Excellgen.com
ASSEMBLY AND ACTIVATION OF ENZYME SSDNA COMPLEXES
Scott W Morrical, Associate Professor
Biochemistryuniversity Of Vermont & St Agric College
85 South Prospect Street
burlington, Vt 05405
Grant 3R01GM048847-04S1 from National Institute Of General Medical Sciences IRG: MBC
Project start date: 1996-10-01
Project end date: 1998-07-31
3R01GM048847-04S1 (1997): $30941