This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. The primary research areas are: 1) analysis of the structure and dynamic behavior of the Ribonuclease H domain of HIV reverse transcriptase; 2) dynamic analysis of DNA pol beta - a model system; 3) studies of the unblocking of primer-terminated strands by nucleotides. We recently determined the first solution structure of the 15 kD RNase H domain of HIV reverse transcriptase, using the [13C,15N]-labeled enzyme. The structure generally replicates the previously determined crystal structure, except that the resonances of the C-terminal helix (helix E) are subject to extreme exchange broadening, precluding a structural determination for this element. We have also performed 15N relaxation studies on the RNase H domain in order to evaluate a previous proposal that the isolated domain is dynamically unstable, and hence inactive. Under the conditions of our studies, we find that the dynamic characteristics, i.e. order parameters, internal diffusion rates, etc. of the RNase H domain are qualitatively similar to the (active) E. coli RNase HI enzyme, indicating that dynamic instability is probably not responsible for the inactivity of the isolated domain. We are currently evaluating the interaction of this domain with nucleotides and other ligands, in order to better understand how ligand binding selects the active conformation. In our studies of the base-excision repair enzyme DNA pol beta, we have now assigned all of the methionine methyl resonances in the [methyl-13C]methionine-labeled enzyme. We are currently characterizing the conformational and dynamic changes that result from complex formation with gapped DNA and nucleoside triphosphates. Observation of the M282 methionine resonance is particularly informative as an indicator of the large, open -> closed conformational transition which results from nucleotide binding, since this residue is located on helix N which undergoes a large conformational change. Finally, we have been interested in understanding the fate of AZTp4A, a dinucleoside tetraphoshate that results from the deblocking of AZT-terminated primers by ATP. We have used 31P NMR to demonstrate that the AZTp4A nucleotide can be converted back into AZT triphosphate (AZTTP) by the enzyme Ap4A hydrolase. The latter enzyme

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES050147-09
Application #
6838372
Study Section
(LSB)
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2003
Total Cost
Indirect Cost
Name
U.S. National Inst of Environ Hlth Scis
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Zheng, Xunhai; Mueller, Geoffrey A; Kim, Kyungmin et al. (2017) Identification of drivers for the metamorphic transition of HIV-1 reverse transcriptase. Biochem J 474:3321-3338
Zheng, Xunhai; Pedersen, Lars C; Gabel, Scott A et al. (2016) Unfolding the HIV-1 reverse transcriptase RNase H domain--how to lose a molecular tug-of-war. Nucleic Acids Res 44:1776-88
London, Robert E (2016) Structural Maturation of HIV-1 Reverse Transcriptase-A Metamorphic Solution to Genomic Instability. Viruses 8:
Zheng, Xunhai; Perera, Lalith; Mueller, Geoffrey A et al. (2015) Asymmetric conformational maturation of HIV-1 reverse transcriptase. Elife 4:
Kirby, Thomas W; Derose, Eugene F; Beard, William A et al. (2014) Substrate rescue of DNA polymerase ? containing a catastrophic L22P mutation. Biochemistry 53:2413-22
Zheng, Xunhai; Pedersen, Lars C; Gabel, Scott A et al. (2014) Selective unfolding of one Ribonuclease H domain of HIV reverse transcriptase is linked to homodimer formation. Nucleic Acids Res 42:5361-77
Horton, Julie K; Stefanick, Donna F; Gassman, Natalie R et al. (2013) Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses. DNA Repair (Amst) 12:774-85
Zheng, Xunhai; Mueller, Geoffrey A; DeRose, Eugene F et al. (2013) Protein-mediated antagonism between HIV reverse transcriptase ligands nevirapine and MgATP. Biophys J 104:2695-705
Zheng, Xunhai; Mueller, Geoffrey A; DeRose, Eugene F et al. (2012) Metal and ligand binding to the HIV-RNase H active site are remotely monitored by Ile556. Nucleic Acids Res 40:10543-53
Zheng, Xunhai; Mueller, Geoffrey A; Cuneo, Matthew J et al. (2010) Homodimerization of the p51 subunit of HIV-1 reverse transcriptase. Biochemistry 49:2821-33

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