Interactions and architecture of human MCM proteins in vitro and in vivo.

摘要

The eukaryotic minichromosome maintenance (MCM) proteins have six subunits, Mcm2–7p. Together they play essential roles in the initiation and elongation of DNA replication. Human MCM proteins are actively expressed in cancer cells but not in non-proliferating normal cells, and they have been suggested to be attractive targets for potential anticancer drugs. It has been known that the six MCM proteins interact and form a ring shaped heterohexameric complex containing one of each individual subunit, and they can also form subcomplexes. However, the structures of these complexes are still unknown. In this study we systematically studied the pairwise interactions between individual human MCM protein subunits by using the yeast two-hybrid system combined with co-immunoprecipitation from the two-hybrid yeast lysates, co-pull down with bacterially expressed recombinant proteins, and in vivo protein-protein crosslinking with a non-cleavable crosslinker in human cells followed by co-immunoprecipitation. With the first two methods we revealed multiple binary interactions among the six human MCM proteins. In human cells, we demonstrated that hMcm2p interacts with hMcm3, 5, and 6p; hMcm3p with hMcm2, 5, and 7p; hMcm4p with hMcm6 and 7p; and hMcm7p with hMcm3 and 4p. We also showed that hMcm6p, but not hMcm2 or 4p has self-interaction to form a homodimer. Based on the results from the human cell experiments, which are also supported by the data from the other assays, we propose a model for the architecture of the human MCM protein heterohexameric complex. This provides the first working model for the arrangement of the human MCM subunits in the heterohexameric complex. By combining the interactions identified in human cells and the data from the other methods we also propose models for the structures of all the subcomplexes including the double trimer of hMcm4/6/7p, which is associated with in vitro ATPase and weak DNA helicase activities. Furthermore, our study is also the first to examine the in vivo pairwise interactions of all six MCM subunits in any eukaryote. Thus, this study may serve as a foundation for understanding the overall architectures and functions of eukaryotic MCM protein complexes and as clues for developing potential anticancer drugs targeted to the human MCM proteins.; The second part of this thesis is to identify potential inhibitors of human MCM proteins. We have used the yeast two-hybrid system to screen a random peptide library that we have generated and found that four peptides could specifically interact with hMcm2p. More experiments are required to determine whether these peptides can inhibit the hMcm2p function and show anticancer potency.