Function of replication initiation proteins ORC and Cdt1

In order for cells to proliferate, they need to follow a precisely regulated cycle of growth and division. Before dividing, a cell needs to make a complete and accurate copy of its genome. Replication must be completed before division begins, and a new round of replication cannot start until the cell has successfully divided. To accomplish this, cells have evolved a complex regulation mechanism to ensure DNA replication occurs once, and only once per cell cycle. Much of this regulation acts at the stage of replication initiation, which controls when and where replication can begin. The pre-Replication Complex (pre-RC) is composed of a variety of proteins that act together to recruit the replication helicases to specific sites on chromatin. The Origin Recognition Complex (ORC) is thought to be responsible for marking origins of replication. We utilized a stable cell line hypomorphic for Orc2 to demonstrate that Orc2 is required for replication initiation in human cells. Surprisingly, we discovered a role for Orc2 in progression of the cell cycle, suggesting that it is not only important for recruiting replication machinery to chromatin, but may also play a role in regulation of cell cycle progression. Furthermore, our data suggests that the role of Orc2 in the cell cycle may be more important than its role in pre-RC formation, as Orc2 hypomorph cells have a G 1 /S transition defect, but fire origins normally. After ORC has loaded, Cdc6 and Cdt1 bind chromatin and together recruit the MCM2-7 helicase. Recent studies have shown that Cdt1 can bind to members of the MCM2-7 complex, suggesting that it may recruit the helicase via this interaction. We have designed a variety of C-terminal truncations and point mutants in Cdt1 that disrupt the interaction with MCM2-7. Interestingly, these MCM2-7 non-binding mutants still retain replication initiation activity, as they are able to induce rereplication to the same extent as wild type Cdt1 when overexpressed in human cells. This suggests that a stable interaction between Cdt1 and MCM2-7 is not required for the loading of MCM2-7.