DURHAM, N.C. -- Biochemists have pinpointed how a flaw in DNA that is central to mutations in cancer and aging fools the cellular enzyme that copies DNA. Their finding explains how oxidative DNA damage -- a process long believed to underlie cancers and aging -- can create permanent genetic damage.
The Duke University Medical Center researchers' findings were published online Aug. 22, 2004, by the journal Nature. The scientists were led by Associate Professor of Biochemistry Lorena Beese, Ph.D., and the paper's lead author was Gerald Hsu, a Duke M.D./Ph.D. student. The other co-authors are Thomas Carell and Matthias Ober of Ludwig Maximillians University in Germany. Their research was supported mainly by the National Cancer Institute.
DNA is a double stranded molecule shaped like a spiral staircase. The two strands of the spiral are linked by sequences of molecular subunits, or bases, called nucleotides. The four nucleotides -- guanine, cytosine, adenine and thymine -- naturally complement one another like puzzle pieces. In normal DNA, a guanine matches with a cytosine, and an adenine with a thymine. However, stray reactive oxidizing molecules in the cell can alter guanine to become an "8-oxoguanine" that can lead to a mismatch.
This mismatch occurs in the process of replicating DNA, which begins when the two strands unzip. A protein enzyme called DNA polymerase then works its way along one "template" strand adding nucleotides to create a new double-stranded DNA. In the replication process, the polymerase draws the DNA strand through a small "active site" -- somewhat like a spaghetti strand being drawn through a Cheerio.
Normally, this "high-fidelity" polymerase accurately adds complementary nucleotides and detects any mistakes that have been made. These mistakes or mismatches reveal themselves as malformations that distort the active site -- like kinks in the spaghetti strand that would clog the Cheerio. Such malformations trigger a repair mechanism to correct the mismatch.
The researchers' initial studies revealed that the polymerase biochemically "prefers" to mismatch an 8-oxoguanine with adenine rather than the correct cytosine. If not detected and corrected, such a mismatch leads to errors in the cell's machinery that can trigger the uncontrolled growth of cancer or the death of cells in aging. However, researchers have long known that the 8-oxoguanine-adenine mismatch seems to readily avoid detection by the polymerase.
"There have been a number of studies of the kinetics and the biochemistry of this mismatch reaction, but it was not understood why this particular lesion evaded detection as well as it does," said Beese. "It is one of a series of such oxidative lesions, but it is considered the most mutagenic, which is why we concentrated on understanding it."
In the experiments, Hsu worked with the particularly sturdy polymerase enzyme from a thermostable strain of the bacterium, Bacillus stearothermophilus, which thrives in geothermal hot springs. He crystallized this enzyme along with a DNA strand that contained an 8-oxoguanine. Because the polymerase retains the ability to synthesize DNA in the crystal, Hsu then added either the correct (cytosine) or incorrect (adenine) nucleotides and observed the results.
Using X-ray crystallography, the researchers were able to deduce with great precision the structure of the protein and the DNA in the crystal. The series of crystals they analyzed constituted snapshots of the polymerase's function as it created both accurate and mutated strands from the template.
The biochemists encountered a surprise when they analyzed the polymerase crystals with either the correct or mismatched nucleotides. "We saw that, ironically, when the polymerase binds the correct cytosine opposite 8-oxoguanine, the structure looked like DNA mispairs," said Beese. "This suggested that the enzyme would stall and not readily proceed with replication.
"But when we put in an incorrect adenine nucleotide, it looked like a normal base pair in how it interacted with the polymerase." The researchers' analyses revealed that the mismatched combination of 8-oxoguanine and cytosine was distorted, like a kink in a spaghetti strand that would jam the active site. However, the mismatched 8-oxoguanine and adenine showed no distortion so would proceed smoothly through the polymerase to be incorporated into the new DNA.
"We were able to extend the replication process to show that there were no distortions that would be detected by the polymerase. This means that the DNA would continue to replicate with this mispair, and that could potentially lead to stable incorporation of a lethal mutation," said Beese.
In further analyses, Hsu confirmed that bacterial polymerase would behave just as did the human polymerase in preferring to incorporate the mismatch and failing to recognize it. Also, they found, if the 8-oxoguanine-cytosine pair manages to pass through the polymerase, the distortion disappears, meaning that the chemically flawed guanine will persist in the DNA strand.
In further studies, Beese and her colleagues are exploring other types of DNA lesions and how they affect replication. These lesions include those caused by major carcinogens. The researchers also have developed a method to synchronize the DNA replication process, so that they can make the equivalent of X-ray crystallographic "movies" of the entire process, to better understand it.
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