image: This is a side view of double hexameric ring complex stacked at the N-terminal end in a twisted and tilted manner. view more
Credit: Division of Life Science, HKUST
HKUST-Tsinghua University Scientists Solve the Structure of the eukaryotic MCM2-7 Complex
Scientists from the Hong Kong University of Science & Technology (HKUST) and Tsinghua University have solved the structure of the MCM2-7 Complex, which plays a key role in destabilizing and unwinding duplex DNA during DNA replication.
More than 60 years ago, when Francis Crick and James D. Watson solved the structure of duplex helical DNA, the genetic material found in all living organisms, they predicted that copying DNA requires the separation of the two complementary strands so each can serve as template for replication of the other. Ever since, the mechanism and enzymes involved in the initial melting (destabilization) of DNA have been an outstanding problem for biologists.
For more than three decades since the role of the MCM2-7 complex was linked to DNA replication by Professor Bik Tye, researchers have tried elucidating the structure of the MCM2-7 Complex without much success. Attempts to crystallize the Complex were futile, and image quality of the Complex has been primitive at best.
The solution to the problem came at the heel of recent advances in Cryo Electron Microscopy (CEM). Using state-of-the art CEM Technology, the team of scientists led by Professor Bik Tye at HKUST, and Professor Ning Gao at Tsinghua University, solved the structure of the MCM2-7 complex at 3.8Å, an astounding resolution that has not been achieved before. New advances in cryo-EM that include more powerful electron microscopes, better detectors, faster cameras and more sophisticated algorithms for image reconstruction allow the resolution of large complex structures to near atomic range.
Their findings were published on the the website of the journal Nature on July 29, 2015. (http://www.nature.com/nature/journal/vaop/ncurrent/full/nature14685.html, news and views: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature14643.html)
The MCM2-7 complex consists of a family of six highly conserved but non-identical protein subunits that form a ring structure that encircles duplex DNA. Each of these protein subunits is highly conserved from yeast to human. Although the study was carried out in yeast, information derived from the yeast complex also applies to human.
"The most striking feature of the MCM2-7 complex structure is that the two rings form a tilted and twisted dimer through their N-terminal domain." Professor Tye says. "The central channel, formed by these two staggered rings, has four constriction points that would restrict the movement of duplex DNA with tight grips and a kink at the interface of the two rings that would deform the bound DNA."
These and other details of the fine structure of the MCM2-7 complex instruct the function of the MCM2-7 complex in DNA melting. First, the deformed DNA at the kinked interface of the two rings would serve as a nucleation center for DNA destabilization. Second the tight grip of duplex DNA at either end by each hexamer would further deform DNA at the nucleation point if rotated against each other. Third, possible rotations between ring structures formed by subdomains of each hexamer would lower the activation energy for DNA destabilization even further. This model proposed that allosteric conformational changes following the activation of the MCM2-7 complex by cell cycle regulated kinases bring about DNA destabilization.
"We have always had the idea and the problem, but adequate expertise to make high resolution images of the Complex has been a hurdle," says Dr. Yuanliang Zhai of HKUST, a co-author of the paper. "Collaboration with Professor Ning Gao from Tsinghua University, whose team provided the CEM technology, was instrumental. The 3-D high resolution structure was reconstructed from more than 80K 2-D images that were selected from over 350K images collected". Dr. Zhai took over a year to perfect purification of large quantities of the Complex to homogeneity.
After the breakthrough, the next step for the researchers is to look into the mechanistic functions of the MCM2-7 complex, which so far have been elusive to many scientists.
"Now that we have a clear picture of the structure of the Complex, we can predict more precisely the function of this universal machine that unwinds duplex DNA for the copying of genetic information," beams Professor Tye.
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This work was supported by the Ministry of Science and Technology of China (2013CB910404 to Ning Gao), the National Natural Science Foundation of China (31422016 to Ning Gao), the Research Grants Council of Hong Kong (GRF664013 and HKUST12/CRF/13G to Yuanliang Zhai) and the Hong Kong University of Science & Technology (Bik K Tye).
Journal
Nature