Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Radiation target analysis is a powerful technique that can be used to determine both the structural and functional sizes of macromolecules. We have used this technique to probe the structure-function relationships of the recombinant forms of HIV-1 reverse transcriptase (RT). For the p66/p51 and p66/p66 dimeric forms of HIV-1 RT, both the structural and functional target sizes corresponded to that of the dimeric protein, indicating that a primary ionization in one subunit of the HIV-1 RT enzyme results in the concomitant polymer scission of both subunits. In contrast to p66/p51 and p66/p66 RT, the individually isolated p51 subunit of HIV-1 RT inactivated as a monomer. However, in the presence of a DNA template/primer substrate, the radiation inactivation analyses of p51 yielded a structural target size corresponding to that of a dimeric protein. This would indicate that the DNA substrate acted as a scaffold or template for p51 RT homodimer formation. In light of this observation, radiation inactivation studies can readily be applied to other DNA polymerase enzymes, such as the murine leukemia virus RT, for which the functional form of the enzyme has yet to be determined.
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PMID:Structure-activity relationships in HIV-1 reverse transcriptase revealed by radiation target analysis. 1293 Oct 6

To investigate how structural changes in the amino acid side chain affect nucleotide substrate selection in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a variety of non-natural tyrosine analogues were substituted for Tyr115 of p66 RT. RT variants containing meta-Tyr, nor-Tyr, aminomethyl-Phe, and 1- and 2-naphthyl-Tyr were produced in an Escherichia coli coupled transcription/translation system. Mutant p66 subunits were reconstituted with wild-type (WT) p51 RT and purified by affinity chromatography. Each modified enzyme retained DNA polymerase activity following this procedure. Aminomethyl-Phe115 RT incorporated dCTP more efficiently than the WT and was resistant to the chain terminator (-)-beta-2',3'-dideoxy-3'-thiacytidine triphosphate (3TCTP) when examined in a steady-state fidelity assay. However, 2-naphthyl-Tyr115 RT inefficiently incorporated dCTP at low concentrations and was kinetically slower with all dCTP analogues tested. Models of RT containing these side chains suggest that the aminomethyl-Phe115 substitution provides new hydrogen bonds through the minor groove to the incoming dNTP and the template residue of the terminal base pair. These hydrogen bonds likely contribute to the increased efficiency of dCTP incorporation. In contrast, models of HIV-1 RT containing 2-naphthyl-Tyr115 reveal significant steric clashes with Pro157 of the p66 palm subdomain, necessitating rearrangement of the active site.
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PMID:Investigating the "steric gate" of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by targeted insertion of unnatural amino acids. 1727 99

Previous studies have demonstrated that nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) act as chemical enhancers of human immunodeficiency virus type 1 (HIV-1) RT dimerization. In the current study, we sought to define the role of key residues (101, 103, 108, 181, 188, 190, 225 and 318) in the NNRTI-binding pocket on HIV-1 RT heterodimer stability. Thirteen mutant RTs were constructed and evaluated for p66/p51 RT heterodimer formation using the well-established yeast two-hybrid assay. We found that the mutations K101A, P225H, Y318F and Y318W decreased RT heterodimer stability whereas K103N, V108I, V108W, Y181C, Y188L, G190A, G190E, G190W and P225W increased RT heterodimer stability. While these results demonstrate that residues that comprise the NNRTI-binding pocket contribute to the stability of p66/p51 HIV-1 RT, they did not suggest any obvious correlation between RT dimer stability and the extent of NNRTI resistance. Remarkably, mutations at residue G190 (A, E, W) in the p66 RT subunit were found to dramatically increase heterodimer stability. Notably, the G190W mutation increased RT dimer stability almost to the same extent as did 5 microM efavirenz. In light of these findings, we characterized the in vitro activity of recombinant RT expressing mutations at G190 in the p66 subunit only and compared them with a wild-type enzyme complexed with efavirenz. We found that while mutations at G190 had a significant effect on both the DNA polymerase and ribonuclease H activity of the enzyme, their phenotypic effects did not mirror those induced by efavirenz-binding to RT.
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PMID:Impact of residues in the nonnucleoside reverse transcriptase inhibitor binding pocket on HIV-1 reverse transcriptase heterodimer stability. 1833 60