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Hepatitis C helicase unwinds DNA in a spring-loaded, 3-step process
Diana Yates, Life Sciences Editor
note: Simulation is available.
photo to enlarge
by L. Brian Stauffer
single-molecule fluorescence analysis, Institute
for Genomic Biology professor Sua Myong, right,
and physics professor Taekjip Ha led a team that
discovered the mechanism by which the hepatitis
C helicase unwinds DNA and RNA for replication.
Ha is also affiliated with the Institute for Genomic
Biology and the Howard Hughes Medical Center.
Ill. — The
process by which genes are duplicated is mysterious and complex, involving
a cast of characters with diverse talents and the ability to play well
with others in extremely close quarters. A key player on this stage is
an enzyme called a helicase. Its job is to unwind the tightly coiled
chain of nucleic acids – the
DNA or RNA molecule that spells out the organism’s genetic code – so
that another enzyme, a polymerase, can faithfully copy each nucleotide
in the code.
Researchers at the University of Illinois, Yale University and the
Howard Hughes Medical Institute have shed new light on how the Hepatitis
C helicase plays this role, using a technique developed at Illinois
that can track how a single molecule of RNA or DNA unwinds. Their research
findings appear tomorrow in the journal, Science.
Getting at the underlying mechanisms of replication is no easy task.
Structural studies involve crystallizing the DNA-protein complexes
to see how they interact. Biochemists look at the agents of a reaction,
the energy used and how much time lapses between steps. Such studies
measure the behavior of hundreds of thousands of molecules at a time,
and the results describe a whole population of reactions.
Using single-molecule fluorescence analysis, the research team tracked
how the hepatitis C helicase, NS3, unwound a duplexed DNA molecule
tagged with a fluorescent label on each strand of its double-stranded
region. (The NS3 helicase is primarily involved in unwinding the single-stranded
RNA of the hepatitis virus, but it can also act on DNA. This suggests
that the helicase plays a role in unwinding double-stranded host DNA
during infection. The duplex created for the experiment included both
single- and double-stranded DNA; fluorescent labels were located in
the double-stranded region.)
the gradually increasing distance between the two marked nucleotides as the
strands separated in an unwinding event, the researchers were able to measure
the rate at which the unwinding occurred. What they found was that the DNA
unwound in discrete jumps: Three nucleotide pairs (base pairs) had to be unhitched
from one another before an unwinding event occurred.
like you’re adding tension to a spring,” said U. of I. physics professor Taekjip Ha, a researcher on the study and an affiliate of the Institute
for Genomic Biology and the Howard Hughes Medical Institute. “You
are loading the spring with small mechanical movements until finally
you have accumulated enough tension on the DNA-protein complex to
cause the rapid unwinding of three base pairs.”
are energetically intensive, requiring the input of adenosine triphosphate
(ATP) a cellular fuel source. The researchers observed that three ATP
molecules were consumed in each unwinding reaction, indicating that three “hidden
steps,” each involving the unhitching of one base pair, occurred
for each unwinding event.
one molecule of ATP contains enough energy to unwind as many as 10
base pairs, the researchers said they were not surprised by the high-energy costs
of the reaction.
work hand in hand with polymerases in replication, so it makes sense
that the helicase would work on one base pair at a time,” said Institute
for Genomic Biology professor Sua Myong, who is lead author on the study. “It’s
a very systematic, one-base-pair translocation that may help the polymerase
accurately copy genes one base at a time.”
must also navigate around a lot of obstacles: proteins and other co-factors
that are involved in replication. This requires extra energy. Ha compared
the energy needs of the NS3 helicase to those of a sport utility vehicle.
not fuel efficient but in principle it could also go off-road, carry
some luggage or maneuver around barriers,” he said. “So it may actually
make sense to develop a low-efficiency motor because then you have
extra energy to do extra work when needed.”
that NS3 is the only helicase in the viral genome, and that it is already
being targeted in pharmaceutical studies to combat Hepatitis C infection. It
also belongs to the largest of four helicase superfamilies, so the new findings
could have relevance across many organisms.
for this research was provided by the National Institute of General
Medical Sciences at the National Institutes of Health.
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