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A Double-Hexameric MCM2-7 Complex Is Loaded Onto Origin DNA During Licensing of Eukaryotic DNA Replication

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A Double-Hexameric MCM2-7 Complex Is Loaded Onto Origin DNA During Licensing of Eukaryotic DNA Replication A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication Cecile Evrina, Pippa Clarkea, Juergen Zecha, Rudi Lurzb, Jingchuan Sunc, Stefan Uhlea,1, Huilin Lic, Bruce Stillmand,2, and Christian Specka,2 aDNA Replication Group, Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 0NN, United Kingdom; bMicroscopy Unit, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany; cBiology Department, Brookhaven National Laboratory, Upton, NY 11973; and dCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 Contributed by Bruce Stillman, October 6, 2009 (sent for review September 22, 2009) During pre-replication complex (pre-RC) formation, origin recog- to form the pre-initiation complex (pre-IC). Binding of pre-IC nition complex (ORC), Cdc6, and Cdt1 cooperatively load the proteins and protein kinase activity stimulates MCM2-7 helicase 6-subunit mini chromosome maintenance (MCM2-7) complex onto activity (13). Pre-IC formation culminates in the recruitment of DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that DNA polymerases and the start of active DNA replication (14). restricts DNA replication to once per cell cycle. During S phase ORC and Cdc6 belong to the AAAϩ family of ATP binding MCM2-7 functions as part of the replicative helicase but within the proteins (4, 15), a family that commonly forms ring- or spiral- pre-RC MCM2-7 is inactive. The organization of replicative DNA shaped structures. A spiral-shaped structure consisting of 5 helicases before and after loading onto DNA has been studied in AAAϩ proteins within the replication factor C (RFC) complex bacteria and viruses but not eukaryotes and is of major importance functions to destabilize the homotrimeric proliferating cell nu- for understanding the MCM2-7 loading mechanism and replisome clear antigen (PCNA) ring during PCNA loading onto DNA. In assembly. Lack of an efficient reconstituted pre-RC system has an analogous scenario it is possible that ORC-Cdc6 could hindered the detailed mechanistic and structural analysis of destabilize the MCM2-7 ring. This would result in ring opening MCM2-7 loading for a long time. We have reconstituted Saccha- during the MCM2-7 loading reaction (4, 16). romyces cerevisiae pre-RC formation with purified proteins and MCM2-7 is the best candidate for the eukaryotic replicative showed efficient loading of MCM2-7 onto origin DNA in vitro. helicase, because it can unwind DNA (17) and travels with the MCM2-7 loading was found to be dependent on the presence of all fork (18). However, MCM2-7 alone is a very weak helicase and pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary requires further proteins (Cdc45 and GINS) or posttranslational structure of MCM2-7 changes during pre-RC formation: MCM2-7 modifications for full activity (17, 19). For a long time, analysis before loading is a single hexamer in solution but is transformed of MCM2-7’s role in pre-RC formation and MCM2-7’s helicase into a double-hexamer during pre-RC formation. Using electron activity was hampered by the fact that MCM2-7 can form microscopy (EM), we observed that loaded MCM2-7 encircles DNA. multiple complexes in vitro. MCM2-7 exists as MCM467, The loaded MCM2-7 complex can slide on DNA, and sliding is not MCM35, MCM2-7, and other subcomplexes. Previous MCM2-7 directional. Our results provide key insights into mechanisms of purifications contained a mixture of different complexes varying pre-RC formation and have important implications for understand- in mass, ranging from single subunit monomers to very large ing the role of the MCM2-7 in establishment of bidirectional aggregated complexes (17, 20, 21). Until now the structure of replication forks. eukaryotic MCM2-7 before, during, and after loading remained poorly understood. helicase ͉ initiation ͉ mini chromosome maintenance ͉ ORC ͉ pre-RC In this study we developed a purification method for MCM2-7 from yeast that enabled reconstitution of pre-RC formation in NA replication is a precisely ordered process that requires vitro. Parallel biochemical and EM approaches showed that Dthe stepwise assembly of the replication machinery (1). The MCM2-7, although a single hexamer in solution, forms a double sites of replication protein assembly occur on DNA replication hexamer that can slide on DNA after loading. These results have origins or autonomous replicating sequences (ARS). In eukary- important implications for understanding how DNA is licensed otic cells, origins are recognized by a conserved 6-protein origin and how replication forks are formed. recognition complex (ORC) (2). ORC is bound to DNA throughout the cell cycle and during late M/early G1 phase Results recruits the Cdc6 protein, which facilitates MCM2-7 loading Purification of a Single-Hexameric MCM2-7 Complex. Loading of (3–5). Cdt1 forms a complex with MCM2-7 during late M phase MCM2-7 onto DNA during pre-RC formation is inhibited by (6). Within the nucleus Cdt1 recruits MCM2-7 to the ORC- B-type cyclin-dependent kinases (22). To obtain an MCM2-7 Cdc6-DNA complex via an interaction with Orc6 to form a complex competent for pre-RC formation, we expressed prereplication complex (pre-RC) (5). Cdc6 ATP hydrolysis is required for MCM2-7 loading, and Orc1 ATP hydrolysis pro- motes the release of the MCM2-7 complex from ORC to finish Author contributions: C.E., P.C., R.L., J.S., H.L., and C.S. designed research; C.E., P.C., R.L., J.S., the process of pre-RC formation and DNA licensing (7, 8). and C.S. performed research; C.E., J.Z., S.U., and C.S. contributed new reagents/analytic tools; C.E., P.C., J.Z., R.L., J.S., H.L., B.S., and C.S. analyzed data; and C.E., P.C., J.Z., R.L., J.S., Although only 2 MCM2-7 hexamers are needed to establish H.L., B.S., and C.S. wrote the paper. bidirectional replication forks at an origin, between 10 and 20 The authors declare no conflict of interest. MCM2-7 complexes are loaded onto each replication origin 1Present address: MBM ScienceBridge GmbH, Hans-Adolf-Krebs-Weg 1, 37077 Go¨ttingen, during G1 in animal cells (9). The additional loaded MCM2-7 Germany. complexes function to protect cells from replicative stress (10, 2To whom correspondence may be addressed. E-mail: [email protected] or christian. 11). Once the MCM2-7 complex is loaded onto DNA it becomes [email protected]. resistant to high salt, suggesting that it is physically linked to This article contains supporting information online at www.pnas.org/cgi/content/full/ DNA (12). During the G1/S transition the pre-RC is remodeled 0911500106/DCSupplemental. 20240–20245 ͉ PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0911500106 Downloaded by guest on September 30, 2021 A Co-expression of MCM2-7 in yeast and G1 arrest A HA-Mcm3 IP and HA peptide elution MCM2-7 / origin Superdex 200 gelfiltration Cdt1 + MCM2-7 ORC Cdc6 0.17 0.08 0.04 0.67 Concentration of MCM2-7 on Centricon (100K) 0.34 -Cdt1 pre-RC + high salt pre-RC + high -ORC -DNA pre-RC –Cdc6 load -MCM2-7 B C Mcm6 Mcm3 232 kD 440 kD 1338 kD 158 kD 669 kD void load Mcm2 Mcm4 1 2 3- 456789 10- 1112131415 Mcm7 Mcm5 S S 21 345678910 11 12 13 14 15 16 B γ γ C ATP ATP ATP ATP Fig. 1. MCM2-7 purification and characterization. (A) Strategy for the salt wash: + -- + expression and purification of MCM2-7. (B) Two micrograms of purified mut ARS1 mut ARS1 ARS1 ARS1 MCM2-7 was fractionated on a Superose 6 column. Arrows indicate fraction- salt wash - + - + ation of marker proteins (see Materials and Methods). Load (lane 1) and elution fractions (lanes 2–16) were separated by 7.5% SDS-PAGE and the proteins visualized by silver staining. MCM2-7 peaked at the 669-kDa marker (lane 11), which is consistent with their calculated mass of 605 kDa. (C) Purified MCM2-7 was separated on a 10% SDS-PAGE. 1234 MCM2-7 in yeast cells arrested in G1 phase when B-type 12 34 cyclin-dependent kinases were inactive (Fig. 1A). We used an BIOCHEMISTRY HA-tagged Mcm3 subunit to enrich for a population of MCM2-7 Fig. 2. In vitro reconstitution of pre-RC formation. (A) Loading of MCM2-7 devoid of MCM467, a well-known contaminating subcomplex is dependent on ORC, Cdc6, Cdt1, and DNA. Pre-RC assembly was performed with 6 nM pUC19ARS1, 40 nM ORC, 80 nM Cdc6, 40 nM Cdt1, and 40 nM that has a similar size as MCM2-7 and has helicase activity (23). MCM2-7. Lanes 1–3: 10% load of each protein used in the reaction. Lanes 4–8: The HA affinity-purified material was then applied to a gel- 1 component was absent from the reaction: DNA, ORC, Cdc6, Cdt1, or filtration column to remove the smaller MCM35 subcomplex. MCM2-7. Complete pre-RC reaction (lane 9) and salt-extracted pre-RC reaction The final MCM2-7 preparation was analyzed on a gel-filtration (lane 10). Lanes 11–15: quantification of the number of hexameric MCM2-7 column and eluted in the same fraction as the 669-kDa marker per origin DNA. (B) In vitro pre-RC formation is ATP dependent. The reactions protein (Fig. 1B). This fractionation result is consistent with the were carried out as in A with ATP (lanes 1 and 2) or with the nonhydrolyzable ␥ calculated molecular mass of 605 kDa for MCM2-7.
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