Cdc6 ATPase Activity Regulates ORC Cdc6 Stability and the Selection of Specific DNA Sequences as Origins

DNA replication, as with all macromolecular synthesis steps, is controlled in part at the level of initiation. Although the origin recognition complex (ORC) binds to origins ofDNAreplication, it does not solely determine their location. To initiate DNA replication ORC requires Cdc6 to target initiation to specific DNA sequences in chromosomes andwithCdt1 loads the ring-shaped mini-chromosome maintenance (MCM) 2–7 DNA helicase component onto DNA. ORC and Cdc6 combine to form a ringshaped complex that contains six AAA subunits. ORC and Cdc6 ATPase mutants are defective in MCM loading, and ORC ATPasemutants have reduced activity in ORC Cdc6 DNA complex formation. Here we analyzed the role of the Cdc6 ATPase on ORC Cdc6 complex stability in the presence or absence of specific DNA sequences. Cdc6ATPase is activated byORC, regulates ORC Cdc6 complex stability, and is suppressed by origin DNA. Mutations in the conserved origin A element, and to a lesser extent mutations in the B1 and B2 elements, induce Cdc6 ATPase activity and prevent stable ORC Cdc6 formation. By analyzing ORC Cdc6 complex stability on various DNAs, we demonstrated that specific DNA sequences control the rate of Cdc6 ATPase, which in turn controls the rate of Cdc6 dissociation from the ORC Cdc6 DNA complex. We propose a mechanism explaining how Cdc6 ATPase activity promotes origin DNA sequence specificity; on DNA that lacks origin activity, Cdc6 ATPase promotes dissociation of Cdc6, whereas origin DNA down-regulates Cdc6 ATPase resulting in a stable ORC Cdc6 DNA complex, which can then promote MCM loading. This model has relevance for origin specificity in higher eukaryotes.