DNA replication fork visualised

An American team have built the first model to decipher what goes on at the ‘replication fork’ during DNA duplication. Rockefeller University researchers led by Michael O’Donnell reconstructed at the molecular level the biochemical events known to occur but difficult to study in detail. “We were able to purify and reconstitute the central components that propel the eukaryotic replication fork, which for the first time enables us to study the process and its regulation by the cell in fine detail,” said O’Donnell, head of the Laboratory of DNA Replication. “What is more exciting, I believe, is that this opens up some pressing questions in a number of fields of study, including epigenetics and DNA repair.” The replication fork is assembled as a complex of numerous proteins, including an 11-subunit collective called CMG which unwinds and separates the DNA into individual strands. Each of the strands acts as a template for the daughter copy, which is synthesised by two polymerase enzymes – polymerase epsilon and polymerase delta (Pol epsilon and delta). O’Donnell’s team wanted to determine how these enzymes attached to DNA to match its complementary subunit to the chain. The two DNA strands fit together head to tail – 5’ end to the 3’ end – and new DNA can only be synthesised in one direction (5’ to 3’). This occurs at different paces, resulting in a leading strand – Pol epsilon – and a lagging strand, Pol delta, being synthesised in opposite directions. Researchers bought together the essential enzymes with a set of nucleotides and a linear model of duplex DNA to create their simple model. They found Pol epsilon needs CMG to attach to DNA securely – even with an excess of Pol delta, CMG chose Pol epsilon without fail. Pol delta, binds very strongly to another accessory protein – the PCNA clamp, a ring shaped protein that encircles DNA. Only when the clamp is attached to the lagging strand does Pol delta bind strongly to PCNA. The model could help reveal how epigenetic changes occur, and what happens when the replication fork encounters an area of DNA damage as it travels down the length of DNA. “As a research tool, our model could allow scientists to better understand what occurs in DNA replication, and what goes wrong in disease,” said O’Donnell. The findings are published in Nature Structural and Molecular Biology. Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork