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. 2009 Dec 1;106(48):20240-5.
doi: 10.1073/pnas.0911500106. Epub 2009 Nov 12.

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

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Free PMC article

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

Cecile Evrin et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
MCM2-7 purification and characterization. (A) Strategy for the expression and purification of MCM2-7. (B) Two micrograms of purified MCM2-7 was fractionated on a Superose 6 column. Arrows indicate fractionation 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.
Fig. 2.
Fig. 2.
In vitro reconstitution of pre-RC formation. (A) Loading of MCM2-7 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 MCM2-7. Lanes 1–3: 10% load of each protein used in the reaction. Lanes 4–8: 1 component was absent from the reaction: DNA, ORC, Cdc6, Cdt1, or MCM2-7. Complete pre-RC reaction (lane 9) and salt-extracted pre-RC reaction (lane 10). Lanes 11–15: quantification of the number of hexameric MCM2-7 per origin DNA. (B) In vitro pre-RC formation is ATP dependent. The reactions were carried out as in A with ATP (lanes 1 and 2) or with the nonhydrolyzable ATP analogue ATPγS (lanes 3 and 4). (C) In vitro pre-RC formation is sequence specific. The reactions were carried out as in A with a WT ARS1 (lanes 1 and 2) or with mutant DNA (A- B2- B3-) (lanes 3 and 4).
Fig. 3.
Fig. 3.
MCM2-7 can slide off DNA ends. The reactions were carried out as in Fig. 2A, but 4 different DNA variants were used. As indicated in the figure, linear ARS1 fragments with either one end (lanes 1, 2, 5, and 6) or both ends (lanes 3 and 7) blocked with streptavidin and a circular plasmid pUC19ARS1 (lanes 4 and 8) were used. Salt washes are indicated. MCM2-7 was only retained on linear DNA when both ends were blocked.
Fig. 4.
Fig. 4.
Gel filtration analysis of the oligomerization state of MCM2-7 before and after loading onto DNA. (A) MCM2-7 is a single hexamer in solution. MCM2-7 was incubated with DNA and treated with DNase I (see Materials and Methods). The sample was separated on a Superose 6 column. Fractions (lanes 1–14) were separated by 7.5% SDS-PAGE and analyzed by Western blotting using an anti-Mcm2 antibody. (B) MCM2-7 forms a double hexamer in the pre-RC. Reactions were prepared as described in Fig. 2A (lane 10). DNase I mediated release of the protein–DNA complexes, and analyses were done as in A. (C) Calibration of a Superose 6 column. The marker proteins used are described in Materials and Methods. The x axis was plotted as the log of the molecular weight in kDa and the y axis as the Kav value [Kav = (Ve − Vo)/(Vt − Vo)] according to the manufacturer's instructions.
Fig. 5.
Fig. 5.
EM analysis of MCM2-7 before and after loading. (A) MCM2-7 before loading. MCM2-7 was diluted in ATPγS-containing buffer and visualized by negative staining with uranyl acetate. (B) Salt-washed MCM2-7 after loading. Samples were prepared as described in Materials and Methods. The protein–DNA complexes were stained as in Fig. 5A. (C) Salt-washed MCM2-7 after loading. Samples were prepared as described in Fig. 5B but visualized by rotary shadowing with platinum; dsDNA seems to pass through the center of a ring-shaped MCM2-7 complex.
Fig. 6.
Fig. 6.
Speculative model of MCM2-7 loading and activation. (A) During pre-RC formation ORC-Cdc6-DNA is joined by a Cdt1-MCM2-7 complex. The reaction is dependent on all pre-RC protein factors and ATP hydrolysis, but it is currently not known how double-hexameric MCM2-7 assembles at an origin. (B) During pre-IC formation double-hexameric MCM2-7 is activated by kinases and proteins; exact roles of each are unknown. Three models for potential helicase activity at the replication fork are shown. The model on the left suggests that 1 ssDNA strand passes inside the MCM2-7 hexamer to drive movement similar to the papillomavirus E1 helicase (38); the middle model predicts a separation of the MCM2-7 double hexamer with dsDNA through its middle and is commonly referred to as the ploughshare model (39). The model on the right is similar to a proposed SV40 T-antigen DNA unwinding model (28), whereby the double hexamer pumps dsDNA toward the center and ssDNA exits through pores within the middle of the complex.

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