This proposal will focus on analysis of the catalytic mechanism and DJA template interactions of the human immunodeficiency virus-I (HIV) DNA poluymerase. This virus is the etiologic agent of acquired immunodeficiency syndrome (AIDS). Recombinant HIV-I polymerase has been obtained from Genetics Institute (Boston, MA). DNA binding properties of HIV polymerase that could relate to DNA synthesis efficiency will be analyzed using structurally defined DNA molecules. Experiments will assess dependence of binding of 3'OH termini or cofactors such as dNTPs. They will be performed under conditions where synthetic and RNase H activities are differentially inhibited. Distribution of polymerase between primer termini and single-stranded regions of DNA will be measured. Template strand switching during processive DNA synthesis will be studied with regard to the role of RNase Hand template requiremtns for switching to occur. Processivity, an inherent property of a polymerase, also will be studied in response to potentially therapeutic anti-viral drugs. Using specifically primed phage DNA templates, positions on the template that act as barriers to synthesis by the polymerase will be determined. Results will be correlated to particular sequences or secondary structures. The fidelity of DNA synthesis catalyzed by HIV polymerase will be studied using an M13mp21acZ-alpha forward mutational assay system. It will be determined whether generation of errors correlates with positions of pauses in DNA synthesisl. Tjhe potential for the host cell to attenuate misincorporation by HIV polymerase will be addressed by fidelity analyses performed in the presence of calf DNA polymerase delta II, a high M., nuclear polymerase, having a non- dissociable 3' to 5' exonuclease. Finally, a study of the role of HIV polymerase in recombination will be undertaken. Specific experiments will address the ability of HIV polymerase to bind and synthesize on two templates simultaneously. Novel variations of the M13 mutational assay will be used to quantitate HIV poluymerase-mediated recombinational events. Results will provide fundamental insights into the properties of HIV polymerase, the enzyme that replicates the HIV genome, and a primary target protein for AIDS therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI027054-01A1
Application #
3141112
Study Section
(ARR)
Project Start
1989-09-30
Project End
1992-07-31
Budget Start
1989-09-30
Budget End
1990-07-31
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Rochester
Department
Type
School of Medicine & Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
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DeStefano, J J; Mallaber, L M; Fay, P J et al. (1993) Determinants of the RNase H cleavage specificity of human immunodeficiency virus reverse transcriptase. Nucleic Acids Res 21:4330-8
DeStefano, J J; Bambara, R A; Fay, P J (1993) Parameters that influence the binding of human immunodeficiency virus reverse transcriptase to nucleic acid structures. Biochemistry 32:6908-15
Buiser, R G; Bambara, R A; Fay, P J (1993) Pausing by retroviral DNA polymerases promotes strand transfer from internal regions of RNA donor templates to homopolymeric acceptor templates. Biochim Biophys Acta 1216:20-30
DeStefano, J J; Buiser, R G; Mallaber, L M et al. (1992) Parameters that influence processive synthesis and site-specific termination by human immunodeficiency virus reverse transcriptase on RNA and DNA templates. Biochim Biophys Acta 1131:270-80
DeStefano, J J; Mallaber, L M; Rodriguez-Rodriguez, L et al. (1992) Requirements for strand transfer between internal regions of heteropolymer templates by human immunodeficiency virus reverse transcriptase. J Virol 66:6370-8
DeStefano, J J; Buiser, R G; Mallaber, L M et al. (1991) Human immunodeficiency virus reverse transcriptase displays a partially processive 3' to 5' endonuclease activity. J Biol Chem 266:24295-301
DeStefano, J J; Buiser, R G; Mallaber, L M et al. (1991) Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled. J Biol Chem 266:7423-31
Buiser, R G; DeStefano, J J; Mallaber, L M et al. (1991) Requirements for the catalysis of strand transfer synthesis by retroviral DNA polymerases. J Biol Chem 266:13103-9