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.
Beese and her colleagues are working with a consortium of Southern states to obtain state funding that would allow researchers in the region access to the synchrotrons for basic biomedical, engineering and physics studies.
Journal
Nature