In order to study the replication fork, O'Donnell and his laboratory needed to recreate the process in a simple model. In a test tube, they brought together the essential enzymes with a set of nucleotides (DNA building blocks) and a linear molecule of duplex DNA.
Pol epsilon, they found, does not attach very well to the DNA on its own. It requires the presence of the CMG complex to attach securely. Even in an excess of Pol delta, CMG chose Pol epsilon without fail. Pol delta, however, binds very strongly to another accessory proteinthe PCNA clampa ring shaped protein that encircles DNA. Only when the PCNA clamp is on the lagging strand does Pol delta strongly bind to PCNA. Even when the researchers added a 20 to 1 excess of Pol epsilon, PCNA only would work with Pol delta on a lagging strand model DNA.
"As a research tool, our model could allow scientists to better understand what occurs in DNA replication, and what goes wrong in disease states," O'Donnell says.
To create his replication fork model, O'Donnell used enzymes from yeast. Like human cells, yeast cells are eukaryotic, meaning a membrane encloses their nucleus. Prokaryotic cells, like bacteria, evolved a separate (although similar) method for replicating DNA. The eukaryotic machinery, from single-celled amoeba to humans, are remarkably conserved through evolution, which allows for high confidence that the replication fork model also represents what occurs in human cells.
"For much of my career, I studied the replication fork in prokaryotes, thinking that perhaps what I learned could be applied to create new types of antibiotics that would stop the replication process in its tracks," O'Donnell says. "Now I study the replication fork in eukary
|Contact: Franklin Hoke|