Difference: AneelAggarwal (1 vs. 13)
Revision 1301 Apr 2009 - Main.SherryllJones
Public information on Grants associated with NYSBC | ||||||||
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| > > | Grant Number: 1R01CA138546-01 Project Title: Structural bases of high fidelity of DNA polymerase delta
PI Information: Name Email Title AGGARWAL, ANEEL K. aneel.aggarwal@mssm.edu PROFESSOR
Abstract: DESCRIPTION (provided by applicant): Accurate DNA replication is crucial for the maintenance of genomic stability and for the suppression of mutagenesis and carcinogenesis. DNA polymerase d (Pold) is a high fidelity polymerase that plays an indispensable role in replication from yeast to humans. Pold from the yeast S. cerevisiae is comprised of three subunits, Pol3, Pol31, and Pol32. Pol3 is the catalytic subunit of the holoenzyme, encoding both the polymerase and the 3' to 5' exonuclease proofreading functions. Mutations in either the polymerase or the exonuclease domain of Pol3 that lower the fidelity of Pold cause cancers in mice and humans. For example, mutations that map to Pol3 have been identified in cancer cell lines and in sporadic colon cancers. Here, we propose structural, biochemical, and genetic studies on yeast Pold that are crucial for understanding the action mechanisms of this high fidelity polymerase. We will: 1) Determine the crystal structures of Pol3 in the polymerizing and editing modes. The structures will provide a mechanistic understanding of the basis for the high selectivity of Pol3 for the correct nucleotide, and will yield insights into the conformational transitions that underlie its fidelity. 2) We will use the structural information to make mutations that a) alter the fidelity of DNA synthesis, and b) affect the transition to the editing mode. Together, these mutations will test specific hypotheses that are inferred from the structures to form the basis of nucleotide selection and to contribute to active site switching. We will examine the effects of these mutations on Pold function by both biochemical and genetic means. 3) To understand the contributions that the Pol31 and Pol32 subunits make to Pold structure and function, we will carry out biochemical and structural studies on the Pold holoenzyme. The effects of Pol31 and Pol32 on the DNA binding proficiency and on the processivity of DNA synthesis by Pold will be determined, and pre-steady state kinetic analyses will be carried out to identify the contributions that different steps of the polymerization reaction make to the high fidelity of Pold. In addition, we will determine the structure of Pold holoenzyme in ternary complex with DNA and dNTP. A comparison of the ternary structures of Pol3 and Pold holoenzyme will be invaluable for deciphering the contributions of Pol31 and Pol32 to Pold function. The combined structural, biochemical, and genetic approaches proposed here will be important for defining the action mechanisms of Pold's polymerizing and proofreading functions and for delineating the structural bases of its high fidelity. PUBLIC HEALTH RELEVANCE: The aim of this study is to uncover the mechanisms underlying the high fidelity of DNA polymerase 4 (Pol4), which is crucial for maintaining genomic stability and the suppression of carcinogenesis. Mutations that lower the fidelity of Pol4 cause cancers in mice and humans.
Public Health Relevance: The aim of this grant is to uncover the mechanisms underlying the high fidelity of DNA polymerase 4 (Pol4), which is crucial for maintaining genomic stability and the suppression of carcinogenesis. Mutations that lower the fidelity of Pol4 cause cancers in mice and humans.
Thesaurus Terms: There are no thesaurus terms on file for this project.
Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 100296574 Fiscal Year: 2009
Department: STRUCTURAL AND CHEMICAL BIOLOGY Project Start: 01-MAR-2009 Project End: 31-JAN-2014 ICD: NATIONAL CANCER INSTITUTE IRG: MSFE
Grant Number: 5R01GM062947-08 Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES PI Information: Name Email Title AGGARWAL, ANEEL K. aneel.aggarwal@mssm.edu PROFESSOR Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: RNA binding protein, arthropod genetics, developmental genetics, embryogenesis, gene induction /repression, genetic regulation, genetic translation, invertebrate embryology, messenger RNA, molecular assembly /self assembly, protein structure, transcription factor genetic regulatory element, posttranscriptional RNA processing, protein protein interaction Drosophilidae, analytical ultracentrifugation, fluorescence resonance energy transfer, nuclear magnetic resonance spectroscopy, yeast two hybrid system Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 100296574 Fiscal Year: 2008 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2010 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | |||||||
| Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION
Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements.
Thesaurus Terms:
gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor
DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction
crystallization, nuclear magnetic resonance spectroscopy
Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU
OF NEW YORK UNIVERSITY
NEW YORK, NY 10029
Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1
Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: There are no thesaurus terms on file for this project Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | ||||||||
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Personal Preferences (details in TWikiVariables)
Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: There are no thesaurus terms on file for this project Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | ||||||||
Revision 1101 Apr 2008 - Main.DavidCowburn
Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: There are no thesaurus terms on file for this project Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES | ||||||||
| Changed: | ||||||||
| < < | Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this projectyiyiu are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. | |||||||
| > > | Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. | |||||||
| Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | ||||||||
Revision 1001 Apr 2008 - Main.DavidCowburn
Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: | ||||||||
| Changed: | ||||||||
| < < | There are no thesaurus terms on file for this projectyiyiu. | |||||||
| > > | There are no thesaurus terms on file for this project | |||||||
| Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU
OF NEW YORK UNIVERSITY
NEW YORK, NY 10029
Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1
Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this projectyiyiu are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | ||||||||
Revision 921 Mar 2008 - Main.DavidCowburn
Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: | ||||||||
| Changed: | ||||||||
| < < | There are no thesaurus terms on file for this project. | |||||||
| > > | There are no thesaurus terms on file for this projectyiyiu. | |||||||
| Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU
OF NEW YORK UNIVERSITY
NEW YORK, NY 10029
Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1
Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES | ||||||||
| Changed: | ||||||||
| < < | Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. | |||||||
| > > | Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this projectyiyiu are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. | |||||||
| Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | ||||||||
Revision 808 Feb 2008 - Main.DavidCowburn
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| Changed: | ||||||||
| < < |
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| > > |
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Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | ||||||||
Revision 708 Feb 2008 - Main.DavidCowburn
Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 |
Revision 621 Nov 2006 - Main.DavidStokes
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Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | ||||||||
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Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | |||||||
| > > |
Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | |||||||
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| Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 | ||||||||
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| Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | ||||||||
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| Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | ||||||||
Revision 313 Jun 2005 - Main.LisaHickey
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| > > | Public information on Grants associated with NYSBC Grant Number: 2R01AI041706-06 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: THE ROLE OF IRF PROTEINS IN GENE TRANSCRIPTION Abstract: DESCRIPTION (provided by applicant): The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1. Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements. Thesaurus Terms: gene expression, gene induction /repression, genetic regulation, genetic transcription, interferon beta, protein isoform, protein structure function, transcription factor DNA binding protein, genetic regulatory element, immune system, immunity, phosphorylation, protein protein interaction crystallization, nuclear magnetic resonance spectroscopy Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-JUN-1998 Project End: 30-NOV-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1 Grant Number: 2R01GM062947-05 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: STRUCTURE & ASSEMBLY OF TRANSLATION REPRESSOR COMPLEXES Abstract: DESCRIPTION (provided by applicant): Translation regulation plays a vital role in the lives of most organisms. It provides an important checkpoint in the pathways for cell growth and differentiation, and a link to the pathology of several diseases. A prominent example of translational regulation in early fly embryogenesis is the repression of maternal hunchback mRNA by Pumilio, Nanos, and Brain tumor. The Nanos protein is only synthesized at the posterior of the embryo due to the translational repression of its own maternal mRNA by Smaug. Pumilio and Nanos are also required for the translation repression of maternal Cyclin B mRNA. Our long-term objective is to uncover the structure and mechanism of assembly of these translational regulators. Specific aims are: 1. Test our basic hypothesis that two Pumilio molecules bind to a single hunchback regulatory element. This will accomplished through a combination of structure and genetics. 2. Identify specific residues in Brain tumor that interact with Nanos. 3. Cocrystallize Pumilio and Nanos in a ternary complex with hunchback mRNA. 4. Determine the structure of Pumilio Puf domain bound to Cyc B mRNA. We will first define the minimal element in the Cyclin B mRNA that binds Pumilio with high affinity and confers regulation in vivo. 5. Elucidate the structure of a SAM domain/RNA complex by NMR methods. 6. Guided by structure, isolate a Smaug dominant negative for the identification of potential corepressors. Together, these structural and genetic experiments will provide a molecular basis for the translation regulatory events that organize the body pattern along the anterior-posterior axis. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-MAY-2001 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 5R01GM044006-15 PI Name: AGGARWAL, ANEEL K. PI Email: aggarwal@inka.mssm.edu PI Title: PROFESSOR Project Title: RECOGNITION AND CLEAVAGE OF DNA BY RESTRICTION ENZYMES Abstract: DESCRIPTION (provided by applicant): Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI? and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI? and Bglll: BstYI. We will also manipulate the specificity of BamHI? with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI? bound to one non-cognate DNA sequence. We will now determine structures of BamHI? bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules. Thesaurus Terms: DNA, chemical cleavage, enzyme mechanism, enzyme structure, restriction endonuclease DNA binding protein, active site, enzyme activity, enzyme substrate complex, hybrid enzyme, hydrolysis, nucleic acid sequence, structural biology X ray crystallography, crystallization, fluorescence resonance energy transfer Institution: MOUNT SINAI SCHOOL OF MEDICINE OF NYU OF NEW YORK UNIVERSITY NEW YORK, NY 10029 Fiscal Year: 2005 Department: PHYSIOLOGY AND BIOPHYSICS Project Start: 01-APR-1990 Project End: 31-MAR-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | |||||||
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