In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization

Abstract

The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2-7 onto DNA. Helicase loading involves two MCM2-7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2-7 double hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A motifs, which are predicted to affect ATP binding, influence the ORC-Cdc6 interaction and MCM2-7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects MCM2-7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant. Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading or in releasing ORC from loaded MCM2-7. To determine whether Cdc6 regulates MCM2-7 double hexamer formation, we analysed complex assembly. We discovered that inhibition of Cdc6 ATPase restricts MCM2-7 association with origin DNA to a single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2-7 hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs modulate MCM2-7 recruitment, show that ATPase activity is required for MCM2-7 hexamer dimerization and demonstrate that MCM2-7 hexamers are recruited to origins in a consecutive process.

Figure 1.

Schematic representation of conserved AAA+ ATPase motifs. A simplified AAA+ structure including the conserved Walker A, Walker B, sensor-1, arginine finger and sensor-2 is shown. Hydrophobic amino acids are abbreviated as h. The general AAA+ consensus sequence of the Walker A and Walker B motifs is shown.

Figure 2.

The influence of Cdc6 mutants on pre-RC formation. Pre-RC assembly was performed as described in the methods section. A 30% load of the pre-RC proteins is shown in lanes 1–6. The use of the mutants is indicated in the figures. The gel was silver stained to visualize the proteins; however, the smallest subunit of the Orc1–6 complex stains only weakly. (A) Cdc6, Cdc6 K114E (Walker A) and Cdc6 R332E (sensor-2) were used in pre-RC assays in the presence of ATP (lanes 8–16) or ATPγS (lane 7). Pre-RCs were washed with a low salt buffer (lanes 7–9, 11, 12, 14 and 15) or a high salt buffer (lanes 10, 13 and 16). (B) Cdc6, Cdc6 E224G (Walker B) and Cdc6 N263A (sensor-1) were used in pre-RC assays in the presence of ATP (lanes 8–16) or ATPγS (lane 7). Pre-RCs were washed with a low salt buffer (lanes 7–9, 11, 12, 14 and 15) or a high salt buffer (lanes 10, 13 and 16).

Figure 3.

Pre-RC complexes crosslinked in solution. During pre-RC formation with ATPγS, an ORC–Cdc6–Cdt1–MCM2–7 complex is formed that is labelled OCCM. During pre-RC formation with ATP, an MCM2–7 double hexamer is formed that is labelled dHex MCM2–7. The single MCM2–7 hexamer is labelled sHex MCM2–7. A small amount of single hexameric MCM2–7 can be detected in the high salt-washed pre-RC ATP owing to destabilization of the complex. (A) Analysis of crosslinked pre-RC reactions using silver staining. Purified ORC and MCM2–7 (lanes 1 and 2), crosslinked purified ORC and MCM2–7 (lanes 3 and 4), crosslinked high salt-washed pre-RC ATP (lane 5), crosslinked pre-RC ATPγS (lane 6) and low salt-washed crosslinked pre-RC ATP (lane 7) are shown. (B) Purified Cdc6, Cdt1, ORC and MCM2–7 (lanes 1, 5, 9 and 14); purified crosslinked ORC and MCM2–7 (lanes 10 and 15); crosslinked pre-RC ATP (lanes 2, 4, 6, 8, 11, 13, 16 and 18); and crosslinked pre-RC ATPγS (lanes 3, 7, 12 and 17) were analysed by Cdc6, Cdt1, Orc3 and Mcm2 Western blot.

Figure 4.

Pre-RC complexes on DNA. (A) Experimental outline for (B) and (C). Pre-RC assays were assembled in the presence of ATPγS and ATP. When tagged and untagged MCM2–7 were used, equimolar amounts of each complex were combined in pre-RC reactions. Complexes were released from DNA via restriction digest, and DNA-bound complexes were immune precipitated (IP) and together with input and supernatant (Sup) analysed by Western blotting with anti-Mcm2, anti-Cdt1, anti-Orc3 and anti-Cdc6 antibodies. (B) Co-immunoprecipitation of MBP-tagged and untagged MCM2–7 in the presence of ATPγS. (C) Co-immunoprecipitation of high salt-washed MBP-tagged and untagged MCM2–7 in the presence of ATP. Electron micrographs of metal-shadowed protein–DNA complexes with (D) ORC–Cdc6, (E) pre-RC ATP, (F) pre-RC Cdc6 E224G and (G) pre-RC ATPγS. Electron micrographs of negative-stained samples of (H) pre-RC ATP—(double hexamers are circled in white) and (I) pre-RC ATPγS (ORC–Cdc6–Cdt1–MCM2–7 complexes are circled in white).

Figure 5.

Analysis of the role of ORC4R in pre-RC formation. (A) ATPase activity of ORC and ORC4R in the absence (columns 1 and 2) and presence of DNA (columns 3 and 4). (B) The stability of pre-RCs formed in the presence of ORC (lanes 1–6) or ORC4R (lanes 7–12). Pre-RCs were formed in the presence of low salt buffer (lanes 1 and 7) or with buffer containing 100, 200, 300, 400, 500 mM sodium chloride, respectively (lanes 2–6 and 8–12). (C) Pre-RC complexes were crosslinked in solution. Pre-RC ATP (lanes 1, 5, 6, 10, 11, 15, 16 and 20), pre-RC ATPγS (lanes 2, 7, 12 and 17) and pre-RC with ORC4R (lanes 3, 4, 8, 9, 13, 14, 18 and 19). (D) Electron micrographs of metal-shadowed pre-RCs formed with ORC (upper picture) and ORC4R (lower picture). (E) Electron micrographs of uranyl acetate-stained pre-RC samples prepared with ORC (upper picture) and ORC4R (lower picture).

Figure 6.

Structural analysis of conserved Cdc6 ATP-binding and hydrolysis motifs. (A) Crystal structure of S. solfataricus Cdc6 and (B) an I-Tasser-predicted S. cerevisiae structure of Cdc6. The sequence of S. cerevisiae Cdc6 (amino acids 73–513) was submitted to the I-TASSER server for protein structure prediction. A close-up view of the of S. solfataricus (C) and the predicted S. cerevisiae Cdc6 (D) ATP-binding motif showing the conserved Walker A (K114) and sensor-2 (R332) motifs. The S. solfataricus (E) and the predicted S. cerevisiae (F) Walker B (E224) and sensor-1 (N263) motifs of Cdc6 are shown.

Figure 7.

Blocked Cdc6 ATP hydrolysis leads to formation of an ORC–Cdc6–Cdt1–MCM2–7 complex, and ORC4R promotes double hexamer formation. A model of the MCM2–7-loading process (A–D). (A) ATP–ORC is bound to DNA. (B) ATP–Cdc6 associates with ORC. (C) We show that in the absence of ATP hydrolysis, only one MCM2–7 hexamer associates with ORC–Cdc6. This complex could contain multiple Cdt1 molecules. (D) ATP hydrolysis promotes the recruitment of a second Cdt1–MCM2–7 complex potentially with the help of a second ORC–Cdc6 complex. In consequence, MCM2–7 becomes loaded, forms a stable double hexamer encircling DNA and ORC, Cdc6 and Cdt1 become released from MCM2–7. Orc1 ATPase may function to reactivate ORC for a new round of pre-RC assembly.