Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

How DNA Copying Enzyme "Stops the Presses" for Repair Synthesizing Enzyme

31.03.2004


Lorena S. Beese, Ph.D.
PHOTO CREDIT: Duke University Medical Center


Biochemists have performed detailed structural studies that reveal for the first time how an enzyme key to DNA replication stalls when an error occurs, to allow it to be corrected. Without such instantaneous braking, such mistakes in DNA replication would wreak havoc on DNA replication, killing the cell.

To their surprise, the scientists observed how the enzyme, DNA polymerase, retains a "short-term memory" of mismatches, in some cases halting itself past the point of the mismatch, so that the repair machinery can go to work. They also found that the mismatch structures differed dramatically from those deduced from previous indirect biochemical studies.

In an article in the March 19, 2004, issue of the journal Cell, Duke University Medical Center biochemists Sean Johnson and Lorena Beese, Ph.D., described how they had conducted detailed structural analyses of DNA polymerase as it encountered each of the 12 possible kinds of mismatches possible in DNA replication.



In such replication, the polymerase sequentially attaches DNA units called bases along a single-stranded template DNA. The result is like constructing one rail of a spiral staircase, using the other rail as a guide; and the polymerase "translocates" the template strand through its active site like a thread through the eye of a needle.

In this replication process, the polymerase normally guides the template strand and assembles the complementary, growing "primer" strand by pairing each base with the correct counterpart -- always pairing adenine with thymine and cytosine with guanine.

When mismatches occur, the polymerase must instantly halt itself, triggering the mismatch repair machinery to launch into action, before replication can continue. This stalling is thought to occur because the polymerase-DNA structure is distorted by the mismatched bases, causing it to shut down.

The problem, said Beese, who is an associate professor of biochemistry, is that the critical molecular details of how such distortion acted to brake the polymerase have remained unknown.

"For 40 years, there have been biochemical studies trying to understand how polymerase achieves such a high fidelity of replication," said Beese. "It was known that the polymerase stalled, but it wasn’t known why. However, these studies represent the first direct observation of the structural details of mismatches and how they interact with the polymerase. And they show why and how stalling occurs."

In their studies, Beese and graduate student Johnson used the analytical technique of X-ray crystallography. In this widely used technique, intense X-ray beams are directed through a crystal of a protein to be analyzed, and the pattern of diffractions analyzed to deduce the structure of the protein.

The first steps in their studies were to first crystallize the polymerase with a segment of DNA containing each type of mismatched pair of nucleotides. Importantly, said Beese, the loosely associated crystals were so constructed that the polymerase could actually carry out several replication steps within the crystal.

"We have been able to replicate and translocate up to six base pairs in the crystal -- to my knowledge the biggest such motion ever seen in a crystal," said Beese. Using this approach, the Duke biochemists engineered the polymerase to be error-prone, so that they could produce crystals with mispaired bases inserted in the active site.

What’s more, they were able to move the mismatch away from the active site and still detect the distortion of the polymerase structure that would indicate the polymerase was "sensing" a mismatch. Thus, the enzyme could "remember" a mismatch after it had occurred.

"What was surprising about this finding is that prior to the study a mismatch was thought to induce only very small local distortions right around the mismatch," said Beese. "But what we saw is that the polymerase amplifies this distortion back to the active site." However, cautioned, Beese, the full details of the stalling mechanism under all possible conditions remain to be worked out. So, there could be other details of the stalling mechanism that could affect understanding of this "memory," she said.

Significantly, said Beese, she and Johnson discovered that both the growing primer strand and the template are involved in the stalling process.

"Although each mismatch is different, we saw that it isn’t just on the primer side that the structure is disrupted by a mismatch, but also on the template side, and sometimes both. And we also saw a mechanism we hadn’t expected at all, which is that some mismatches just get stuck and don’t translocate."

Although Beese emphasized that their studies are quite basic, such findings could help explain how the polymerase-triggered repair system is affected by DNA damage from carcinogenic chemicals.

The next steps in their research, said Beese, will be to instantaneously capture the polymerase in the act of processing a mismatch. The researchers plan to use flashes of ultraviolet light to unleash "caged" chemicals that trigger replication -- and at the same time use flashes of X-rays to illuminate the crystal. This approach may allow the researchers to make a movie of the polymerase during the synthesis and mismatch detection process.

Dennis Meredith | dukemed news
Further information:
http://dukemednews.org/news/article.php?id=7499

More articles from Life Sciences:

nachricht Fine organic particles in the atmosphere are more often solid glass beads than liquid oil droplets
21.04.2017 | Max-Planck-Institut für Chemie

nachricht Study overturns seminal research about the developing nervous system
21.04.2017 | University of California - Los Angeles Health Sciences

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

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

Im Focus: Deep inside Galaxy M87

The nearby, giant radio galaxy M87 hosts a supermassive black hole (BH) and is well-known for its bright jet dominating the spectrum over ten orders of magnitude in frequency. Due to its proximity, jet prominence, and the large black hole mass, M87 is the best laboratory for investigating the formation, acceleration, and collimation of relativistic jets. A research team led by Silke Britzen from the Max Planck Institute for Radio Astronomy in Bonn, Germany, has found strong indication for turbulent processes connecting the accretion disk and the jet of that galaxy providing insights into the longstanding problem of the origin of astrophysical jets.

Supermassive black holes form some of the most enigmatic phenomena in astrophysics. Their enormous energy output is supposed to be generated by the...

Im Focus: A Quantum Low Pass for Photons

Physicists in Garching observe novel quantum effect that limits the number of emitted photons.

The probability to find a certain number of photons inside a laser pulse usually corresponds to a classical distribution of independent events, the so-called...

Im Focus: Microprocessors based on a layer of just three atoms

Microprocessors based on atomically thin materials hold the promise of the evolution of traditional processors as well as new applications in the field of flexible electronics. Now, a TU Wien research team led by Thomas Müller has made a breakthrough in this field as part of an ongoing research project.

Two-dimensional materials, or 2D materials for short, are extremely versatile, although – or often more precisely because – they are made up of just one or a...

Im Focus: Quantum-physical Model System

Computer-assisted methods aid Heidelberg physicists in reproducing experiment with ultracold atoms

Two researchers at Heidelberg University have developed a model system that enables a better understanding of the processes in a quantum-physical experiment...

Im Focus: Glacier bacteria’s contribution to carbon cycling

Glaciers might seem rather inhospitable environments. However, they are home to a diverse and vibrant microbial community. It’s becoming increasingly clear that they play a bigger role in the carbon cycle than previously thought.

A new study, now published in the journal Nature Geoscience, shows how microbial communities in melting glaciers contribute to the Earth’s carbon cycle, a...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Expert meeting “Health Business Connect” will connect international medical technology companies

20.04.2017 | Event News

Wenn der Computer das Gehirn austrickst

18.04.2017 | Event News

7th International Conference on Crystalline Silicon Photovoltaics in Freiburg on April 3-5, 2017

03.04.2017 | Event News

 
Latest News

New quantum liquid crystals may play role in future of computers

21.04.2017 | Physics and Astronomy

A promising target for kidney fibrosis

21.04.2017 | Health and Medicine

Light rays from a supernova bent by the curvature of space-time around a galaxy

21.04.2017 | Physics and Astronomy

VideoLinks
B2B-VideoLinks
More VideoLinks >>>