DNA replication is the process where cells make a precise copy of their DNA before cell department. An essential part of the complex DNA duplication equipment is a molecular motor called CMG, which has the essential task of separating the 2 strands of the DNA double helix so that they can be copied. “Understanding how CMG moves along DNA is crucial for our understanding of DNA duplication,” says Daniel Ramírez Montero, researcher and first author of the publication. We used optical tweezers to hold the CMG-containing DNA still for easier visualization, and then took motion pictures of CMG as it moved along the DNA. In addition, they made the unanticipated discovery that CMG can move randomly along the DNA when a crucial molecule called ATP is missing; furthermore, they reveal that subsequent re-binding of ATP allows CMG to tightly hold onto the DNA, thereby halting its random movement.
Diagram of the experimental setup: (Top) Schematic of a CMG-containing DNA molecule kept in location with optical tweezers (red beams) while a scanning laser (green beam) is taking a photo of it; CMG motors are depicted in blue. (Bottom) Actual picture of a fluorescently identified CMG molecular motor (green area) on an unlabelled DNA particle held in location with optical tweezers. Credit: Adapted from Ramirez Montero, et al, Nature Communications, 2023.
TU Delft scientists have developed a brand-new technique to put together and image the molecular motor CMG in DNA replication, using unmatched resolution and insights into the duplication process. This development could lead to a much better understanding of congenital diseases and cancer development.
DNA duplication is the process where cells make a specific copy of their DNA prior to cell division. A key part of the elaborate DNA duplication machinery is a molecular motor called CMG, which has the essential job of separating the 2 strands of the DNA double helix so that they can be copied.
DNA duplication is fundamental to life, as the faithful transmission of genetic information in between generations of cells is important for the survival and health of all living organisms. It is brought out by an elaborate cellular equipment, called the replisome, that is made up of protein foundation. A key part of this equipment is CMG, the molecular motor that powers the replisome during DNA duplication..
CMG has the essential job of separating the two strands of the DNA double helix, so that the message that they encode can be checked out and copied. “Understanding how CMG moves along DNA is vital for our understanding of DNA replication,” states Daniel Ramírez Montero, researcher and very first author of the publication. Since errors in this procedure can lead to hereditary disorders or cancer, studying DNA duplication is very essential.
Motion on movie.
A group of TU Delft researchers led by Spinoza laureate Professor Nynke Dekker, in collaboration with Francis Crick Institute group leader Dr. John Diffley, has established a method to bring out this securely controlled process outside of the cell and measure the motion of specific CMG molecular motors. The researchers extracted all 36 proteins from cells to build up CMG on DNA.
” With this brand-new technique, we had the ability to envision the movement of individual CMGs made from scratch. We used optical tweezers to hold the CMG-containing DNA still for much easier visualization, and then took movies of CMG as it moved along the DNA. By doing this, we could measure its movement at the single-molecule level for the very first time,” explains Ramírez Montero..
Example of CMG motors moving along a DNA molecule kept in location by an optical trap. Credit: Taken from Ramírez Montero, et al., Nature Communications, 2023.
New insights.
Using their bottom-up technique, which integrates cutting-edge biochemistry and biophysics, the group of researchers was able to directly visualize the movement of specific CMG motors assembled from scratch for the very first time, and determine this movement with unmatched resolution. In addition, they made the unforeseen discovery that CMG can move arbitrarily along the DNA when an essential particle called ATP is missing; furthermore, they show that subsequent re-binding of ATP allows CMG to securely hold onto the DNA, thereby stopping its random movement. This halting is necessary, as it most likely assists in the activation of CMG, a critical process in the initiation of DNA duplication..
This work will lead the way for additional research studies that may discover unidentified information of key procedures in DNA duplication. These discoveries can in turn get us closer to understanding how cells manage to faithfully transfer their hereditary info with every cellular division, along with to better understand the mistakes in the process that can contribute to the development of congenital diseases or cancer. Ramírez Montero: “Biological systems can look disorderly and really complex at first sight, but by observing them at this resolution, we can understand the simple and elegant physics behind them.”.
Recommendation: “Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation” by Daniel Ramírez Montero, Humberto Sánchez, Edo van Veen, Theo van Laar, Belén Solano, John F. X. Diffley and Nynke H. Dekker, 14 April 2023, Nature Communications.DOI: 10.1038/ s41467-023-37093-9.
