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Structural studies of HIV-1 reverse transcriptase: resistance to AZT via ATP-mediated excision
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Title
Structural studies of HIV-1 reverse transcriptase: resistance to AZT via ATP-mediated excision
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Title
Resistance to AZT via ATP-mediated excision
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Tu
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Xiongying
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Xiongying Tu
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author
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Arnold
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Eddy
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Advisory Committee
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Eddy Arnold
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chair
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Sarafianos
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Stefan
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Advisory Committee
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Stefan G Sarafianos
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internal member
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Olson
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Wilma
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Advisory Committee
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Wilma K Olson
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Jones
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Roger
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Advisory Committee
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Roger A Jones
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internal member
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Hughes
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Stephen
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Advisory Committee
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Stephen H Hughes
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Rutgers University
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degree grantor
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Graduate School - New Brunswick
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theses
OriginInfo
DateCreated
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2008
DateOther
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2008-05
Language
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English
PhysicalDescription
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electronic
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application/pdf
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text/xml
Extent
xx, 246 pages
Abstract
AZT, 3'-azido-2',3'-deoxythymidine, is a widely used anti-AIDS drug. Incorporation of AZTTP, the triphosphate form of AZT, into a growing DNA chain by the human immunodeficiency virus (HIV) reverse transcriptase (RT) blocks chain elongation. HIV develops resistance to AZT by acquiring mutations in reverse transcriptase (RT) that enhance ATP-mediated excision of AZTMP. This excision frees the end of the primer strand, making it possible for RT to continue viral DNA synthesis. The product of the excision reaction is AZT adenosine dinucleoside tetraphosphate (AZTppppA). In order to understand the structural bases of this resistance mechanism, five different HIV-1 RT complex structures involved in this ATP-mediated excision were determined using a cross-linking strategy and X-ray crystallography.
The structures of wild-type and AZT-resistant HIV-1 RT with a dsDNA and an AZTppppA (excision product complex) were determined to 3.15 and 3.2 Å resolution, respectively. Superposition of these two structures reveals that the ATP binds differently to wild-type RT and AZT-resistant RT. Primary resistance mutations do not improve a pre-existing site but create a new, higher-affinity binding site for ATP. The primary AZT-resistance mutations K70R and T215Y make significant contributions to ATP-binding, whereas, secondary resistance mutations assist the binding of ATP to RT. These structures also provide insights into antagonism between AZT-resistance mutations and other nucleoside-analog resistance mutations, including K65R and K70E.
The structure of AZT-resistant HIV-1 RT with a pre-translocation AZTMP-terminated DNA was determined to 3.7 Å. This structure has the fingers subdomain in a closed configuration instead of an open configuration found in the reported structure of the wild-type HIV-1 pre-translocation complex. The pre-translocation complex with the fingers in a closed configuration may be the preferred acceptor for ATP. This structure also implies polymerization/pyrophosphorolysis may occur without major opening of the fingers.
The structures of the AZT-resistant HIV-1 RT with a post-translocation AZTMP-terminated DNA and the unliganded AZT-resistant HIV-1 RT were determined to 2.9 and 2.65 Å resolution, respectively. Structural comparisons of these two complexes and excision product complexes suggest that the side chains of the two primary resistance mutations may undergo conformational changes that should be induced by the ATP.
The structures of wild-type and AZT-resistant HIV-1 RT with a dsDNA and an AZTppppA (excision product complex) were determined to 3.15 and 3.2 Å resolution, respectively. Superposition of these two structures reveals that the ATP binds differently to wild-type RT and AZT-resistant RT. Primary resistance mutations do not improve a pre-existing site but create a new, higher-affinity binding site for ATP. The primary AZT-resistance mutations K70R and T215Y make significant contributions to ATP-binding, whereas, secondary resistance mutations assist the binding of ATP to RT. These structures also provide insights into antagonism between AZT-resistance mutations and other nucleoside-analog resistance mutations, including K65R and K70E.
The structure of AZT-resistant HIV-1 RT with a pre-translocation AZTMP-terminated DNA was determined to 3.7 Å. This structure has the fingers subdomain in a closed configuration instead of an open configuration found in the reported structure of the wild-type HIV-1 pre-translocation complex. The pre-translocation complex with the fingers in a closed configuration may be the preferred acceptor for ATP. This structure also implies polymerization/pyrophosphorolysis may occur without major opening of the fingers.
The structures of the AZT-resistant HIV-1 RT with a post-translocation AZTMP-terminated DNA and the unliganded AZT-resistant HIV-1 RT were determined to 2.9 and 2.65 Å resolution, respectively. Structural comparisons of these two complexes and excision product complexes suggest that the side chains of the two primary resistance mutations may undergo conformational changes that should be induced by the ATP.
Note
(type = degree)
Ph.D.
Note
(type = bibliography)
Includes bibliographical references (p. 236-245).
Subject
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Topic
Chemistry and Chemical Biology
Subject
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Topic
HIV (Viruses)
Subject
(ID = SUBJ3);
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Topic
Reverse transcriptase
Subject
(ID = SUBJ4);
(authority = ETD-LCSH)
Topic
AZT (Drug)
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Graduate School - New Brunswick Electronic Theses and Dissertations
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rucore19991600001
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http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.17402
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ETD_974
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Identifier
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doi:10.7282/T3GM87N3
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ETD doctoral
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The author owns the copyright to this work.
Copyright
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Copyright protected
Availability
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Open
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Xiongying Tu
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Rutgers University. Graduate School - New Brunswick
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Non-exclusive ETD license
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Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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