Kinetic rates of nucleotide incorporation and stepping statistics of HIV-1 reverse transcriptase (HIV-RT) during DNA polymerization.

摘要

Human Immunodeficiency Virus (HIV) is a pandemic that is growing rapidly throughout the world. HIV is transmitted from person to person via direct sexual contact, either homosexual or heterosexual, by blood or blood products, and from mother to child. Today, there are more then 40 million people living with HIV throughout the world with this disease, and each year more and more people become infected. HIV Reverse Transcriptase (HIV-RT) is the polymerase responsible for replicating the HIV genome, and is the subject of this dissertation. Dynamic single molecule fluorescence microscopy has been utilized to study the kinetic behavior of adsorbed HIV-RT. HIV-RT is the enzyme that converts viral RNA into ds-DNA for the HIV virus. This multifunctional enzyme performs three catalytic activities essential to viral replication: RNA-template directed DNA polymerization, Ribonuclease H (RNase H) activity, and DNA-template directed DNA polymerization. Single-base nucleotide incorporation rates have been determined by stalling and restarting various polymerases, but intrinsic processive rates have been difficult to obtain, particularly for polymerases with low processivity such as HIV-RT. The intrinsic processive DNA-dependent polymerization of HIV-RT is approximately Possionian (i.e. each nucleotide is added sequentially) with a rate of about one hundred bases per second at 21°C for the dGTP nucleotide. Variation of DNA template length allows independent verification of this rate constant, and overall polymerization times have been found to scale linearly. From the same set of experiments, based on the stepping statistics of polymerization, we also estimate the rates for HIV-RT early termination and final release of completely replicated primer-template DNA. Other nucleotide incorporations (dCTP, dATP, dTTP) have been studied and rates of replication have recently been determined.