TWO HEADS AND THREE TOOLS are sometimes better than one. An HHMI professor and a colleague who mentor HHMI-supported undergraduates are using the tools of molecular biology, biochemistry, and biophysics to solve a scientific puzzle.

What has their attention is the mysterious mechanism that enables DNA replication in simian virus 40 (SV40), a mammalian model for that vital process. “We’ve taken what began as a biochemical and molecular genetic approach, then used structural biology to learn about protein interactions, and then returned to biochemistry to validate our structural model in a functional way,” said Ellen Fanning, an HHMI professor at Vanderbilt University, in Nashville, Tennessee.

Fanning and Walter Chazin, director of Vanderbilt’s Center for Structural Biology, reported their findings in the April 2005 issue of Nature Structural & Molecular Biology, published online March 27, 2005.

The scientists sought the mechanism by which single-stranded DNA (ssDNA) breaks free from the chains of its binding protein to allow repair or replication, a process that is not well understood. Fanning and Chazin found structural and biochemical evidence for that mechanism, providing a model of this early step in DNA processing in mammalian cells.

Every organism has an ssDNA-binding protein for DNA replication and repair pathways. In eukaryotes (organisms whose cells have a nucleus), it is called replication protein A (RPA). One of the common functions of RPA in DNA processing pathways is facilitating “hand-off,” a process that ensures that the correct proteins move into place along the ssDNA to begin DNA processing.

RPA plays an important protective role for ssDNA. RPA binds with at least a dozen different repair and replication proteins. The question has been how RPA gets dislodged, allowing various enzymes access to the DNA for necessary processing.

Using SV40 as a model system, the scientists mapped atomic-level interaction on the surfaces of proteins involved in DNA processing. They used biochemical and genetic tools to determine how the interactions of those proteins promote synthesis of small segments of RNA known as primers, which are required for initiation of DNA replication.

“This provides a testable model for how the ssDNA-binding protein can be displaced from single-stranded DNA to allow a DNA processing pathway,” Fanning said. “This is a general phenomenon that happens throughout all DNA processing pathways.”

Photo: Irving Geis ©2000 HHMI