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ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA

Abstract

Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC–Cdc6 complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.

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Figure 1: ORC and Cdc6 form a pre-RC–like complex on DNA.
Figure 2: Analysis of ATP binding and hydrolysis mutants in ORC and Cdc6.
Figure 3: ORC-Cdc6 interaction analyzed by glycerol gradient sedimentation.
Figure 4: Three-dimensional structures of the yeast ORC.
Figure 5: Analysis of AAA+ signatures within the proteins Orc1, Orc2, Orc3, Orc4, Orc5 and Cdc6.

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Acknowledgements

We thank A. Stenlund for comments on the manuscript, S.P. Bell for ORC mutants and P. Wendell for technical assistance. This work was supported by a grant from the US National Institutes of Health (GM45436). H.L. acknowledges support from Brookhaven National Laboratory LDRD project number 05-112 and US Department of Energy grant KP1102010. C.S. was a fellow of the Leukemia and Lymphoma Society.

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Correspondence to Bruce Stillman.

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Supplementary information

Supplementary Fig. 1

Extended footprint formation is salt sensitive at low ORC concentrations. (PDF 415 kb)

Supplementary Fig. 2

Gel-shift assay of P32 labeled ARS1 DNA with ORC and Cdc6. (PDF 83 kb)

Supplementary Fig. 3

ATP and ADP dependent footprint of ORC (PDF 225 kb)

Supplementary Fig. 4

Gel-shift assay with ORC and Cdc6 in the presence of ATP, ATPγS or ADP. (PDF 78 kb)

Supplementary Fig. 5

DNase I footprint with ORC and Cdc6 in the presence of 1 mM ADP. (PDF 374 kb)

Supplementary Fig. 6

DNase I footprint with ORC and Cdc6 with ATP and ATPγS. (PDF 632 kb)

Supplementary Fig. 7

ORC-Cdc6 interaction analyzed by glycerol gradient sedimentation in the presence of ADP. (PDF 580 kb)

Supplementary Fig. 8

Superposition of the ORC map with the ORC-Cdc6 map. (PDF 399 kb)

Supplementary Video 1

Three-dimensional structures of the yeast ORC in the presence of ATPγS, as described in Figure 4. (MOV 1138 kb)

Supplementary Video 2

Three-dimensional structures of the yeast ORC-Cdc6 complex in the presence of ATPγS. (MOV 1087 kb)

Supplementary Methods (PDF 85 kb)

Supplementary References (PDF 60 kb)

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Speck, C., Chen, Z., Li, H. et al. ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. Nat Struct Mol Biol 12, 965–971 (2005). https://doi.org/10.1038/nsmb1002

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