Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication
- Van Andel Research Institute, United States
- Imperial College London, United Kingdom
- Brookhaven National Laboratory, United States
- Stony Brook University, United States
Figures
Figure 1 with 1 supplement
The Cdc6 Walker B catalytic residue is essential in yeast but is easily bypassed by intragenic suppressor mutations.
(A) Structure of the Pyrobaculum aerophilum Cdc6 orthologue with bound ADP (Liu et al., 2000) highlighting the ATP binding pocket and key residues. The ‘DD’ Walker B residues are colored red and the …
Figure 1—figure supplement 1
Growth properties of various cdc6 Walker B mutants.
For (A) and (B), M4466 (W303-1A, cdc6Δ::ura3) containing only the indicated cdc6 alleles on pMW71 (Supplementary file 2) were spotted onto YPD plates in a 10-fold dilution series and incubated at …
Figure 2 with 1 supplement
Cdc6 ATPase activity is not required for MCM loading in vitro.
(A) Silver-stained 10% SDS gel with molecular weight standards and 100 ng of the indicated purified Cdc6 proteins. (B) Cdc6 ATPase assays. (C) MCM loading assay using the purified proteins shown on …
Figure 2—figure supplement 1
MCM loading by ORC-Cdc6-E224Q gives rise to a heterogeneous mixture of intermediates by EM.
(A) Negative stain-EM and (B) cryo-EM of complexes formed with ORC, Cdc6-E224Q, and MCM-Cdt1 proteins revealed a heterogeneous mixture of OCCM (ORC-Cdc6-Cdt1-MCM) and OCM (ORC-Cdc6-MCM) complexes; …
Figure 3
Cdc6 ATPase mutants promote MCM origin binding in yeast.
(A) Cell synchronization protocol for MCM ChIP. (B) PCR products amplified in base pairs surrounding ARS305. (C) All the cdc6 ATPase mutants tested promote MCM binding to origins similar to the …
Figure 4 with 1 supplement
Cdc6 removal after MCM loading bypasses ATPase requirement and is sufficient to allow DNA replication.
(A) Synchronization protocol (left) and flow cytometry profiles (right) of yeast strains M378 (cdc6-1), M4455 (cdc6-1 GAL1p-CDC6::LEU2), and M4531 (cdc6-1 GAL1p-cdc6-E224Q::LEU2). Asynchronous cells …
Figure 4—figure supplement 1
Overproduction of wild-type or Cdc6-NQ proteins does not cause a G1-arrest.
(A) Cell synchronization protocol (right) and flow cytometry profiles (left) of yeast strains M378 (cdc6-1), M4455 (cdc6-1 GAL1p-CDC6::LEU2), and M4513 (cdc6-1 GAL1p-cdc6-NQ::LEU2). Asynchronous …
Figure 5 with 2 supplements
Cdc6-E224G ATPase mutant is also defective in G1 progression but its degradation promotes DNA replication.
(A) Synchronization protocol (left) and flow cytometry profiles (right) of yeast strains M378 (cdc6-1) and M4766 (cdc6-1 GAL1p-cdc6-E224G::LEU2). Asynchronous cells were arrested in G2/M for 3 hr …
Figure 5—figure supplement 1
Growth phenotypes of cdc6 mutants altering additional residues that potentially affect ATP hydrolysis or ATP sensing.
Mutational summary of Box VI, sensor 1, Box VII (R-finger), Box VII', and Box VIII (sensor 2) regions of Cdc6. Yellow highlighting indicates that the mutation gave a lethal phenotype in yeast at …
Figure 5—figure supplement 2
Expression of the Cdc6-N263A ATPase mutant protein is dominant negative for growth, causes a G1 block, but loads functional MCM that can promote DNA replication after Cdc6-N263A degradation.
(A) pFJ235 (pRS416 Gal1p-CDC6) and mutant derivatives pFJ237 (Gal1p-cdc6-E224Q), pFJ404 (Gal1p-cdc6-N263A), and pFJ412 (Gal1p-cdc6-N263L) (Supplementary file 2) were transformed into wild-type …
Additional files
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Supplementary file 1
Yeast strains used in this study.
- https://doi.org/10.7554/eLife.05795.013
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Supplementary file 2
Plasmids used in this study.
- https://doi.org/10.7554/eLife.05795.014
