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 CHAMPAIGN -- 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.)
By tracking 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.
"It's 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."
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Such reactions 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.
Although 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.
"Helicases 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."
The helicase 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.
"It's 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."
Myong noted 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.
Funding for this research was provided by the National Institute of
General Medical Sciences at the National Institutes of Health.
[Text copied from
University
of Illinois news release]
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