Title

Stable complex formation by HIV-1 reverse transcriptase

Date of Award

2006

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Walter A. Scott - Committee Chair

Abstract

The formation of RT complex induced by the next complementary dNTP was observed and is referred to as the +1 complex. It was identified as a stable by various biochemical assays and forms cross-links at a specific position in the template upon UV treatment.A unique RT complex formed in the presence of PFA (phosphonoformic acid or foscarnet, a pyrophosphate analog) was also observed 1 nt upstream compared to the +1 complex, indicating that the PFA complex stays in a pre translocated position whereas the +1 complex stays in a post-translocated position.Another unique, stable complex formed with the dNTP complementary to the +2 base of the template, named the +2 complex, was detected. The +2 complex as predominantly positioned 1 nt downstream from the +1 complex which places RT in a "hyper-translocated" position. The +2 complex is less stable than the +1 and PFA complexes. Differences in positioning of the +1, +2 and PFA complexes on DNA indicate that binding of the +1 dNTP, the +2 dNTP or PFA can control positioning of RT on DNA.One of the distinctive characteristics of the +1, +2 and PFA complexes is the tight interaction of the downstream border of RT on the template DNA. Furthermore, the specific interaction between RT and the single-stranded portion of the template was identified as the +2 base of the template in the +1 complex, which is consistent with the crystal structure of Huang et al. (1998). More importantly, the interaction of RT with the +1 base of the template observed in the PFA cross-linked product reinforced the conclusion that the PFA complex captures RT in a "closed" conformation and is positioned 1 nt upstream on DNA compared to the +1 complex.The characterization of stable complex formation by HIV-1 RT induced by the binding of dNTPs or PFA yielded new insights into the mechanistic properties of HIV-1 RT that are most likely involved in conformational change, translocation, fidelity, processivity and possible product inhibition by PFA.

Keywords

Biology, Molecular

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:3243102

Link to Full Text

Share

COinS