Biomaterial Database

DNA secondary structure effects on DNA synthesis catalyzed by HIV-1 reverse transcriptase. 1998

Z Suo, and K A Johnson

Department of Biochemistry and Molecular Biology, the Pennsylvania State University, University Park, Pennsylvania 16802, USA.

The effect of DNA secondary structure on polymerization catalyzed by human immunodeficiency virus (HIV-1) reverse transcriptase (RT) was studied using a synthetic 66-nucleotide DNA template containing a stable hairpin structure. Four RT pause sites were identified within the first half of the hairpin stem. Additionally, five weak pause sites within the second half of the stem and the loop of the hairpin were identified at low temperatures. These weak pause sites were relocated to the site of the first few stem base pairs of two new hairpins formed due to a change in DNA secondary structure. Each pause site was correlated with a high free energy barrier of melting the stem base pair. Pre-steady state kinetic analysis of single nucleotide incorporation showed that polymerization at each pause site occurred by both a fast phase (10-20 s-1) and a slow phase (0. 02-0.07 s-1) during a single binding event. The reaction amplitudes of the fast phase were small (4-10% of enzyme sites), whereas the amplitudes of the slow phase were large (14-40%) at the pause sites. In contrast, only a single phase with a large reaction amplitude (32-50%) and a fast nucleotide incorporation rate (33-87 s-1) was observed at the non-pause sites. DNA substrates at all sites had similar dissociation rates (0.14-0.29 s-1) and overall binding affinity (16-86 nM). These results suggest that the DNA substrates at pause sites were bound in both productive and non-productive states at the polymerase site of RT. The non-productively bound DNA was slowly converted into a productive state upon melting of the next stem base pair without dissociation of the DNA from RT.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008956	Models, Chemical	Theoretical representations that simulate the behavior or activity of chemical processes or phenomena; includes the use of mathematical equations, computers, and other electronic equipment.	Chemical Models,Chemical Model,Model, Chemical
D008957	Models, Genetic	Theoretical representations that simulate the behavior or activity of genetic processes or phenomena. They include the use of mathematical equations, computers, and other electronic equipment.	Genetic Models,Genetic Model,Model, Genetic
D009690	Nucleic Acid Conformation	The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.	DNA Conformation,RNA Conformation,Conformation, DNA,Conformation, Nucleic Acid,Conformation, RNA,Conformations, DNA,Conformations, Nucleic Acid,Conformations, RNA,DNA Conformations,Nucleic Acid Conformations,RNA Conformations
D009838	Oligodeoxyribonucleotides	A group of deoxyribonucleotides (up to 12) in which the phosphate residues of each deoxyribonucleotide act as bridges in forming diester linkages between the deoxyribose moieties.	Oligodeoxynucleotide,Oligodeoxyribonucleotide,Oligodeoxynucleotides
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D004279	DNA, Viral	Deoxyribonucleic acid that makes up the genetic material of viruses.	Viral DNA
D013816	Thermodynamics	A rigorously mathematical analysis of energy relationships (heat, work, temperature, and equilibrium). It describes systems whose states are determined by thermal parameters, such as temperature, in addition to mechanical and electromagnetic parameters. (From Hawley's Condensed Chemical Dictionary, 12th ed)	Thermodynamic
D014158	Transcription, Genetic	The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION.	Genetic Transcription
D054303	HIV Reverse Transcriptase	A reverse transcriptase encoded by the POL GENE of HIV. It is a heterodimer of 66 kDa and 51 kDa subunits that are derived from a common precursor protein. The heterodimer also includes an RNAse H activity (RIBONUCLEASE H, HUMAN IMMUNODEFICIENCY VIRUS) that plays an essential role the viral replication process.	Reverse Transcriptase, HIV,Reverse Transcriptase, Human Immunodeficiency Virus,Transcriptase, HIV Reverse