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Insertions into the β3-β4 Hairpin Loop of HIV-1 Reverse Transcriptase Reveal a Role for Fingers Subdomain in Processive Polymerization
- Source :
- Journal of Biological Chemistry. 273:7529-7537
- Publication Year :
- 1998
- Publisher :
- Elsevier BV, 1998.
-
Abstract
- Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) displays a characteristic poor processivity during DNA polymerization. Structural elements of RT that determine processivity are poorly understood. The three-dimensional structure of HIV-1 RT, which assumes a hand-like structure, shows that the fingers, palm, and thumb subdomains form the template-binding cleft and may be involved in determining the degree of processivity. To assess the influence of fingers subdomain of HIV-1 RT in polymerase processivity, two insertions were engineered in the beta3-beta4 hairpin of HIV-1NL4-3 RT. The recombinant mutant RTs, named FE20 and FE103, displayed wild type or near wild type levels of RNA-dependent DNA polymerase activity on all templates tested and wild type or near wild type-like sensitivities to dideoxy-NTPs. When polymerase activities were measured under conditions that allow a single cycle of DNA polymerization, both of the mutants displayed 25-30% greater processivity than wild type enzyme. Homology modeling the three-dimensional structures of wild type HIV-1NL4-3 RT and its finger insertion mutants revealed that the extended loop between the beta3 and beta4 strands protrudes into the cleft, reducing the distance between the fingers and thumb subdomains to approximately 12 A. Analysis of the models for the mutants suggests an extensive interaction between the protein and template-primer, which may reduce the degree of superstructure in the template-primer. Our data suggest that the beta3-beta4 hairpin of fingers subdomain is an important determinant of processive polymerization by HIV-1 RT.
- Subjects :
- Models, Molecular
DNA polymerase
Molecular Sequence Data
Mutant
Protein Engineering
Biochemistry
Protein Structure, Secondary
Structure-Activity Relationship
chemistry.chemical_compound
Escherichia coli
Computer Simulation
Amino Acid Sequence
Molecular Biology
Polymerase
biology
Wild type
Cell Biology
Processivity
Protein engineering
Molecular biology
HIV Reverse Transcriptase
Reverse transcriptase
Cell biology
Kinetics
Mutagenesis, Insertional
chemistry
biology.protein
DNA
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 273
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....06ba47c366a97a07a55f0e3875e60fb8