Cells have evolved careful checks to ensure DNA is copied only once, but how they switch on replication at the right moment has been the focus of a thirty-year research question. New work from the Crick has recorded the missing step.
Representation of the structure of the pre-initiation complex, the critical missing step in DNA replication. Credit: Macromolecular Machines Laboratory.
During cell division, DNA must be copied only once to keep the chromosomes physically stable as they are replicated and passed to the next generation. Cells have evolved safety measures to stop DNA replication going awry, essential to avoid cancer.
“One of these fail-safes revolves around the replicative helicase, the enzyme that unwinds the DNA double helix, to expose the two strands for replication,” says Alessandro Costa, who leads the Crick’s Macromolecular Machines Laboratory. “Separating helicase loading onto DNA from its activation prevents DNA being unzipped in the wrong place or at the wrong time.”
Alessandro and colleagues in his lab are interested in how this replicative helicase is switched on when the time is right. He describes his work as making a ‘molecular movie’ of DNA replication, one step at a time. Until now, one critical frame of the movie was missing.
For thirty years, biochemical and genetic studies hinted at the existence of a ‘pre-initiation complex’, a bundle of all the tools needed to build the active replicative helicase. But no one had actually extracted the complex from other proteins in a cell, let alone described what it looks like.
“We knew that three activator proteins attached themselves to the replicative helicase, and split it into two machineries that copy each strand of DNA,” Alessandro says. “But we didn’t know how these two molecular machines were physically built.”
Reported today in Nature, Thomas Pühringer, a researcher in Alessandro’s lab, put this thirty-year query to rest during his PhD, successfully recreating all the steps in yeast proteins in a test tube. To do this, he used a biochemical system originally developed by Alessandro’s collaborator, John Diffley, and his team at the Crick. Thomas then captured the structure of the pre-initiation complex for the first time, using a powerful imaging technique called cryo-electron microscopy. “This was very challenging, as the complex is present for such a fleeting moment,” he says.
AI-based tool AlphaFold then allowed them to fill in the gaps by identifying the most flexible parts of the complex, as these were captured at lower resolution.
“The eureka moment, in this case, was the biochemical and imaging experiments giving us the ‘right’ questions to ask AI, which could help us understand how each subunit interacts with each other, to keep the protein complex together," Alessandro describes. “This took us five years: without AI to complement the experimental work, it would’ve taken a lot longer.”
With this new frame in the movie, the team could identify the functions of so-called ‘firing factors’ in the pre-initiation complex, proteins that help to switch on the replicative enzyme.
“We found that some firing factors work as expected from the genetic experiments,” Thomas adds. “But others had unexpected dual roles, such as one which helps to pull all the tools together at the beginning, but also helps to separate the two DNA strands later on.”
He continues, “This resolves a mystery in the field. There is a firing factor in human DNA replication which is also found in yeast but was thought to have evolved different functions. Our work shows that this is not the case, indicating the yeast and humans start DNA replication using the similar strategies.”
Now that they have captured the missing frame for yeast DNA replication, Alessandro describes the next challenge in the field as recreating the same movie but for multicellular organisms like animals or humans, a richer sequel to the original film. This will advance understanding of how faults in DNA replication can lead to cancer.
“With strong collaborations across between scientists at the Crick and beyond and the increasing power of imaging and AI, I think it won’t be long before we understand how multicellular organisms initiate the process of copying their chromosomes,” he concludes.
