Prevailing DNA Synthesis Model Challenged

According to new study out of Weill Cornell Medicine, CDT1, a protein that prepares DNA for replication, also prevents the replication process from running out of control by preventing CMG helicase from functioning.

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 precisely once before dividing. It is widely believed that in preparation for cell division, the licensing proteins attach to specific regions in the DNA, designating them as replication origins. When the DNA synthesis phase of the cell cycle begins, replication begins only at those licensed sites, and only initiates, or “fires” once, according to the current model.

The findings from this new study are in contrast to that prevailing model. “The same factor that is allowing for this licensing to happen is only degraded after these replication origins have fired,” explains Tobias Meyer, senior author on the study published earlier this month in Molecular Cell. “In principle, the cell could load these licensing machines onto DNA that’s already replicated, so, instead of two copies, you’re getting three or four copies of that segment of the DNA, and these cells would be expected to lose genome integrity and die or become cancerous.”

Figuring out how cells avoid that fate has been tricky. “We needed to be studying events in the first minutes of the DNA synthesis phase of the cell cycle, so it’s a very transient period,” notes first author Nalin Ratnayeke. To solve this difficult experimental problem, Ratnayeke used computer-aided microscopy to monitor thousands of growing cells simultaneously, catching the replicating cells in the act and analyzing 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 a replication origin, but also acts as a brake for DNA replication, preventing CMG helicase from functioning. To start 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 firing phase of the cell cycle have depended on inhibiting licensing factors,” adds Ratnayeke, adding that “the mechanism that we identified here is actually the opposite … the licensing factor CDT1 itself is preventing the progression of DNA synthesis.”

To confirm their results, the scientists collaborated with colleagues at the Medical Research Council, who found that the inhibitory mechanism can be recapitulated in a simplified system that reproduces the entire DNA synthesis process with purified components in a test tube. “That allowed us to reconstitute all the components for DNA synthesis, and to prove that CMG helicase is directly inhibited by CDT1,” says Dr. Meyer.