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Duplication of chromosomes requires numerous proteins acting together to unwind and replicate the two strands of duplex DNA. O’Donnell’s laboratory studies these replication mechanisms with the goal of understanding how the protein gears act together to make copies of DNA, and how they function with repair and recombination factors to ensure that these copies are accurate.

Over the years, research from O’Donnell’s lab has provided an overview of how the replication machine functions in Escherichia coli, yeast, and humans. A circular protein that he and his colleagues refer to as a sliding clamp completely encircles duplex DNA, acting as a mobile tether to hold the replication machine to the chromosome and sliding along behind the machinery for long distances.

The sliding clamps of prokaryote (β) and eukaryote proliferating cell nuclear antigen (PCNA) have similar structure and function. O’Donnell solved the structures of these ring-shaped proteins in collaboration with John Kuriyan’s laboratory (now at the University of California, Berkeley) and showed that they comprise six domains organized on a dimer or trimer surface.

To become attached to DNA, sliding clamps require a multiprotein clamp loader machine that uses adenosine triphosphate (ATP) to open and place the circular clamp on DNA. The lab has studied the detailed workings of these clamp loaders in both prokaryotic (γ complex) and eukaryotic (RF-C) systems. New and recent studies into other, accompanying processes include how the replication machinery interfaces with proteins in repair, DNA damage checkpoint paths, and recombination.

The eukaryotic replisome is much more complex than its prokaryotic counterpart. O’Donnell’s lab recently succeeded in reconstituting the eukaryotic replisome from over 30 different polypeptides—a project that has held his fascination over the past few years. O’Donnell and his colleagues have determined the architecture of the eukaryotic replisome, a feat not yet accomplished in a prokaryotic system. Eukaryotic replisomes must deal with histones, which organize the DNA in eukaryotic cells. The histones also regulate how the genome is expressed in different tissues of an organism, a process referred to as epigenetic inheritance. The replisome structure has implications about how the process of replication deals with histones, and how it may conserve the inheritance of epigenetic information.

Studies of eukaryotic replisome function have also outlined the rules by which distinct DNA polymerases act on the two strands of DNA during replication. The lab is applying techniques such as single-molecule technology and single-particle reconstruction by electron microscopy to understand how the replisome functions with other factors. These studies can be expected to provide new insights into eukaryotic replication, repair, and epigenetic inheritance.