Functional in vitro- and in vivo-characterisation of the ring-shaped, replicative helicase MCM2-7 to identify the role of subunit interfaces in ring-opening, which in turn control loading and release of the helicase from double-stranded DNA.

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The precise duplication of chromosomal DNA is essential for preserving the genetic complement of the cell. Recently, the group of Dr. Speck has reconstituted the first step in DNA replication, loading of the replicative MCM2-7 helicase onto dsDNA, using purified proteins. Reconstitution of this important process, termed pre-replication complex (pre-RC) formation, enables now the detailed characterisation of this reaction. Loading of the ring-shaped MCM2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1. During this reaction the hexameric MCM2-7 ring must be opened to allow entry of the DNA into its central channel. It has been proposed that a weak interface between Mcm2 and Mcm5 could be involved in the loading process, generating a gate for DNA entry. In the proposed project I am testing this hypothesis using the reconstituted pre-RC assembly system and an in vivo approach. I have already fused FK506- and rapamycin-binding protein (FKBP) or FKBP-rapamycin binding (FRB) domains to the individual subunits of MCM2-7 to generate a rapamycin-inducible protein interaction. Initially, it was tested whether those modified proteins are tolerated in yeast. Then, using the reconstituted pre-RC in vitro assay we have shown that the blocking of the gate at the Mcm2/5 interface hinders loading of MCM2-7. In contrast, the same reaction performed in the absence of rapamycin, i.e. with an open gate, allows efficient loading. Also, all other interfaces tested so far remained unaffected by rapamycin. In the future these constructs will be tested in vivo. These studies will focus on their ability to inhibit pre-RC formation as well as the release of MCM2-7 after completion of DNA replication. This work has the potential to identify specific Mcm subunit interfaces as regulators of DNA loading and unloading. Since the complex MCM2-7 is frequently overexpressed in cancer this interface could be an interesting target to regulate MCM activity in vivo.