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 Rainbow colors reveal cell history: Uncovering β-cell heterogeneity
22.09.2017 | DFG-Forschungszentrum für Regenerative Therapien TU Dresden

nachricht The pyrenoid is a carbon-fixing liquid droplet
22.09.2017 | Max-Planck-Institut für Biochemie

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

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

Im Focus: The pyrenoid is a carbon-fixing liquid droplet

Plants and algae use the enzyme Rubisco to fix carbon dioxide, removing it from the atmosphere and converting it into biomass. Algae have figured out a way to increase the efficiency of carbon fixation. They gather most of their Rubisco into a ball-shaped microcompartment called the pyrenoid, which they flood with a high local concentration of carbon dioxide. A team of scientists at Princeton University, the Carnegie Institution for Science, Stanford University and the Max Plank Institute of Biochemistry have unravelled the mysteries of how the pyrenoid is assembled. These insights can help to engineer crops that remove more carbon dioxide from the atmosphere while producing more food.

A warming planet

Im Focus: Highly precise wiring in the Cerebral Cortex

Our brains house extremely complex neuronal circuits, whose detailed structures are still largely unknown. This is especially true for the so-called cerebral cortex of mammals, where among other things vision, thoughts or spatial orientation are being computed. Here the rules by which nerve cells are connected to each other are only partly understood. A team of scientists around Moritz Helmstaedter at the Frankfiurt Max Planck Institute for Brain Research and Helene Schmidt (Humboldt University in Berlin) have now discovered a surprisingly precise nerve cell connectivity pattern in the part of the cerebral cortex that is responsible for orienting the individual animal or human in space.

The researchers report online in Nature (Schmidt et al., 2017. Axonal synapse sorting in medial entorhinal cortex, DOI: 10.1038/nature24005) that synapses in...

Im Focus: Tiny lasers from a gallery of whispers

New technique promises tunable laser devices

Whispering gallery mode (WGM) resonators are used to make tiny micro-lasers, sensors, switches, routers and other devices. These tiny structures rely on a...

Im Focus: Ultrafast snapshots of relaxing electrons in solids

Using ultrafast flashes of laser and x-ray radiation, scientists at the Max Planck Institute of Quantum Optics (Garching, Germany) took snapshots of the briefest electron motion inside a solid material to date. The electron motion lasted only 750 billionths of the billionth of a second before it fainted, setting a new record of human capability to capture ultrafast processes inside solids!

When x-rays shine onto solid materials or large molecules, an electron is pushed away from its original place near the nucleus of the atom, leaving a hole...

Im Focus: Quantum Sensors Decipher Magnetic Ordering in a New Semiconducting Material

For the first time, physicists have successfully imaged spiral magnetic ordering in a multiferroic material. These materials are considered highly promising candidates for future data storage media. The researchers were able to prove their findings using unique quantum sensors that were developed at Basel University and that can analyze electromagnetic fields on the nanometer scale. The results – obtained by scientists from the University of Basel’s Department of Physics, the Swiss Nanoscience Institute, the University of Montpellier and several laboratories from University Paris-Saclay – were recently published in the journal Nature.

Multiferroics are materials that simultaneously react to electric and magnetic fields. These two properties are rarely found together, and their combined...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

“Lasers in Composites Symposium” in Aachen – from Science to Application

19.09.2017 | Event News

I-ESA 2018 – Call for Papers

12.09.2017 | Event News

EMBO at Basel Life, a new conference on current and emerging life science research

06.09.2017 | Event News

 
Latest News

Rainbow colors reveal cell history: Uncovering β-cell heterogeneity

22.09.2017 | Life Sciences

Penn first in world to treat patient with new radiation technology

22.09.2017 | Medical Engineering

Calculating quietness

22.09.2017 | Physics and Astronomy

VideoLinks
B2B-VideoLinks
More VideoLinks >>>