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.

Figure 1

ORC and Cdc6p form a pre-RC like complex on DNA. Important elements of the ARS1 origin of replication are indicated as A, B1, B2 and B3. a, DNase I footprints were done with 2 ng (0.4 nM) of a 290 bp DNA ARS1 fragment labeled with γ-³²P at the 5’-end of either strand (lanes 1–10, T-rich strand of the ARS consensus sequence; lanes 11–20, A-rich strand). Lanes 1 and 11 contain no protein. Lanes 2–5, 7–10, 12–15 and 17–20 contain 3.4, 8.5, 17 and 42.5 nM Cdc6p. Lanes 6–10 and 16–20 contain 40 nM ORC. Regions protected by the addition of Cdc6p are indicated by a vertical bar. Hypersensitive sites are indicated by a star. The arrow points to one region discussed in the text. b, ARS1 sequence including the A, B1 and B2 element. Comparison of in vivo footprint data of G2 and G1 arrested yeast cells and in vitro footprint data with ORC or ORC and Cdc6p. c, DNase I footprint with ORC and Cdc6p at the 2μ origin of replication. Lane 1 contains no protein. Lanes 5–8 contain 40 nM of ORC. Lane 2–4 and 6–8 contain 1.4, 7 and 17 nM Cdc6p. d, Gel-shift assay of P³² labeled ARS1 DNA with ORC and Cdc6p. Protein concentrations are indicated in the figure. Wt and mutant ARS1 sequences are indicated in the figure, A-, B1-,B2-,B3- (858–865, 835–842, 802–808, 756, 758), B1 point-mutant 838A→G, B1- (835–842). e, DNase I footprint with ORC and Cdc6p at the wt and B1 mutant (835–842) ARS1 origin of replication as indicated. Lane 1 and 6 contain no protein. Lanes 2–5 and 7–10 contain 40 nM ORC. Lanes 3–5 and 8–10 contain 1.4, 7 and 17 nM Cdc6p. f, DNase I footprint with ORC and Cdc6p at the wt and B1 point mutant 838A→G ARS1 origin of replication. Protein concentrations as in a. The arrow points to regions discussed in the text. g, DNase I footprint with ORC and Cdc6p at the wt and A- mutant (858–865) ARS1 origin of replication. Protein concentrations as in e.

Figure 2

Analysis of ATP binding and hydrolysis mutants in ORC and Cdc6p. a, DNase I footprint with ORC and Cdc6p ATP binding mutants at the T-rich strand of ARS1 origin of replication. Protein concentrations of ORC and Cdc6p as in Figure 1a. For ORC-1A (lanes 36–38) is shown a titration with 1 µM, 10 µM, 100 µM and 1 mM ATP. b, Gel-shift assay of γ-³²P labeled ARS1 DNA with ORC and Cdc6p ATP binding mutants. Proteins used and concentrations of ORC and Cdc6p are indicated in the figure. c, Gel-shift assay of γ-³²P labeled ARS1 DNA with wt ORC, ORC1 ATP hydrolysis mutants and Cdc6p. Proteins used and concentrations of ORC and Cdc6p are indicated in the figure. d, DNase I footprint with wt ORC, ORC1 ATP hydrolysis mutants and Cdc6p at the ARS1 origin of replication. Protein concentrations as in figure 1a.

Figure 3

ORC - Cdc6p interaction analyzed by glycerol gradient sedimentation. a, The load onto the gradient (lane L) and fractions as well as any material in the pellet (lane B) were analyzed by gel electrophoresis and silver staining. Arrows indicate the sedimentation position of protein standards from a gradient prepared in parallel. Proteins analyzed and adenine nucleotide are indicated. b, Western blot analysis of ORC – Cdc6p peak fractions with an monoclonal αCdc6p antibody.

Figure 4

Three-dimensional structures of the yeast ORC. a, b, c, ORC alone. d, e, f, ORC in complex with the yeast Cdc6p. g, h, i, Density maps of a hexameric archaeal MCM N-termini complex. The MCM complex was generated by filtering the atomic structure to a resolution of 2.5nm. An archaeal Cdc6 ortholog is displayed by pink ribbons. See supplementary movies 1 and 2.

Figure 5

Analysis of AAA+ signatures within the proteins Orc1p, Orc2p, Orc3p, Orc4p and Orc5p and Cdc6p. a, secondary structure prediction of Orc1p, Orc2p, Orc3p, Orc4p, Orc5p and Cdc6p. Amino acids shown are indicated. Red corresponds to predicted α-helical and green to a β-sheet prediction using the SAM-T02 method (Karplus, K. et al. Combining local-structure, fold-recognition, and new fold methods for protein structure prediction. Proteins 53 Suppl 6, 491-6 (2003); http://www.soe.ucsc.edu/research/compbio/HMM-apps/. Deletions of sequence corresponding to no structure alignment are indicated by // and insertions by v. The amino acid sequence of the Walker A and B boxes and their position within the sequence is indicated in the bottom. b, Sequence conversation within the Walker A and B boxes from different species (S. cerevisiae, S. pombe, H. sapiens, M. musculus, D. melanogaster, X. laevis) for Orc1p, Orc2p, Orc3p, Orc4p, Orc5p and Cdc6p are shown in the sequence logo format (Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res 14, 1188-90 (2004) http://weblogo.berkeley.edu/logo.cgi.