Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

How an insidious mutation fools DNA replication

31.08.2004


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.

Dennis Meredith | EurekAlert!
Further information:
http://www.duke.edu

More articles from Life Sciences:

nachricht Microscope measures muscle weakness
16.11.2018 | Friedrich-Alexander-Universität Erlangen-Nürnberg

nachricht Good preparation is half the digestion
16.11.2018 | Max-Planck-Institut für Stoffwechselforschung

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: UNH scientists help provide first-ever views of elusive energy explosion

Researchers at the University of New Hampshire have captured a difficult-to-view singular event involving "magnetic reconnection"--the process by which sparse particles and energy around Earth collide producing a quick but mighty explosion--in the Earth's magnetotail, the magnetic environment that trails behind the planet.

Magnetic reconnection has remained a bit of a mystery to scientists. They know it exists and have documented the effects that the energy explosions can...

Im Focus: A Chip with Blood Vessels

Biochips have been developed at TU Wien (Vienna), on which tissue can be produced and examined. This allows supplying the tissue with different substances in a very controlled way.

Cultivating human cells in the Petri dish is not a big challenge today. Producing artificial tissue, however, permeated by fine blood vessels, is a much more...

Im Focus: A Leap Into Quantum Technology

Faster and secure data communication: This is the goal of a new joint project involving physicists from the University of Würzburg. The German Federal Ministry of Education and Research funds the project with 14.8 million euro.

In our digital world data security and secure communication are becoming more and more important. Quantum communication is a promising approach to achieve...

Im Focus: Research icebreaker Polarstern begins the Antarctic season

What does it look like below the ice shelf of the calved massive iceberg A68?

On Saturday, 10 November 2018, the research icebreaker Polarstern will leave its homeport of Bremerhaven, bound for Cape Town, South Africa.

Im Focus: Penn engineers develop ultrathin, ultralight 'nanocardboard'

When choosing materials to make something, trade-offs need to be made between a host of properties, such as thickness, stiffness and weight. Depending on the application in question, finding just the right balance is the difference between success and failure

Now, a team of Penn Engineers has demonstrated a new material they call "nanocardboard," an ultrathin equivalent of corrugated paper cardboard. A square...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

VideoLinks
Industry & Economy
Event News

“3rd Conference on Laser Polishing – LaP 2018” Attracts International Experts and Users

09.11.2018 | Event News

On the brain’s ability to find the right direction

06.11.2018 | Event News

European Space Talks: Weltraumschrott – eine Gefahr für die Gesellschaft?

23.10.2018 | Event News

 
Latest News

Purdue cancer identity technology makes it easier to find a tumor's 'address'

16.11.2018 | Health and Medicine

Good preparation is half the digestion

16.11.2018 | Life Sciences

Microscope measures muscle weakness

16.11.2018 | Life Sciences

VideoLinks
Science & Research
Overview of more VideoLinks >>>