Marc Wold's Laboratory

Research

The human genome is composed of approximately 4x10⁹ base pairs of DNA. In order to grow, a human cell must precisely duplicate this entire genome during each cell cycle. This requires the concerted action of many proteins to specifically initiate DNA replication at multiple origins and to accurately synthesize large amounts of DNA. The primary interest of our laboratory is to understand the molecular mechanisms of eukaryotic DNA replication and how DNA replication is regulated in the cell.

A small double-strand DNA virus, SV40, has been used as a model system for studying DNA replication and for identifying human factors essential for its replication. One of the factors identified is a multiple subunit, single-strand DNA binding protein called Replication Protein A (RPA). This human protein is required for multiple processes in cellular DNA metabolism including DNA replication, repair, recombination, and cell cycle regulation, and homologs of RPA have been identified in all eukaryotes examined. RPA is composed of three tightly associated subunits of 70, 32, and 14 kDa in size. All three subunits of RPA are required for cellular function; however, each subunit may have a distinct role in RPA function. In addition to binding to single-strand DNA, RPA also interacts with multiple proteins, including replication, repair, and cell cycle regulatory proteins.

We are examining the structure and function of Replication Protein A. To accomplish this, we have developed an expression system that allows for the molecular manipulation, synthesis, and biochemical characterization of RPA. We are also utilizing cell culture to correlate biochemical activities with cellular functions of RPA. This combination of studies will allow for a better understanding of the roles and the molecular mechanisms of RPA function.