Knowing the structure of a complex biological system isn’t nearly enough to understand how it works. It helps to know how the system runs.
In that light, researchers at Rice University have devised a key mechanism by which DNA repeats.
Combining structural experiments and computer simulations, bioscientist Yang Gao, theoretical physicist Peter Volins, graduate student Shikai Jin and their colleagues have uncovered details about how Helicases, a family of ringlike motor proteins, denature DNA during replication. Their work could lead to new targets for disease-fighting drugs.
The synergy between the experiments and the large-scale simulations they describe Proceedings of the National Academy of Science Can become a paradigm for modeling the mechanisms of many complex biological systems.
“These are dynamic processes that cannot be well captured by experimental methods alone. But it is important to show the mechanisms of these helicases, as they are essential for DNA replication, and are also potential drug targets.”
Yang Gao, Assistant Professor, Biosciences, Rice University
Yang Gao is also a CPRIT Scholar in Cancer Research.
The hexameric helicase consists of six sides that self-assemble from peptides into a washer-like ring that separates the parent double strand of DNA into the daughter single strand. So far, researchers have been unable to figure out how the helicase moves as it unwinds the double strand.
Rice simulations reinforce the idea that DNA-binding loops within the six subunits of the helicase form a kind of ladder that moves down the DNA backbone driven by ATP hydrolysis, a process that releases chemical energy stored in ATP molecules.
It was known that ATP in each subunit is pulled towards the NTPase protein to drive the stepping movement. But the researchers did not understand exactly that ATP hydrolysis was the key. The Rice team found that ATP binds tightly to the helicase subunits, but hydrolysis significantly lowers the energy barrier for subunit disassociation, allowing the protein to move.
The researchers noted that since the helicase-DNA complex is so large, there have been only a few attempts to simulate the transfer of helicase from one end of the strand to the other. The rice hybrid of two coarse-grained simulation techniques provided an opportunity to study the process from start to finish.
The simulations revealed several previously unknown intermediate states and indicated interactions involved in the long-distance movement of the helicase. They showed that no more than 12 nucleotides can travel along the backbone at each step of translation.
To look for the mechanism, the team focused on the T7 bacteriophage, a virus that infects bacteria that is often used as a model system. To simulate their helices, known as gp4, the researchers combined two force fields: AWSEM, initially developed by Volins and colleagues to predict how proteins fold, and Open3SPN2, a DNA simulator developed by molecular engineer Juan de Pablo at the University of Chicago. Force fields describe the forces that determine how atoms and molecules move when in contact. (The novel combination of force fields was the subject of a Rice-led 2021 paper in PLOS Computational Biology.)
Both force fields are coarse-grained molecular models based on machine learning that use only a subset of atoms in a system but still provide accurate results while greatly reducing computation time.
“The combination of models enabled GPU acceleration so that we could make our molecular dynamics simulations very fast,” Jin said. “The combined software is now 30 times faster than the versions we used for other studies.”
It helps that T7 is half the size of the helicase in human cells. “In human systems, the helicase consists of six different polypeptide chains, but in T7, it is the one that makes six copies,” he said.
Volins, co-director of the Rice Center for Open, said, “Since our new form of open3spn2 is related to a single strand of DNA, it allows us to analyze the processes where double-stranded DNA normally opens as it is. Happens in the presence of Helicases.” Theoretical biological physics. “The single-stranded DNA force field is novel in itself but it was just a background in this project, where it enables us to look at this process in great detail.”
“The cryo-EM structures we have for these essential complexes are physically accurate, but these systems are dynamic,” Gao said. “They have to move to do their job, and there’s still a lot we want to know about how they do it.
“That is where these computational models can make a big contribution and will, of course, be adapted to other large systems to investigate important questions,” he said.
The paper’s co-authors are Rice graduate students Carlos Bueno and Wei Lu; Postdoctoral associate Xun Chen; and Qian Wang, Hefei, alumnus of the University of Science and Technology of China, and Mingchen Chen of NeoX Biotech, Beijing. Wolynes is the DR Bullard-Welch Foundation Professor of Science and Professor of Chemistry, Biological Sciences and Physics and Astronomy at Rice.
The National Science Foundation (2019745), the National Institute of General Medical Sciences (GM44557), the Cancer Prevention and Research Institute of Texas (RR190446) and the National Institutes of Health (1R35GM142722) supported the research.
Jin, S., and others, (2022) Computationally exploring the mechanism of translation of the bacteriophage T7 gp4 helicase with ssDNA. Proceedings of the National Academy of Science, doi.org/10.1073/pnas.2202239119.