Mechanism of Mcm2-7 Loading
The head-to-head arrangement of Mcm2-7 in the double-hexamer provides a molecular basis for the establishment of bi-directional DNA replication forks at an origin. But how are two Mcm2-7 hexamers loaded cooperatively, yet in opposite orientation by one set of loading factors? Or in other words, how is the symmetry of the Mcm2-7 double hexamer generated from an intrinsically asymmetric origin/pre- RC (Figure 5)? Origin sites in yeast are characterized by asymmetrically arranged conserved sequence motifs, and ORC neither exhibits any structural symmetry nor does it appear to bind symmetrically to origin sites. Do multiple ORC complexes perhaps cooperate to mediate Mcm2-7 loading? Or does Cdc6 function in concert with ORC to mediate the loading of the opposite Mcm2-7 hexamer? Subunit interactions in the pre-RC remain largely undefined. For example, while ORC and Cdc6 likely interact directly during pre-RC assembly, it is not known with which polypeptides Cdc6 interacts directly in the pre-RC. Similarly direct binding partners for the Mcm2-7 subunits in the pre-RC remain to be defined. Structural studies of the pre-RC and its intermediate states and the identification of protein-protein contacts will be essential to address the above questions.
Protein-DNA Interactions During Origin Licensing
The loading of Mcm2-7 around DNA implies that both DNA and Mcm2-7 need to be coordinated by the pre-RC during Mcm2-7 double hexamer formation. Moreover, the loading of two Mcm2-7 hexamers in opposite orientation may require the looping of origin DNA around the pre-RC. While ORC makes extensive contacts via multiple of its subunits to origin DNA, it is unclear whether, for example, Cdc6-and/or Cdt1 are also directly involved in constraining the path of the DNA for loading into the Mcm2-7 double hexamer, as may perhaps be suggested by the presence of potential DNA-binding domains of the helix-turn-helix type in these proteins. Identifying those subunits in the pre-RC that contact DNA and defining and characterizing their respective DNA binding domains will be essential for understanding tire path of the DNA in the pre-RC and for testing the contributions of the various DNA binding activities to Mcm2-7 loading both in vitro and in vivo.
Another fundamental question concerns the fact that extensive protein remodelling is clearly required to generate the Mcm2-7 head-to-head double hexamer from individual Cdtl-Mcm2-7 heptamers. This structural remodelling of Mcm2-7 during loading onto DNA confers a surprisingly robust salt stability to the Mcm2-7 complex, which is otherwise very sensitive to salt conditions. How is this structural remodelling being achieved? The energy derived from nucleotide-binding and hydrolysis is likely to play an important role, since ORC, Cdc6, and Mcm2-7 are all members of the AAA+ family of ATPases. The structurally conserved ATP-binding domains of this class of ATPases form higher-order oligomers that bind and hydrolyze ATP at subunit interfaces, which in turn induces conformational changes within the AAA+ assemblies that drive the molecular remodelling of target substrates. In the pre-RC, for example, one important remodelling event could be the opening and closing around the DNA of the Mcm2-7 ring. Do the AAA+ domains of ORC and Cdc6 thus interact and function analogously to other AAA+ assemblies to mediate Mcm2-7 loading? Alternatively, the ATP-binding domains of ORC and Cdc6 may also rather function as nucleotide-dependent switches that confer directionality to a multi-step reaction such as Mcm2-7 loading.
Mcm2-7 Double Hexamer Mobility
Under high-salt conditions Mcm2-7 double hexamers can dissociate off the ends of linear DNA, but remain stably bound to circular DNA, indicating that the double hexamer can randomly diffuse along DNA. It is currently not clear if Mcm2-7 double hexamers can also slide along DNA under physiological salt conditions. Real-time observations of Mcm2-7 double hexamers on single DNA molecules are required to address this issue. However, following activation the Mcm2-7 helicase needs to track along DNA for processive DNA unwinding, suggesting that activation mechanisms must involve a modulation of the mode of DNA-interaction during the transition from the inactive Mcm2-7 double hexamer to the actively unwinding Mcm2-7 helicase. Another interesting aspect of Mcm2-7 double hexamer mobility on DNA may concern the mechanism of origin choice. Can Mcm2-7 double hexamers move away from their site of loading and get activated at a distance from the loading site? Could other chromatin-associated activities such as those involved in transcription or chromatin-remodelling thus perhaps determine the site of origin firing by pushing Mcm2-7 double hexamers along the chromosome? Alternatively, could inactive Mcm2-7 double hexamers be pushed ahead of actively moving DNA replication forks to be available for potential replication restart mechanisms at sites of replication fork failure? Understanding the dynamic association of Mcm2-7 and associated factors with the DNA or chromatin template will be essential for gaining insight into these possibilities.