ATP Hydrolysis by ORC Catalyzes Reiterative Mcm2-7 Assembly at a Defined Origin of Replication  Jayson L. Bowers, John C.W. Randell, Shuyan Chen, Stephen P. Bell  Molecular Cell  Volume 16, Issue 6, Pages 967-978 (December 2004) DOI: 10.1016/j.molcel.2004.11.038

Figure 1 Mutational Analysis of an Arginine-Finger Motif in ORC4 (A) Amino acid sequence alignments of the AAA+ box VII domain of ORC4 from S. cerevisiae, human, mouse, X. laevis, D. melanogaster, and Z. mays. Bold type indicates residues that are conserved in all six homologs. The conserved arginine residues marked as 1, 2, or 3 were examined in this study. 1 = R263, 2 = R267, and 3 = R271 in the S. cerevisiae sequence. (B) ORC4 arginine finger mutations are lethal. Complementation tests were carried out on three ORC4 arginine finger alleles under the control of their native promoters. JBy9 (ORC4), JBy10 (orc4R267A), JBy11 (orc4R267E), and JBy12 (orc4R267K) were grown on media containing 5-FOA to select for the loss of the URA3 plasmid carrying a wild-type (wt) ORC4 gene. (C) Purification of wt and mutant ORC complexes. Wt and mutant ORC were purified from insect cells coinfected with viruses expressing all six ORC genes. 2 μg each of wt ORC and ORC-4R were separated on a 10% PAG and stained with Coomassie. Molecular Cell 2004 16, 967-978DOI: (10.1016/j.molcel.2004.11.038)

Figure 2 ORC-4R Is Defective in ATP Hydrolysis, but Not ATP Binding (A) ATP hydrolysis by wt and mutant ORC. ATP hydrolysis rates were determined for wt ORC, ORC-4R, and ORC-1A incubated with 10 μM ATP in the presence or absence of ARS1 DNA. (B) ATP binding by ORC-5A and ORC-4R, 5A at the indicated ATP concentrations in the presence or absence of ARS1 DNA. (C) Azido-ATP crosslinking to ORC and ORC-4R. [γ-32P] 8-N3-ATP was incubated with ORC and ORC-4R in the presence or absence of ARS1 DNA. All crosslinking observed was dependent on UV exposure and protein addition (data not shown). Molecular Cell 2004 16, 967-978DOI: (10.1016/j.molcel.2004.11.038)

Figure 3 ORC-4R and Wt ORC Share Identical DNA Binding Specificity (A) DNase I protection of ARS1 DNA by wt ORC and ORC-4R. Reactions were carried out in the presence of 1 mM ATP. (B) The in vivo association of ORC and ORC-4R with origin DNA. ChIP was performed by using a strain with a native copy of ORC4 and a protein A-tagged copy ORC4 or orc4-R267A under the control of a galactose-inducible promoter. Cells were grown in media containing raffinose or raffinose plus galactose and allowed to proceed through three doublings. ChIP was performed by using IgG coupled beads that bind protein A. The association of Orc4-protein A and Orc4R267A-protein A to ARS1 and URA3 was assayed. Molecular Cell 2004 16, 967-978DOI: (10.1016/j.molcel.2004.11.038)

Figure 4 Overexpression of ORC-4R Is Lethal and Disrupts Pre-RC Assembly (A) Strains containing galactose-inducible copies of the indicated ORC4 alleles were tested for viability on noninducing (glucose) or inducing (galactose) media. All tested strains also contained galactose-inducible copies of wt ORC1, ORC2, ORC3, ORC5, and ORC6. (B) ChIP analysis of pre-RC assembly upon overexpression of ORC or ORC-4R. Cells were grown in raffinose and arrested in G2/M with nocodazole for 3 hr. 2% galactose or glucose was added to control expression of ORC or ORC-4R for 2 hr. Cells were then washed and transferred into the same media containing α factor. Mcm2-7 origin binding was determined by ChIP at ARS1 and URA3. Molecular Cell 2004 16, 967-978DOI: (10.1016/j.molcel.2004.11.038)

Figure 5 Mcm2-7 Loading by ORC and ORC-4R (A) Schematic of the pre-RC assembly assay. Extracts from cells overexpressing Orc1-6 and/or Cdc6 were incubated with magnetic beads coupled to 1 pmol 1039 bp ARS1 DNA. After 30 min, the beads were collected and washed, and bead-associated proteins were denatured and analyzed by SDS/PAGE and immunoblotting. (B) Overexpression of both Orc1-6 and Cdc6 is required for maximal pre-RC assembly. Pre-RC assembly reactions were performed with extracts from cells overproducing Orc1-6 (ySC15), Cdc6 (ySC16), or all seven proteins (ySC17). (C) Dependence of pre-RC assembly on origin DNA. Pre-RC assembly assays were performed with ySC17 whole-cell extract (WCE) with wt ARS1 DNA or a mutant DNA (A-) in which the ARS consensus sequence was disrupted via linker insertion (ARS1(858-865) Marahrens and Stillman [1992]). (D) Depletion of ORC from WCE. 0.4 ml ySC17 WCE was passed through two sequential S sepharose columns by centrifugation. Total extract proteins from WCE or ORC-depleted extract (DE) were analyzed by SDS/PAGE and immunoblotting. (E) Reconstitution of Mcm2-7 loading by recombinant ORC. Pre-RC assembly assays were performed with ySC17 WCE, DE, or DE reconstituted with 2 pmol wt ORC (DE+ORC). Half of each reaction was analyzed by SDS/PAGE and immunoblotting. Purified protein standards consisted of 0.5, 1, 2, 5, 10, 20, and 50 fmol Mcm2-7 and 5, 10, 20, 50, 100, 200, and 500 fmol Orc1-6 and Cdc6. (F) Mcm2-7 complexes loaded in the pre-RC assembly reaction are resistant to salt extraction. Pre-RCs formed in ySC17 WCE were incubated for 5 min on ice in buffer containing 500 mM NaCl prior to washing and analysis as before. Molecular Cell 2004 16, 967-978DOI: (10.1016/j.molcel.2004.11.038)

Figure 6 ATP Hydrolysis by ORC Stimulates Pre-RC Assembly (A) Schematic of pre-RC assembly reactions with recombinant ORC. ORC was preincubated with nucleotide and bead-coupled ARS1 DNA. Beads were washed prior to the addition of ORC-depleted ySC17 whole cell extracts. Pre-RC assembly was monitored by immunoblotting as before. (B) Pre-RC assembly by ORC and ORC-4R. Assembly reactions were performed with WCE, extracts depleted of ORC as in Figure 5 (DE), or DE after prebinding of ORC or ORC-4R to bead-coupled ARS1 DNA (DE+ORC: WT or 4R). (C) Mcm2-7 complexes loaded by ORC-4R are resistant to salt extraction. Pre-RCs were formed with ORC-4R in ORC-depleted extracts as for (B) and incubated in buffer containing 500 mM NaCl as for Figure 5F. (D) Inhibition of pre-RC assembly by ORC bound ATPγS. For each time course, 12 pmol ORC (wt or 4R) was incubated for 10 min in 120 μl binding buffer with 5 mM ATP or ATPγS and then added to magnetic beads coupled to 6 pmol ARS1 DNA in binding buffer. After 10 min, beads were washed and incubated with 120 μl ORC-depleted extract in a 240 μl assembly reaction. At the indicated times following addition of ORC-depleted extract, 40 μl were removed from the reaction. Beads were collected and washed, and associated proteins were analyzed by immunoblotting. Molecular Cell 2004 16, 967-978DOI: (10.1016/j.molcel.2004.11.038)

Figure 7 ATP Hydrolysis by ORC Is Required for Reiterative Mcm2-7 Loading (A) Outline of experimental approach. (B) Isolation of Mcm2-7-associated protein-DNA complexes in WCE. A timecourse of pre-RC assembly was analyzed by immunoblotting. “Input” shows proteins assembled on DNA beads and “Mcm2-7 IP” shows proteins precipitated with an anti-Mcm2-7 antiserum after DNA cleavage with Mfe I at each time point. (C) Quantitation of DNA and Mcm2-7 proteins present in the Mcm2-7 immunoprecipitates shown in (B). (D) Isolation of Mcm2-7-associated protein-DNA complexes in ORC-complemented extracts. ORC-depleted extracts complemented with either wt ORC, ORC-4R, or lacking ORC were allowed to assemble pre-RCs on bead bound DNA for 20 min. Proteins associated with DNA beads (“input”) or precipitated with Mcm2-7 antisera after DNA cleavage by Mnase (“Mcm2-7-IP”) were analyzed by immunoblotting. (E) Quantitation of origin DNA present in the Mcm2-7 immunoprecipitates shown in (D). Molecular Cell 2004 16, 967-978DOI: (10.1016/j.molcel.2004.11.038)