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 Building a brain, cell by cell: Researchers make a mini neuron network (of two)
23.05.2018 | Institute of Industrial Science, The University of Tokyo

nachricht Research reveals how order first appears in liquid crystals
23.05.2018 | Brown University

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

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

Im Focus: LZH showcases laser material processing of tomorrow at the LASYS 2018

At the LASYS 2018, from June 5th to 7th, the Laser Zentrum Hannover e.V. (LZH) will be showcasing processes for the laser material processing of tomorrow in hall 4 at stand 4E75. With blown bomb shells the LZH will present first results of a research project on civil security.

At this year's LASYS, the LZH will exhibit light-based processes such as cutting, welding, ablation and structuring as well as additive manufacturing for...

Im Focus: Self-illuminating pixels for a new display generation

There are videos on the internet that can make one marvel at technology. For example, a smartphone is casually bent around the arm or a thin-film display is rolled in all directions and with almost every diameter. From the user's point of view, this looks fantastic. From a professional point of view, however, the question arises: Is that already possible?

At Display Week 2018, scientists from the Fraunhofer Institute for Applied Polymer Research IAP will be demonstrating today’s technological possibilities and...

Im Focus: Explanation for puzzling quantum oscillations has been found

So-called quantum many-body scars allow quantum systems to stay out of equilibrium much longer, explaining experiment | Study published in Nature Physics

Recently, researchers from Harvard and MIT succeeded in trapping a record 53 atoms and individually controlling their quantum state, realizing what is called a...

Im Focus: Dozens of binaries from Milky Way's globular clusters could be detectable by LISA

Next-generation gravitational wave detector in space will complement LIGO on Earth

The historic first detection of gravitational waves from colliding black holes far outside our galaxy opened a new window to understanding the universe. A...

Im Focus: Entangled atoms shine in unison

A team led by Austrian experimental physicist Rainer Blatt has succeeded in characterizing the quantum entanglement of two spatially separated atoms by observing their light emission. This fundamental demonstration could lead to the development of highly sensitive optical gradiometers for the precise measurement of the gravitational field or the earth's magnetic field.

The age of quantum technology has long been heralded. Decades of research into the quantum world have led to the development of methods that make it possible...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

VideoLinks
Industry & Economy
Event News

Save the date: Forum European Neuroscience – 07-11 July 2018 in Berlin, Germany

02.05.2018 | Event News

Invitation to the upcoming "Current Topics in Bioinformatics: Big Data in Genomics and Medicine"

13.04.2018 | Event News

Unique scope of UV LED technologies and applications presented in Berlin: ICULTA-2018

12.04.2018 | Event News

 
Latest News

Research reveals how order first appears in liquid crystals

23.05.2018 | Life Sciences

Space-like gravity weakens biochemical signals in muscle formation

23.05.2018 | Life Sciences

NIST puts the optical microscope under the microscope to achieve atomic accuracy

23.05.2018 | Physics and Astronomy

VideoLinks
Science & Research
Overview of more VideoLinks >>>