Title

HIV-1 reverse transcriptase: Conformational change and fidelity

Date of Award

1995

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Walter A. Scott, Committee Chair

Abstract

DNA polymerase binds DNA and nucleotide substrates and goes through a conformational change to catalyze the formation of a phosphodiester bond between the alpha-phosphate of the nucleotide substrate and the 3$\prime$-OH of the primer. If the DNA substrate lacks the hydroxyl group at the 3$\prime$-terminus of the primer, the polymerase conformational change that is induced by the binding of DNA and nucleotide substrates can still happen but the phosphodiester bond cannot be formed. Therefore, the polymerase is trapped in a low energy state and forms a stable ternary complex with the DNA and nucleotide substrates, and this complex is called the dead-end complex (DEC). The formation of DEC is the direct result of polymerase conformational change. Our study has demonstrated that HIV-1 reverse transcriptase (HIV-RT) can form DEC. By using the optimal reaction conditions determined through the characterization of the polymerase activity of HIV-RT on a natural DNA template/primer (M13/M13a), and two pairs of synthetic oligonucleotide template/primers, we determined the structural requirements for DNA and nucleotide substrates to induce the DEC formation. For DNA substrate, the double-strand portion of the template/primer must be at least 18 base pairs (the optimal is 25 base pairs), the single-strand portion must be two bases or longer, and the nucleotide at the 3$\prime$-terminus of the primer must be complementary to the template. The polydeoxyadenosine regions and most of the deoxyadenosine sites in the template are not favored for HIV-RT DEC formation. For nucleotide substrates, the purine or pyrimidine base must be complementary to the next position on the template, the 2$\prime$-position of the ribose must have a hydrogen instead of hydroxyl group, and the nucleotide must contain a triphosphate group. The chemical or structural variations at other portions of the nucleotide structure have less influence on HIV-RT DEC formation. The significance of these structural requirements for the functions of the enzyme is discussed and a model of how a polymerase recognizes and binds with the DNA and nucleotide substrates is proposed.In addition, we also investigated the effects of two non-nucleoside inhibitors on the DEC formation and showed that the influence on HIV-RT conformational change is at least one mechanism by which the enzyme activity is inhibited.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9536895