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Dr. Victor Serebrov Post-doc
victor.serebrov@yale.edu
Exploring
Physical and Kinetic Characteristics of Unwinding by NS3 Helicase from
Hepatitis C Virus
RNA and DNA helicases belonging to the family of DExH/D box proteins form
a very large group of proteins associated with virtually all processes
involving nucleic acids. They act as chemo-mechanical machines that utilize
the energy of NTPs to unwind or otherwise manipulate RNA or DNA. Like
other known molecular motors, many helicases are processive, i.e. they
can translocate along duplexes in a stepwise manner without dissociation,
unwinding a certain number of base pairs per step as they translocate.
The mechanism of such translocation/unwinding and its coupling with NTP
hydrolysis is yet to be understood. Thus far, only a small number of helicases
have been well characterized as molecular motors with known step size,
functional oligomeric state and details on energy coupling. In particular,
determination of the step size and number of NTP molecules hydrolyzed
per step has been a challenging task, partially due to difficulties in
detecting transient states of unwinding that correspond to partially unwound
duplexes. Our laboratory has been exploring mechanisms of unwinding by
RNA helicases. We have developed a new combinatorial approach to address
the mechanistic aspects of RNA unwinding. This approach incorporates random
nicks in a polynucleotide duplex, thereby generating a library of substrates
with varying lengths. When the helicase passes a nick, the unwound strand
dissociates from the substrate, thus allowing us to detect partially unwound
duplexes. After subjection to single-cycle unwinding, the products are
resolved by electrophoresis. By applying this analysis to NS3, we have
independently established the "physical" and "kinetic" step size for unwinding
of RNA (18 base pairs, in each case), which we relate to the stoichiometry
of the functional, translocating species (the NS3 dimer). Having obtained
microscopic unwinding rate constants at each position along the duplex,
we demonstrate that NS3 unwinds RNA through a highly coordinated cycle
of fast ripping and local pausing that occurs with regular spacing along
the duplex substrate, much like the stepping behavior of cytoskeletal
motor proteins. The next step of our study will involve exploring the
mechanistic behavior of the NS3 on different types of polymers (such as
DNA) and in complex with other components of the replication machinery
(i.e. the NS4A co-factor and NS5B polymerase). |
