Building on earlier work exploring the complex choreography by which intricate cellular proteins interact with and copy DNA prior to cell division, scientists at the U.S. Department of Energy's Brookhaven National Laboratory and collaborators have captured a key step-molecular images showing how the enzyme that unwinds the DNA double helix gets drawn to and wrapped around its target. Details of the research, published in the journal Nature Structural & Molecular Biology, enhance understanding of an essential biological process and may suggest ways for stopping cell division when it goes awry.
Protein machinery involved in DNA replication caught in action: The "origin recognition complex" (yellow), already activated by an initiation factor (brown), grabs onto the helicase core (purple blue) to load the helicase ring onto the DNA double helix (red). The background is a cryo-electron micrograph of many of these complexes (dark) frozen in ice.
Credit: Courtesy Brookhaven National Laboratory
"This was truly collaborative work where molecular biology expertise from Christian Speck's lab at Imperial College, London, Bruce Stillman's group at Cold Spring Harbor Laboratory, and the cryo-electron microscopy expertise at Brookhaven were all essential," said Huilin Li, a biologist at Brookhaven Lab and Stony Brook University and a lead author on the paper.
"Our work is aimed at understanding the molecular details and mechanism of DNA replication at a fundamental level," said Li, "But our findings could have important implications, possibly pointing to new ways to fight cancer, because irregularities in DNA duplication and uncontrolled cell division are hallmarks of the disease."
The current research picks up where a study conducted last year left off [see: http://www.bnl.gov/newsroom/news.php?a=11391]. That research determined the structure of a piece of protein machinery called the "origin recognition complex" (ORC), which identifies and binds to DNA-replication "start" sites. When joined by a replication initiation factor, the ORC undergoes conformational changes that set in motion the whole replication process. The new study reveals how this previous structure recruits and interacts with the enzyme that eventually unwinds the DNA double helix into two separate strands.
"What we've uncovered in this study was a kind of missing link-what happens to this helicase enzyme before it encircles the DNA and starts unwinding the two strands," Li said.
Speck, Group Head at the MRC Research Institute in London, commented, "Our international collaboration has now revealed how the different protein components are assembled to generate a helicase loading complex. It is fascinating to see for the first time the architecture of this molecular machine."
Catching the molecular machinery in action is no simple task. Intermediate protein structures exist on fleeting timescales, and the interactions take place at the atomic level. Researchers working in Speck and Stillman's labs used tools of molecular biology and biochemistry to slow down the process. They purified and then remixed together pieces of the protein puzzle (including the origin recognition complex, the replication initiator, the core of the helicase, and other components) and a slow-acting energy agent so the energy-requiring reaction is unable to proceed to completion. Like dancers paused in place by a sudden stop of music, the molecular components "froze" partway through the helicase recruitment/assembly process.
Jingchuan Sun at Brookhaven then literally froze the samples, embedding them in ice, and took tens of thousands of pictures with a cryo-electron microscope. He then used computer software to reconstruct the 3-D structure from the 2-D electron microscope pictures.
"The 3-D reconstruction gave us a snapshot of the elusive intermediate structure," Sun said.
Comparing the new structure (components of the helicase bound to the origin recognition complex) with the structures of the ORC produced last year revealed conformational changes. Binding of the helicase core components appears to shift the ORC into a spiral conformation that closely matches the spiral shape of double-stranded DNA.
"This shape-shifting of the ORC appears to be an important step in facilitating binding of the ring-shaped helicase to the DNA," Sun said.
The scientists also note that the spiral-shaped ORC is similar to another spiral protein complex that loads a different ring structure to keep DNA polymerase enzymes from falling off the DNA while synthesizing new strands to complete the replication process.
"Both of these complexes were discovered in the Stillman lab nearly two decades ago. It's rewarding to see now that these two energy-requiring protein machines form similar spiral structures to recruit and load their 'cargo' onto DNA for these crucial steps in the replication process," said Li.
Said co-author Stillman, president of Cold Spring Harbor Laboratory, "It is amazing how two seemingly separate steps in the process of duplicating our genome are so similar in their biochemical mechanism. Using the advanced microscope facilities at Brookhaven Lab has once again generated a surprising result."
This research was funded by the National Institutes of Health (GM45436, GM74985), the U.K. Medical Research Council, the Japan Society for the Promotion of Science, and the Uehara Memorial Foundation. Huilin Li and the EM facility at Brookhaven Lab are partially supported by Brookhaven National Laboratory institutional funding via his joint appointment with Stony Brook University.
Scientific paper: "Architecture of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 on DNA reveals similarity to DNA polymerase clamp loading complexes"
Study Reveals How Protein Machinery Binds and Wraps DNA to Start Replication
One of ten national laboratories overseen and primarily funded by the Office of Science of the U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies and national security. Brookhaven Lab also builds and operates major scientific facilities available to university, industry and government researchers. Brookhaven is operated and managed for DOE's Office of Science by Brookhaven Science Associates, a limited-liability company founded by the Research Foundation for the State University of New York on behalf of Stony Brook University, the largest academic user of Laboratory facilities, and Battelle, a nonprofit applied science and technology organization. Visit Brookhaven Lab's electronic newsroom for links, news archives, graphics, and more, or follow Brookhaven Lab on Twitter.
Karen McNulty Walsh | EurekAlert!
Cryo-electron microscopy achieves unprecedented resolution using new computational methods
24.03.2017 | DOE/Lawrence Berkeley National Laboratory
How cheetahs stay fit and healthy
24.03.2017 | Forschungsverbund Berlin e.V.
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...
Enzymes behave differently in a test tube compared with the molecular scrum of a living cell. Chemists from the University of Basel have now been able to simulate these confined natural conditions in artificial vesicles for the first time. As reported in the academic journal Small, the results are offering better insight into the development of nanoreactors and artificial organelles.
Enzymes behave differently in a test tube compared with the molecular scrum of a living cell. Chemists from the University of Basel have now been able to...
20.03.2017 | Event News
14.03.2017 | Event News
07.03.2017 | Event News
24.03.2017 | Materials Sciences
24.03.2017 | Physics and Astronomy
24.03.2017 | Physics and Astronomy