Mapping the DNA binding site of HIV-1 integrase using fluorescent oligonucleotides and fluorescence polarization.
Item
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Title
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Mapping the DNA binding site of HIV-1 integrase using fluorescent oligonucleotides and fluorescence polarization.
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Identifier
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AAI3127928
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identifier
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3127928
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Creator
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Voloshchuk, Natalya.
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Contributor
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Adviser: Lesley Davenport
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Date
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2004
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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 | Biology, Molecular
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Abstract
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The binding site of HIV-1 integrase (HIV-1 IN) for viral DNA has been characterized using double and single-stranded 21-base pair model oligonucleotide substrates of the U5 and U3 LTR termini, labeled with fluorescent guanosine pteridine analogs at various positions along the specific sequence. HIV-1 IN recognizes viral DNA ends specifically during the 3'-processing reaction prior to the non-specific chromosomal integration step. At 20°C, the 3'-processing activity of HIV-1 integrase is negligible and binding of the protein with the oligonucleotide sequences may be studied exclusively. Two pteridine probes, 3-MI and 6-MI, have been site-selectively inserted along the U5 sequence and all exhibit rotational sensitivity on binding to HIV-1 IN. Varying the position of the fluorescent probe along the viral DNA sequence permits mapping of contact sites between HIV-1 IN and the model substrate using steady-state fluorescence emission anisotropy (EA). Apparent KD values, obtained from direct EA titrations, reveal relatively high affinity interactions between the protein and double-stranded oligonucleotide substrates (∼10-7 M) fluorescently labeled on either the plus or minus strand. For the U5 end a 2-fold preference for binding to positions 2 and 5 on the plus strand of the oligonucleotide over the negative strand was determined. Indeed, the KDapp values determined from the negative strand 21-base pair model viral substrates showed less selectivity, similar to a control oligonucleotide with random sequence. Lower binding affinities are observed for single-stranded over duplex substrates. Here, determined affinities are relatively invariant to the fluorophore position along the length of the single-stranded oligonucleotide suggesting no specificity for binding. In agreement with other in vitro systems, the U3 end 21-mer oligonucleotide appears to be less active than the U5 end short oligonucleotide as reflected by lower binding affinity and reduced activity of HIV-1 integrase with the U3 end model oligonucleotide. There was no U5 or U3 end preference observed for HIV-1 IN interaction with longer oligonucleotides (41 bp) over shorter oligonucleotides (21bp) for this system. The influence of the divalent cations, Mg2+, Mn2+ and Zn2+ on the binding to the protein, revealed that Mn2+ is required for binding, which correlates with the activity observed for this protein.
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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.
