Our focus is on eukaryotic DNA replication and its control, with emphasis on the identification of the protein components involved in these processes. In general, eukaryotic replication proceeds along pathways that have been conserved from yeast to mammalian cells, and strikingly, as we gain more detailed information, appears to extend to prokaryotes.
All initiation events commence with the formation of a multimeric complex at the origin of initiation. In SV40 DNA replication, the origin binding protein is SV40 T antigen, an 82 kDa protein that multimerizes at the origin in the presence of ATP. In eukaryotes, the origin binding protein is a six subunit complex, the origin recognition complex (ORC). In S. cerevisiae, ORC is bound to origins of replication throughout the cell cycle . In G1, ORC recruits additional replication proteins, including Cdt1, Cdc6p and the MCM family of proteins, to form a pre-replicative complex (Pre-RC). This is followed by the addition of Mcm10p, Cdc45 and a number of other proteins, including the replicative polymerases to the complex. At the initiation of S-phase, Cdc6p is degraded and the MCM proteins and Cdc45p are found to move with the replication fork. Similar observations have been made in Xenopus extracts with proteins and complexes homologous to those found in S. cerevisiae.
Our laboratory has divided eukaryotic replication into multiple stages, and we are studying enzymes that participate in both the initiation and elongation stages.
