Joachim J. Li
Regulation of the Initiation of DNA Replication in Saccharomyces cerevisiae
The initiation of DNA replication is tightly coordinated with other event in the eukaryotic cell cycle. To insure that S phase alternates with M phase, reinitiation is prevented at replication origins until the cell cycle is completed. A family of six structurally similar initiation proteins called the Mcm proteins has been implicated in this block to reinitiation. Tight association of the Mcm proteins with chromatin in G1 phase is somehow thought to prime the cell for initiation; during the course of replication, however, these proteins gradually dissociate from chromatin and remain dissociated for the remainder of the cell cycle, thereby preventing reinitiation.
In the budding yeast Saccharomyces cerevisiae, changes in the subcellular localization of Mcm proteins are observed in addition to changes in chromatin association: the proteins concentrate in the nucleus at the beginning of G1 phase, then disperse throughout the cell during S phase and remain dispersed until the next G1 phase. Clearly this change in subcellular distribution could also contribute to the block to reinitiation. Whether the dispersal thoughout the cell simply reflects a lack of chromatin association and consequently an inability to anchor Mcm proteins in the nucleus or is an additional layer of control is not known, however.
To examine the control of Mcm protein localization in S. cerevisiae, we have fused green fluorescent protein (GFP) to one of these proteins, Cdc47, and shown that the fusion protein can functionally substitute for its wild-type counterpart in both cell growth and replication initiation. We note that should Mcm localization simply reflect Mcm chromatin association, this system would provide a convenient method for monitoring this chromatin association in real time during the cell cycle. We are currently analyzing the role of other initiation proteins (including other Mcm proteins) in regulating Cdc47-GFP localization. Of special interest is whether Cdc6, an initiation protein required to load Mcm proteins on chromatin, is also needed to localize Cdc47-GFP in the nucleus. If not, it would suggest that the control of Mcm localization is separable from that of Mcm chromatin association.