A protein that prepares DNA for replication also prevents the replication process from getting out of hand, according to a new study by researchers at Weill Cornell Medicine. The work, published Jan. 5 in molecular cellsolves a mystery that has long puzzled biologists.
The cells of humans and all other higher organisms use a complex system of checkpoints and “licensing” proteins to ensure that they replicate their genomes exactly once before dividing. In preparation for cell division, the licensing proteins attach to specific regions in the DNA, thus referred to as origins of replication. When the DNA synthesis phase of the cell cycle begins, replication begins only at those licensed sites and, according to the current model, is started or ‘fired’ only once.
However, that model missed a crucial point. “The same factor that causes these licensing to occur only breaks down after these origins of replication are activated,” said senior author Dr. Tobias Meyer, the Joseph Hinsey Professor of cell and developmental biology at Weill Cornell Medicine. “Basically, the cell could load these licensing machines onto DNA that has already replicated, so instead of two copies you get three or four copies of that segment of the DNA, and these cells would be expected to lose genome integrity and die or develop cancer .”
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Figuring out how cells avoid that fate has been tricky. “We had to study events in the first minutes of the DNA synthesis phase of the cell cycle, so it’s a very transient period,” said first author Nalin Ratnayeke, a graduate student who worked on this project at both Stanford University and Weill. Cornell Medicine in Dr. Meyer. The lab moved to Weill Cornell Medicine in 2020. To solve this difficult experimental problem, Ratnayeke used computer-aided microscopy to monitor thousands of growing cells simultaneously, catch the replicating cells in the act and analyze the activities of their licensing and replication factors.
The work revealed that a well-known licensing factor, CDT1, not only licenses a segment of DNA to become an origin of replication, but also acts as a brake on DNA replication, preventing an essential replication enzyme called CMG helicase from functioning. To begin synthesizing DNA, the cell’s enzymes must first break down CDT1. “Previously proposed mechanisms for coordinating this transition from the licensing phase of the cell cycle to the activation phase of the cell cycle depended on inhibitory licensing factors,” Ratnayeke said, adding that “the mechanism we identified here is actually the opposite… licensing factor CDT1 itself prevents the progression of DNA synthesis.”
To confirm their results, the scientists teamed up with colleagues at the Medical Research Council in Cambridge, UK, who discovered that the inhibitory mechanism can be summarized in a simplified system that reproduces the entire DNA synthesis process using purified components in a test tube. “That allowed us to reconstruct all components for DNA synthesis and prove that CMG helicase is directly inhibited by CDT1,” said Dr. Meyer, who is also a professor of biochemistry and a member of the Sandra and Edward Meyer Cancer Center at Weill Cornell medicine.
Because errors in replication licensing can kill cells or create cancer, the results provide new insight into cell health and disease. “Future work to mechanistically identify what’s going on with Cdt1 inhibition will provide more insight into the biophysics of how CMG helicase functions, and pinpoint specific regions of this complex that can be targeted using drugs,” said Ratnayeke.
Reference: Ratnayeke N, Baris Y, Chung M, Yeeles JTP, Meyer T. CDT1 inhibits CMG helicase in early S phase to separate origin licenses from DNA synthesis. molecular cell. 2023;83(1):26-42.e13. doi: 10.1016/j.molcel.2022.12.004
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