In previous studies we have analyzed ORC-Cdc6 complex formation in the presence and absence of DNA and showed that Cdc6 confers increased sequence specificity to the ORC-Cdc6 complex (37
). We also showed that ORC and Cdc6 form a ring-shaped complex with a surface of similar dimension and shape to the ring-shaped surface of the MCM-hexamer, which likely functions as a MCM loading machine prior to initiation of DNA replication (38
). In this study we concentrated on analyzing the mechanism by which Cdc6 increases DNA sequence specificity or origin recognition and stability of the ORC-Cdc6-DNA complex, both of which are relevant to understanding why origins of DNA replication locate to specific regions within chromosomes for pre-RC formation and MCM loading (31
Six out of the seven proteins within the ORC-Cdc6 complex are predicted to belong to the class of AAA+ proteins (38
). ATP binding and hydrolysis frequently regulate the function of AAA+ proteins (40
). In our previous study we focused on the role of ORC ATP binding and ORC ATPase in regulating ORC-Cdc6 complex (38
). In this study, the contribution of Cdc6 ATPase on ORC-Cdc6 interaction and sequence specificity was investigated. Mutants interfering with Cdc6 ATPase have been characterized in vivo
, Cdc6 E224G has a mutation in the highly conserved Walker B motif (31
) and is dominant lethal when over-expressed in yeast. Cdc6 N263A (42
) has a mutation in sensor 1, is temperature sensitive and lethal when over-expressed. In the absence of DNA we found that Cdc6 addition to ORC resulted in rapid Cdc6-induced ATP hydrolysis and dissociation from ORC. Cdc6-ADP, which is produced during ATP hydrolysis, is not capable of binding to ORC (38
). Cdc6 mutants defective for ATPase did not show increased ATPase in the presence of ORC and resulted in stable complex formation. The Cdc6 E224G mutant actually inhibited ORC ATPase activity, perhaps explaining why this mutant is a dominant lethal. The biological function of ORC induced Cdc6 ATPase in the absence of DNA is likely the forced disassembly of the complex to ensure that the ORC-Cdc6 complex only forms on origin DNA (38
In the presence of origin DNA a stable ORC-Cdc6 complex was formed (31
) and Cdc6 ATPase activity was induced, but this low level ATPase could originate from partial binding of the ORC-CDC6 complex to regions outside the origin, which are unable to suppress Cdc6 ATPase. In the presence of non-origin DNA, however, the Cdc6 ATPase activity was much higher and the complex was not stable on DNA. Mutations in conserved genetic elements of ARS resulted in activation of ATPase activity, which was dependent on Cdc6 ATPase. The A element represents the primary binding site for ORC (11
), the sequence of A elements are most conserved within different ARS and point mutants between the A-element have drastic phenotypes on plasmid stability (11
). Mutations in the A element have also been shown to reduce ORC binding (11
). We suggest that Cdc6 activated ATPase and consequently disassembly of the ORC-Cdc6-DNA complex is a reason for the low plasmid stability of ARS A box mutants, in addition to the reduced affinity of ORC for DNA. Similarly, mutations in the B elements are important for origin function, however in this case, only simultaneous deletion of at least two of the B elements result in nonfunctional origins (12
). Mutations in the B1 element reduce slightly the affinity of ORC for origin DNA (13
) and mutations in the B2 element can be compensated by Cdc6 over-expression (15
). We found that mutations in the B elements resulted in increased Cdc6 ATPase activity, which promotes disassembly of the complex. Cdc6 over-expression may be sufficient to force ORC-Cdc6 complex formation on the defective origin and therefore could compensate for B2 mutations.
The ARS1 A838G
mutation is of particular interest, since this mutant binds to ORC well, but is partially defective in the Cdc6-dependent extended footprint on the ARS1
origin DNA (38
) and has reduced ability to suppress the Cdc6-induced ATPase activity of the ORC-Cdc6 complex. We therefore suggest that specific nucleotide sequences modulate origin utilization by targeting the ORC-Cdc6 complex rather than ORC alone, again suggesting that Cdc6 contributes to origin selection.
It is known that origins with weak ORC binding sites can still function efficiently (46
). We suggest that these DNA sequences bind the ORC-Cdc6 complex more efficiently to promote pre-RC formation. Furthermore, when Cdc6 is over-expressed in certain circumstances, re-replication of only a subset of origins of DNA replication occurs (47
). Since Cdc6 contributes to origin selection and utilization, this may be one reason why not all origins are re-replicated when Cdc6 is present in excess.
Cdc6 ATPase within the ORC-Cdc6-DNA complex, first described by Randell et al. (31
), regulates the stability of the ORC-Cdc6 complex. The function of the ORC-Cdc6 complex is to load in cooperation with Cdt1 the potential MCM-helicase onto DNA to form a pre-replication complex (pre-RC) (49
). Based on our new findings that Cdc6 ATPase controls the stability of ORC-Cdc6 complex on DNA, we suggest that it is the stability of the ORC-Cdc6 complex on origin DNA, but not on non-origin DNA that regulates directly the location in chromosomes and amount of MCM loading. Cdc6 ATPase contributes to the selection of DNA sequences that can promote MCM loading and hence formation of a pre-RC. The ORC-Cdc6 complex stability depends on several factors, many of which are hardwired into the DNA sequence at origins, namely the A and B elements, but we envision that chromatin structure might also affect complex stability.
It has been shown that Cdc6 availability is further regulated by association with cyclin (50
) and Cdc6 destruction is regulated by ubiquitin mediated proteolysis (51
). Cyclin-Cdc6 interaction and proteolysis of Cdc6 regulate the overall availability of Cdc6 during the cell division cycle, but when Cdc6 is available to bind to ORC prior to pre-RC assembly, DNA sequence information is a significant determinant in modulating Cdc6 activity and hence where on the DNA pre-RCs are formed. Since subtle changes in the DNA sequence influence Cdc6 ATPase activity, Cdc6-ORC complex stability can also be represented as having a specific probability at any given DNA sequence and therefore contributes to selection of functional origins. This fits well with the recent data in fission yeast in which origins of DNA replication are selected in a stochastic manner dependent on DNA sequence and do not function every cell cycle (54
). We suggest that Cdc6 (cdc18 in S. pombe
) ATPase activity influences those DNA sequences that are selected as origins of DNA replication and even the stochastic firing of origins in each S phase. Such a scenario may exist for the selection of sites of DNA replication in the chromosomes of plant and animal cells where ORC binding is not known to be sequence specific. In fact Cdc6 is a rate limiting component for the initiation of DNA replication in vertebrate cells (36
) and we suggest that it is the interaction between ORC and Cdc6 and the subsequent ATPase activities of these proteins that determines when and where in chromosomes initiation of DNA replication takes place.
Cdc6 ATPase activity within the ORC-Cdc6 complex is suppressed by origin DNA when compared to the activity in the absence of DNA and activation of Cdc6 ATPase, due to non-origin DNA, leads to disassembly of Cdc6 from DNA. It is possible that ORC bound to non-origin DNA has an altered conformation, which stimulates Cdc6 ATPase upon complex formation. This results in ADP-Cdc6 which cannot interact with ORC and is released from the ORC-DNA complex. Once ADP-Cdc6 is in solution it has to be recharged with ATP. This could be a passive reaction or actively modulated by a protein. On the other hand a specific protein present during pre-RC assembly might modulate Cdc6 ATPase to promote several rounds of MCM loading. Alternatively, a protein may stimulate yeast Cdc6 ATPase activity on origin DNA to promote dissociation of Cdc6 from the origin before its destruction at the G1/S phase transition and hence hinder re-replication in a single cell cycle.