Rockefeller scientists first to reconstitute the DNA ‘replication fork’

PUBLIC RELEASE DATE:

8-Jul-2014

Contact: Franklin Hoke fhoke@rockefeller.edu 212-327-8998 Rockefeller University

When a cell divides, it must first make a copy of its DNA, a fundamental step in the life cycle of cells that occurs billions of times a day in the human body. While scientists have had an idea of the molecular tools that cells use to replicate DNAthe enzymes that unzip the double-stranded DNA and create "daughter" copiesthey did not have a clear picture of how the process works.

Now, researchers at Rockefeller University have built the first model system to decipher what goes on at the "replication fork"the point where DNA is split down the middle in order to create two exact copies. The findings are specific to eukaryotic cells, the defining feature of which is that the DNA is contained within a nucleus. All multicellular life forms, including humans, are eukaryotes. The researchers' findings, which may have profound implications for the study of cell division and human disease, appeared July 6 in the journal Nature Structural and Molecular Biology.

"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," says the paper's senior author Michael O'Donnell, head of the Laboratory of DNA Replication at Rockefeller University. "What is more exciting, I believe, is that this opens up replication-fork biology to biochemical study by many labs, providing a new tool to unravel some pressing questions in a number of fields of study, including epigenetics and DNA repair." O'Donnell is Anthony and Judith Evnin Professor at Rockefeller and a Howard Hughes Medical Institute investigator.

According to O'Donnell, the team's techniques may allow researchers to reconstruct at the molecular level biochemical events that are known to occur but were difficult or impossible to study in detail. For example, scientists know that epigenetic informationinheritable information that is not encoded by the DNA sequence, but instead lies in modifications to proteins associated with the DNAis passed along to the daughter cells after DNA replication. Yet exactly how that occurs remains a mystery. Another unknown is what happens when the replication fork encounters an area of damaged DNA as it travels down the length of DNA.

"Diseases, such as cancer, often arise from DNA damage or defects in episomal inheritance, so these findings could have direct relevance to these fields," O'Donnell says. "There are plenty of hypotheses about the mechanics of DNA replication, but until now the process could not be studied using a defined system with pure proteins."

The replication fork is assembled as a complex of numerous proteins, one of which is an 11-subunit collective called CMG that unwinds and separates the DNA into two individual strands. The emerging replication fork looks much like a zipper opening, with CMG in the role of a zipper slider and the individual strand appearing like the two rows of teeth of the open zipper.

Each of these strands then becomes the templates for daughter copies. The act of synthesizing a new complementary strand to match the templates is performed by two different polymerase enzymes, which match each complementary subunit of DNA (the nucleotide "letters" that make up the genetic code) to the chain, resulting in a new double-stranded DNA molecule. These enzymes are known as polymerase epsilon (Pol epsilon) and polymerase delta (Pol delta), and the O'Donnell laboratory sought to examine how they attach to DNA to perform their task.

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Rockefeller scientists first to reconstitute the DNA 'replication fork'