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DURHAM, N.C. -- Duke University Medical Center biochemists
have made the startling discovery that an enzyme that copies
DNA in living cells can also be made to operate when held in
place in crystal form.

Their achievement opens the way for understanding the finest
details of how the intricate DNA-copying enzyme -- called DNA
polymerase -- manages to reproduce DNA with the impeccable
accuracy necessary for all living things to grow and reproduce
nearly flawlessly.

By shining X-rays through the actively functioning crystals,
the biochemists have taken snapshots that catch the enzyme in
the biochemical act of copying DNA. Soon they will make movies.
The scientists' images also are revealing details of how
cancer-causing chemicals interfere with the copying
process.

The biochemists, led by assistant professor of biochemistry
Lorena Beese, published their discovery in the Jan. 15 issue of
Nature, in an article titled "Visualizing DNA Replication in a
Catalytically Active Polymerase at 1.8 Angstrom Resolution."
Besides Beese, authors of the paper are graduate student James
Kiefer and postdoctoral associate Chen Mao of Duke, and
researcher Jeffrey Braman of Stratagene of La Jolla, Calif.

The scientists' work was supported by the American Cancer
Society, the Searle Scholar Foundation, and the North Carolina
Biotechnology Center.

Enzymes are proteins that are the workhorses of the cell,
catalyzing the multitude of chemical reactions that underlie
all cell functions. The molecules that enzymes act upon are
called substrates.

The DNA polymerase that the Duke researchers studied is part
of a complex molecular assembly line that is central to all
cell division. A cell preparing to divide first unzips its
double-stranded DNA into a single strand, to prepare for DNA
copying. DNA polymerase then attaches to one strand, using it
as a template. The polymerase works its way along the strand,
chemically stitching into place DNA units called nucleotides,
to form a second DNA strand.

After each attachment of the correct nucleotide the enzyme
"translocates," shifting to the next unit, like a stockbroker
reading a ticker tape. Besides merely copying DNA, the
polymerase also exercises exacting quality control, carefully
proofreading its work and halting production the instant it
detects an error, so that repair enzymes can step in.

Despite decades of study, many details of this sophisticated
and critical DNA replication process remain mysterious, Beese
said. To understand those mysteries, she and her colleagues use
an analytic technique called X-ray crystallography to visualize
the polymerase structure. In this technique, scientists shine
an intense X-ray beam through a crystal of protein. The crystal
diffracts the beam into a multitude of spots, and using a
computer, the scientists can deduce the structure of the
protein from the pattern of spots. In past studies, Beese and
her colleagues used the technique to produce high-resolution
structures of DNA polymerases alone.

But in the new work, they sought to study the structure of a
polymerase with a stand of DNA captured in its "active site"--
the pocket in the molecule where DNA assembly takes place.

They crystallized a polymerase that Braman of Stratagene had
isolated from a recently identified strain of a bacterium
called Bacillus stearothermophilus that is found in hot springs
in Idaho. The biochemists found this new polymerase seemed to
produce superior crystals with DNA incorporated. But they were
quite unprepared for the discovery that the enzyme would retain
its catalytic activity in crystal form.

"We were excited when we got our first crystals of enzyme
with DNA incorporated," said Beese. "But we were also puzzled,
because only part of the structural data agreed with our
pre-conceived ideas of what we expected to see." To further
explore the reaction, the biochemists next introduced into the
crystal a chemically active form of nucleotide -- called a
nucleoside triphosphate -- that the polymerase would normally
stitch into the DNA chain. Their aim was merely to capture an
X-ray snapshot of the "complex" of DNA and nucleotide held in
place by the enzyme.

"Initially we were very disappointed after we solved our
structure, because we didn't see triphosphate at the active
site," Beese said. "But we realized that the nucleotide had
actually incorporated and translocated in the crystal. It was
thrilling, because we knew then that the enzyme retained its
catalytic activity in the crystal.

"There is an element of good fortune in how the polymerase
molecules are arranged in the crystal, so as to have empty
space coincide with the space where the DNA wants to go," Beese
said. "This has enabled us to do the experiments that people
have wanted to do for years on this enzyme."

Beese said that other researchers have occasionally reported
that other, very different, enzymes retained chemical activity
in crystal form. "But those reactions didn't involve large
motions of substrates," she said. "In this enzyme, you don't
just have chemistry happening, you also have the DNA product
moving by quite a distance, and that was really quite
unprecedented."

The researchers' X-ray studies of the polymerase operating
in the crystal have revealed important details about its DNA
copying machinery. The polymerase enzyme -- shaped roughly like
a hand, with the DNA nestled in the palm -- is adaptable enough
to grab and hold any DNA molecule, the biochemists found.

However, in copying DNA, the enzyme is exquisitely
engineered to incorporate only the correct nucleotide pieces
into the DNA puzzle. The biochemists also are seeking to
understand how the polymerase recognizes mismatches between the
template and the new strand -- like misshapen pieces jammed
into a puzzle -- and contorts itself to halt the DNA copying
process to allow corrections.

"I think this is the most critical thing to understand --
how the enzyme can distinguish between a correct base pair and
an incorrect base pair," said Beese. "An incorrect base pair
leads to mutations, which can result in terrible genetic
diseases such as cancers in some cases."

The researchers have already done experiments in which they
introduced mismatches, finding their predictions confirmed how
the enzyme changes its shape to halt production. The Duke
biochemists also have launched experiments in which they
introduce cancer-causing chemicals into the polymerase crystal
and obtain snapshots revealing precisely how the carcinogenic
compounds bind themselves to the polymerase, perturbing its
relationship with the DNA and causing errors.

"Our first glimpses of the binding of carcinogens with
polymerase show very dramatic changes in the polymerase," Beese
said. "I think these experiments will give us profound insights
into the molecular basis of these compounds'
carcinogenicity."

While such insights won't lead directly to new cancer
treatments, Beese cautions, "a greater basic understanding of
the cancer process often leads to insights about how to affect
it."

The biochemists also will perform experiments to sort out
the chemical steps by which polymerase copies DNA, Beese said.
"The structural details of how the process proceeds are
unclear," she said. "We know translocation must occur, but
whether it happens before or after the nucleotide is bound,
nobody has ever tracked."

To reveal the detailed process, the scientists plan to
produce movies by taking series of high-speed X-ray snapshots
of the enzyme in action. To trigger all the polymerase
molecules to act at once, they plan to infuse into the crystals
light-activatable nucleoside triphosphates, and activate the
nucleotides with bursts of laser light. They may also conduct
their experiments at low temperatures, in hope that the DNA
copying process slows down enough to enable them to capture the
individual steps.

But if the reaction still proceeds too rapidly to be
visualized, the Duke biochemists may have to resort to the
X-ray equivalent of strobe flash photography -- using short
bursts of high-intensity

X-rays available at synchrotrons such as those at Brookhaven
National Laboratory and the Advanced Photon Source near
Chicago. Synchrotrons use high-energy particle beams to produce
the most intense X-rays available to scientists.