Helicase enzymes are best known for “unzipping” DNA for replication, but have many other functions for DNA repair and maintenance. The Illinois team focused on a particular bacterial helicase called PcrA involved in preventing unwanted recombination.
A DNA double helix consists of two strands twisted around each other. When one strand is damaged or breaks, the surrounding area is degraded, leaving a single-stranded region. Specialized proteins then start the process of recombination – rebuilding the second strand using the intact DNA as a template.
“Recombination is essential for DNA repair, but if it runs amok, it causes problems,” said U. of I. physics professor Taekjip Ha. “This helicase controls recombination by removing recombination proteins from the DNA.”
Using a technique called single molecule fluorescence resonance energy transfer (FRET), Ha and his team were able to identify one of the mechanisms that PcrA uses to regulate recombination. The system uses two dyes that change in relative intensity depending on their proximities to one another. The researchers attached the two dyes to the opposite ends of the single-stranded DNA tail.
Helicases are motor proteins, a class of enzymes that use chemical energy to move along a DNA molecule like a train on a track. But using FRET, the researchers observed the two dyes gradually moving closer to each other, then flying apart, repeatedly. Instead of moving along the single-stranded tail, PcrA binds at the point of the break, where the double- and single-stranded regions meet. Then, it uses its motor function to “reel in” the tail, like a fisherman pulling in a rope.
“By combining the structure-specific binding of the enzyme to the DNA and the motor function, the enzyme can reel in the DNA and in the process kick off recombination proteins,” said Ha, who also is a Howard Hughes Medical Institute investigator.
When PcrA reaches the end of its DNA rope, it releases it and starts the reeling in process over again, removing any additional problematic proteins that have bound to the damaged DNA as it reels.
By using FRET, a technique Ha developed, the team also was able to answer another question about PcrA: How consistent is its motor function? Researchers agree that on average, PcrA moves one DNA unit, called a base pair, for each unit of cellular energy it uses, called ATP. But because researchers traditionally study the enzyme in relatively large samples, broad distributions of data have led to conflicting views on whether the helicase moves in uniform steps or those of varying lengths – even up to six base pairs per ATP.
Since FRET is a single-molecule technique, the researchers were able to document a single enzyme’s function, step by step, and found that PcrA does, in fact, move in uniform steps of one base pair per ATP.
Next, the team plans to create a reaction environment more similar to that in vivo, using three and four colors of FRET dyes to measure activities of multiple proteins simultaneously. They are also working toward understanding why helicase moves only in one direction.
“This is an ideal marriage of a new technology and an interesting biological problem,” Ha said.
The team published its findings in the Aug. 20 edition of the journal Cell. Team members included U. of I. graduate students Jeehae Park and Kyung Suk Lee; bioengineering professor Sua Myong; Anita Niedziela-Majka and Timothy Lohman, of the Washington University School of Medicine in St. Louis; and Jin Yu, of the University of California at Berkeley. The National Institutes of Health and the National Science Foundation supported this work.
Liz Ahlberg | University of Illinois
Unique genome architectures after fertilisation in single-cell embryos
30.03.2017 | IMBA - Institut für Molekulare Biotechnologie der Österreichischen Akademie der Wissenschaften GmbH
Transport of molecular motors into cilia
28.03.2017 | Aarhus University
The Institute of Semiconductor Technology and the Institute of Physical and Theoretical Chemistry, both members of the Laboratory for Emerging Nanometrology (LENA), at Technische Universität Braunschweig are partners in a new European research project entitled ChipScope, which aims to develop a completely new and extremely small optical microscope capable of observing the interior of living cells in real time. A consortium of 7 partners from 5 countries will tackle this issue with very ambitious objectives during a four-year research program.
To demonstrate the usefulness of this new scientific tool, at the end of the project the developed chip-sized microscope will be used to observe in real-time...
Astronomers from Bonn and Tautenburg in Thuringia (Germany) used the 100-m radio telescope at Effelsberg to observe several galaxy clusters. At the edges of these large accumulations of dark matter, stellar systems (galaxies), hot gas, and charged particles, they found magnetic fields that are exceptionally ordered over distances of many million light years. This makes them the most extended magnetic fields in the universe known so far.
The results will be published on March 22 in the journal „Astronomy & Astrophysics“.
Galaxy clusters are the largest gravitationally bound structures in the universe. With a typical extent of about 10 million light years, i.e. 100 times the...
Researchers at the Goethe University Frankfurt, together with partners from the University of Tübingen in Germany and Queen Mary University as well as Francis Crick Institute from London (UK) have developed a novel technology to decipher the secret ubiquitin code.
Ubiquitin is a small protein that can be linked to other cellular proteins, thereby controlling and modulating their functions. The attachment occurs in many...
In the eternal search for next generation high-efficiency solar cells and LEDs, scientists at Los Alamos National Laboratory and their partners are creating...
Silicon nanosheets are thin, two-dimensional layers with exceptional optoelectronic properties very similar to those of graphene. Albeit, the nanosheets are less stable. Now researchers at the Technical University of Munich (TUM) have, for the first time ever, produced a composite material combining silicon nanosheets and a polymer that is both UV-resistant and easy to process. This brings the scientists a significant step closer to industrial applications like flexible displays and photosensors.
Silicon nanosheets are thin, two-dimensional layers with exceptional optoelectronic properties very similar to those of graphene. Albeit, the nanosheets are...
20.03.2017 | Event News
14.03.2017 | Event News
07.03.2017 | Event News
30.03.2017 | Physics and Astronomy
30.03.2017 | Studies and Analyses
30.03.2017 | Life Sciences