In the research reported in Nature Structural & Molecular Biology, 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 or 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 "handoff," 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. "You don't want to have naked single-stranded DNA lying around in a cell," explained Fanning. "It will get tangled, make hairpins within itself, get chewed up by nucleases. Ss binding proteins keep ssDNA straight and accessible to the right processing enzyme."
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. Fanning and Chazin have developed a working model to answer that question.
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.
In the SV40 system, three key proteins interact. The viral protein T antigen (Tag) interacts with RPA and an enzyme known as DNA polymerase-primase (pol-prim). Tag is a helicase, or DNA un
Contact: Jennifer Donovan
Howard Hughes Medical Institute