PA) - Researchers at the University of Pennsylvania School
of Medicine have determined the structure of an important
smallpox virus enzyme and how it binds to DNA. The enzyme, called
a topoisomerase, is an important drug target for coming up with
new ways to fight smallpox. The researchers present their findings
in the August 4 issue of Molecular Cell.
“This enzyme is one of the most closely studied DNA-modifying
enzymes in biology,” says Frederic D. Bushman, PhD,
Professor of Microbiology, one of the senior authors. “The
structure of the DNA complex has been long-awaited.” DNA-modifying
enzymes bind to specific sequences in the genetic code to aid in
the many steps of DNA replication.
The smallpox virus is one of the most easily transmissible infectious
diseases known to humans, resulting in up to 30 percent mortality.
The efficiency with which it spreads, combined with the deadly nature
of the disease, has raised fears that smallpox could be revived
for use in bioterrorism. Knowing the exact three-dimensional structure
of smallpox virus proteins could help researchers design antiviral
agents, but few structures of whole viral proteins exist.
Poxviruses are large viruses that contain two strands of DNA and
replicate themselves entirely in the cytoplasm of infected cells.
Poxviruses do not take over the genetic machinery inside the nucleus
of the host cell, as many viruses do. Because of this strategy,
poxviruses encode many of the enzymes they need to replicate their
own genes, and hence reproduce. One of these enzymes is a topoisomerase,
which is used by the virus to relieve the excessive twisting of
DNA strands that normally occurs during DNA replication and transcription
of the viral genes. Upon initial infection, the poxviruses come
already equipped with some proteins, including topoisomerases, to
The structure was determined in a collaborative effort between the
Bushman lab and the lab of the other senior author Gregory
D. Van Duyne, PhD, Professor of Biochemistry and Biophysics
and an Investigator with the Howard Hughes Medical Institute (HHMI).
Using purified topoisomerase enzyme that had been expressed in bacterial
cells, they bound the enzyme to short segments of DNA that contained
the viral topoisomerase’s specific recognition sequence. They
then determined the three-dimensional structure of the topoisomerase-DNA
complex using X-ray crystallography.
One of the primary differences between the viral topoisomerase enzyme
and the closely related human enzyme that functions in the nucleus
of all human cells is that the viral enzyme only relaxes supercoiled
DNA when it binds to specific DNA sequences. The structure of the
poxvirus topoisomerase-DNA complex provides some important clues
about how this recognition and activation mechanism works.
“The more the viral enzyme differs from the human nuclear
enzyme, the more likely it is that inhibitors could be developed
that are specific to the viral enzymes,” says Bushman.
Knowing the three-dimensional structure of the smallpox virus topoisomerase-DNA
complex will also facilitate the design of agents to combat poxvirus
infections. Topoisomerases are some of the most widely targeted
proteins by drugs that are intended to inhibit growth of the cell.
Drugs that target topoisomerases generally stabilize an intermediate
of the enzyme’s reaction in which one of the DNA strands is
broken. If these breaks are not repaired, the DNA cannot be replicated
and the cell dies.
In the case of smallpox virus, the hope is that drugs targeted to
the viral topoisomerase enzyme will prevent viral replication through
a similar mechanism. The X-ray structure provides a template for
designing small molecules that could stabilize the broken DNA in
the intermediate form, thereby killing smallpox virus particles.
Study co-authors are Kay Perry and Young Hwang, both from Penn.
The research was supported by HHMI and the National Institutes of
Health through the Middle Atlantic Regional Center of Excellence.
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