The goal of this proposal is to establish the structural, kinetic and thermodynamic basis for the fidelity and efficiency of DNA replication. Analysis of HIV reverse transcriptase will allow definition of the structural constraints that ultimately may limit the ability of the virus to avoid an appropriate combination of nucleoside analogs in the treatment of AIDS and other viral infections. Analysis of the mitochondrial DNA polymerase will define the origins of the toxicity of nucleoside analogs thought to be due to incorporation of the analogs into mitochondrial DNA. Studies on T7 DNA polymerase and HIV RT have shown that the selectivity of the polymerase is a function of a two-step nucleotide binding reaction involving a nucleotide-induced change in conformation from an """"""""open"""""""" to a """"""""cloned"""""""" state of the ternary E.DNA.dNTP complex. Based upon the crystal structure of HIV RT complexed with DNA, a working model has been formulated proposing the movement of the """"""""fingers"""""""" domain of the polymerase into the major groove of DNA in response to the binding of a correct base pair.
The specific aims of this proposal are to: (1.) Identify the protein structural domains involved in the conformational change leading to tight nucleotide binding and rapid polymerization; specific photo-affinity labeling will be used to localize amino acids which are brought into close contact with the DNA major groove following the conformational change. (2.) Transient state kinetic analysis of site- directed mutants will be employed to further define the contacts made in the """"""""closed"""""""" conformational state and to quantify the contributions of individual amino acids. The kinetics of the conformational change will be examined by stopped-flow methods using fluorescently labeled nucleotides, DNA and protein. (3.) The effects of DNA structure on the conformational change and the kinetics of polymerization will be examined. In particular, the role of DNA bends will be investigated, and the mechanisms of frameshift mutagenesis will be examined by measuring the kinetics of extension over premutational frameshift intermediates. (4.) The effect of RNA secondary structure on the kinetics of polymerization catalyzed by RT will be investigated and the effect of the nucleocapsid protein on the ability of the polymerase to read through hairpins will be assessed. (5.) Genes for the mitochondrial polymerase will be cloned and conditions will be optimized for the overexpression and purification of active protein. The mechanism and fidelity of the mitochondrial polymerase will be established by kinetic measurements.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM044613-09
Application #
2873511
Study Section
Special Emphasis Panel (ZRG1-AARR-1 (01))
Project Start
1991-04-01
Project End
2003-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
9
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78712
Qian, Yufeng; Kachroo, Aashiq H; Yellman, Christopher M et al. (2014) Yeast cells expressing the human mitochondrial DNA polymerase reveal correlations between polymerase fidelity and human disease progression. J Biol Chem 289:5970-85
Johnson, Kenneth A (2013) A century of enzyme kinetic analysis, 1913 to 2013. FEBS Lett 587:2753-66
Estep, Patricia A; Johnson, Kenneth A (2011) Effect of the Y955C mutation on mitochondrial DNA polymerase nucleotide incorporation efficiency and fidelity. Biochemistry 50:6376-86
Lee, Young-Sam; Johnson, Kenneth A; Molineux, Ian J et al. (2010) A single mutation in human mitochondrial DNA polymerase Pol gammaA affects both polymerization and proofreading activities of only the holoenzyme. J Biol Chem 285:28105-16
Johnson, Kenneth A (2010) The kinetic and chemical mechanism of high-fidelity DNA polymerases. Biochim Biophys Acta 1804:1041-8
Batabyal, Dipanwita; McKenzie, Jessica L; Johnson, Kenneth A (2010) Role of histidine 932 of the human mitochondrial DNA polymerase in nucleotide discrimination and inherited disease. J Biol Chem 285:34191-201
Lee, Young-Sam; Lee, Sujin; Demeler, Borries et al. (2010) Each monomer of the dimeric accessory protein for human mitochondrial DNA polymerase has a distinct role in conferring processivity. J Biol Chem 285:1490-9
Brandis, John W; Johnson, Kenneth A (2009) High-cell density shake-flask expression and rapid purification of the large fragment of Thermus aquaticus DNA polymerase I using a new chemically and temperature inducible expression plasmid in Escherichia coli. Protein Expr Purif 63:120-7
Lee, Young-Sam; Kennedy, W Dexter; Yin, Y Whitney (2009) Structural insight into processive human mitochondrial DNA synthesis and disease-related polymerase mutations. Cell 139:312-24
Lee, Harold R; Helquist, Sandra A; Kool, Eric T et al. (2008) Importance of hydrogen bonding for efficiency and specificity of the human mitochondrial DNA polymerase. J Biol Chem 283:14402-10

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