Biophysical studies on DNA binding and *transcriptional activities of Myc -Max -Mad network.
Item
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Title
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Biophysical studies on DNA binding and *transcriptional activities of Myc -Max -Mad network.
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Identifier
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AAI3204995
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identifier
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3204995
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Creator
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Banerjee, Anamika.
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Contributor
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Adviser: Dixie J. Goss
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Date
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2006
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biophysics, General | Biology, Molecular
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Abstract
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The members of the Myc/Max/Mad network comprise a wide range of eukaryotic transcription factors. Max is at the center of this network and can initiate activation or repression of transcription depending on whether its heterodimeric partner is Myc or Mad. The consensus sequence on the promoter DNA recognized by Myc-Max-Mad network of proteins is a hexameric palindrome CACGTG, also called an E Box.;Fluorescence anisotropy has been used to determine the dissociation constants for DNA-protein and protein-protein interactions of this pathway. These data indicates that Max has higher affinity for DNA binding, however the heterodimeric partner of Max confers specificity of binding. At the level of protein-protein interactions, Myc-Max forms the most stable dimer followed by Mad-Max and Max-Max. These proteins might follow either a monomeric or a dimeric pathway to assemble on DNA. Investigation of the entire thermodynamic cycle of monomer-dimer pathway revealed that the equilibrium constants for first monomer binding to DNA is ∼10nM, while the second monomer binding equilibrium is ∼2-5muM. This reduced binding affinity of the second monomer may result from a conformational change that monomers are believed to undergo upon binding and also might be essential for reassortment of Max's partner.;The effect of poly ions on protein dimerization and the temperature dependence of this effect were investigated. Negatively charged polymers like poly-L-Glutamic acid reduced the Kds for all three protein-protein dimers. Determination of the thermodynamic parameters both in the absence and in the presence of negative polymers, shows that the protein-protein interaction is characterized by negative favorable enthalpy and negative unfavorable entropy.;Fluorescence Resonance Energy Transfer (FRET) of DNA labeled at either terminus was used to investigate change in DNA conformation upon binding of these homo or heterodimers. The results indicate that only Max binding can bend DNA (∼65°) by asymmetric neutralization of charges on DNA's phosphate backbone, but the other two heterodimers Myc-Max and Mad-Max cannot. Myc-Max has additional contacts with the phosphate backbone through residues specific to Myc. The contacts are absent in Max-Max-DNA complex. It is possible that these contacts are reason that DNA does not under go conformation change due to binding of the heterodimers. Finally it was shown that binding affinity of Max increased ∼15 fold towards prebent DNA.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
