Heads of Laboratories
The elegant structure of duplex DNA suggests that the replication process would be simple. Duplication of the chromosomes, however, requires numerous proteins acting together to unwind and replicate the two strands of duplex DNA. Dr. 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 those copies are accurate.
Over the years, research from Dr. O’Donnell’s lab has provided an overview of how the replication machine functions in Escherichia coli, and several of its features have been found to be common to yeast and humans. A circular protein, which 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 as it functions. The sliding clamps of prokaryote (β) and eukaryote proliferating cell nuclear antigen (PCNA) have similar structure and function. Dr. 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. Once on DNA, the clamp binds the replication machinery and slides along behind it, constantly holding it to the chromosome for long distances.
To become attached to DNA, sliding clamps require a multiprotein clamp loader machine that uses adenosine triphosphate (ATP) to open the circular clamp and place it onto DNA. The detailed workings of how these clamp loaders function have been one of the lab’s aims in both prokaryotic (γ complex) and eukaryotic (RF-C) systems. Biochemical studies by Dr. O’Donnell’s group, combined with crystal structure information from Dr. Kuriyan’s laboratory, show that these clamp loaders are circular heteropentamers of crescent-shaped subunits that act like a hand with fingers; ATP binding powers the hand to manipulate the ring-shaped clamp onto DNA, and ATP hydrolysis allows the ring to close. Understanding the process by which two DNA polymerases cooperate with one clamp loader and a hexameric ring-shaped helicase to simultaneously synthesize both strands of duplex DNA is a project that has also held Dr. O’Donnell’s fascination over the past few years. He and his colleagues have learned about three point switches between primase, the clamp loader and DNA polymerase and the processes by which sliding clamps are recycled and regulated.
New and recent studies into other, accompanying processes include how the replication machinery interfaces with proteins in repair, DNA damage checkpoint paths and recombination. For example, the sliding clamps interact directly with MutS and MutL, and the O’Donnell lab is studying how it functions in mismatch repair. The β and PCNA clamps also bind several other proteins, indicating that these clamps and clamp loaders are at the center of many DNA metabolic processes. Dr. O’Donnell’s lab is initiating a project on the role of these proteins in recombinative repair in which the replication fork encounters a lesion that must be fixed by recombination and repair proteins. Scientists had widely anticipated that a replication fork would collapse upon encountering a lesion, but Dr. O’Donnell’s group found that this is not the case. Instead, blocks on the lagging strand are bypassed entirely by the replication machinery. Further, the replication fork helicase itself is capable of encircling either one of two strands, and in the latter mode it is capable of branch migration of Holliday junctions, making it a likely candidate as a central actor in recombinative repair of a stalled replication fork.
The O’Donnell lab is also studying how replication origins are activated for DNA synthesis, with the expectation that studies in the yeast system will lead to information on the overall control of chromosome replication.
Dr. O’Donnell received his Ph.D. at the University of Michigan, where he worked under Charles Williams Jr. on electron transfer in the flavoprotein thioredoxin reductase. He performed postdoctoral work on E. coli replication with Arthur Kornberg and then on herpes simplex virus replication with I. Robert Lehman, both in the biochemistry department at Stanford University. Dr. O’Donnell then became a member of the faculty of Weill Cornell Medical College in 1986 and an investigator at the Howard Hughes Medical Institute in 1992 before moving to Rockefeller in 1996. Dr. O’Donnell is a member of the National Academy of Sciences.
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