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polymerases are very dynamic enzymes that need to control simultaneously
parameters such as specificity, fidelity, and efficacy together with interac-
tions with different types of substrates and partners. Fluorescently labeled
oligonucleotides were used to investigate the mechanism of several DNA
or RNA polymerases as well as to monitor the binding of regulatory pro-
teins. 37 RNA or DNA polymerase activity has been probed by measuring
changes in extrinsic fluorescence at the steady-state and kinetic levels as well
as by fluorescence anisotropy as the size of the binder is significantly smaller
than that of the protein. 18
The reverse transcriptase (RT) of HIV (human immunodeficiency virus)
is a key enzyme in the virus replication cycle and catalyzes a chain of reac-
tions to convert the single-stranded HIV RNA genome into a double-
stranded DNA for further integration in the cell host genome. This requires
that RT is able to discriminate between different nucleic acids and to place
them correctly in one of the three catalytic sites for RNA-dependent DNA
synthesis, DNA-dependent DNA synthesis, and RNAse-H. RT structure
and mechanism have been investigated in detail by combining steady-state
transient kinetic, and FRET single-molecule assay using dyes attached to the
enzyme or to its different partners: tRNA, viral-RNA, and DNA primer/
template. 38-41 These investigations have shown that catalysis of RT is
dependent on the binding orientation of the substrate, which adopts
opposite conformations for DNA and RNA duplexes ( Fig. 4.9 ). The
large change in dye fluorescence upon binding of primer/template
oligonucleotide to RT was used to follow this interaction at both the
steady-state and pre-steady-state levels ( Fig. 4.9 ). Stopped-flow
technology has been developed for real-time fluorescence kinetic
analysis. 42-44 A stopped-flow apparatus is a rapid mixing device used to
study the chemical kinetics of a reaction in solution, and stopped-flow
measurements involve the rapid mixing of two or more solutions
( Fig. 4.9 ). The dead time, corresponding to the time between the end of
mixing and the beginning of the observed kinetics of the reaction, ranges
between 0.5 and 2 ms. In most cases, protein/partner interactions cannot
be analyzed as a simple first-order or second-order reaction, as they often
combine binding and conformational events that can be discriminated
depending on the rate of each step. In the case of HIV RT, experimental
use of fluorescently labeled double-stranded oligonucleotide for rapid
kinetics studies has allowed for a detailed characterization of the
mechanism and structure of the enzyme during initiation of replication.
Kinetic analysis of
the Fluorescein phosphoramidite (FAM) - labeled
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