Stabilisation of half MCM ring by Cdt1 during DNA insertion

Origin licensing ensures precise once per cell cycle replication in eukaryotic cells. The Origin Recognition Complex, Cdc6 and Cdt1 load Mcm2-7 helicase (MCM) into a double hexamer, bound around duplex DNA. The complex formed by ORC-Cdc6 bound to duplex DNA (OC) recruits the MCM-Cdt1 complex into the replication origins. Through the stacking of both complexes, the duplex DNA is inserted inside the helicase by an unknown mechanism. In this paper we show that the DNA insertion comes with a topological problem in the stacking of OC with MCM-Cdt1. Unless an essential, conserved C terminal winged helix domain (C-WHD) of Cdt1 is present, the MCM splits into two halves. The binding of this domain with the essential C-WHD of Mcm6, allows the latching between the MCM-Cdt1 and OC, through a conserved Orc5 AAA-lid interaction. Our work provides new insights into how DNA is inserted into the eukaryotic replicative helicase, through a series of synchronized events.

• GST-Cdc6. We followed the protocol described by Frigola et al., 2013 with minor changes. The preScission protease (GE Healthcare) was incubated overnight instead of 2h at 4ºC. Also, the peak fractions resulting from the hydroxyapatite elution were not concentrated using a centricon before being aliquoted.
• HIS-Mcm2. The pellet was resuspended in 80ml of buffer D+1mM PMSF, and 1mM lysozyme (hereafter, lysis buffer). Cells were kept on ice and disrupted using sonication (Labsonic). The sample was centrifuged at 10000rpm for 30min at 4ºC with a Thermo Scientific Fiberlite F13-14x50cy rotor. The soluble phase containing the his6-tagged Mcm2 was incubated on a 4ml His Trap FF beads (Generon) at 4ºC for 30min. Beads were washed with 20 column volumes (CVs) of buffer D+10mM imidazole. The protein was eluted with buffer D+200mM imidazole. His6-SUMO tag from Mcm2 was removed by digestion with 500ng of Ulp1 protease on ice for 15min. 5ml of untagged protein was subjected to fractionation over a HiLoad TM 16/600 SuperdexTM 200pg (GE Healthcare) pre-equilibrated in buffer D. Peak fractions were pooled, diluted 1:4 with buffer E, and concentrated over a 1ml MonoQ TM 5/50 GL column, using an elution gradient of 0.17-0.7M NaCl in buffer F, over 1CV. Peak fractions containing Mcm2 were aliquoted, snap-frozen in liquid nitrogen, and stored at -80ºC.
• HIS-Mcm3. Basically, like HIS-Mcm2 with minor changes. The lysis buffer also contained 2 tablets of protease inhibitor cocktail (Roche). Moreover, the protein was diluted 1:5 with buffer E before being applied to the MonoQ TM 5/50 GL column, and the elution was performed with a step from 0.14 to 0.7M NaCl in buffer F.
• HIS-Mcm4DC. The starting cultures were 1.5L in volume. To purify this protein, a similar protocol to the purification of HIS-Mcm2 was followed, except that before applying the protein to the MonoQ TM 5/50 GL column a dilution of 1:7 with buffer E was made. Also, the column was eluted with a liner 0.1-0.5M NaCl gradient in buffer F over 5CVs.
• HIS-Mcm4 C-WHD. The starting cultures were 0.75L in volume. The purification was performed as described for HIS-Mcm2 until the Ulp1 digestion, because the protein is smaller, and therefore, HIS tag and Ulp1 cannot be separated from the protein of interest by gel filtration. So, after the protease cleavage, a 1:10 dilution was made with buffer D to dilute the imidazole. Then, the protein was loaded onto a 5ml HisTrap TM FF crude (GE Healthcare) column pre-equilibrated in buffer D+10%glycerol. The flow-through containing the Mcm4 C-WHD was collected, snap-frozen in liquid nitrogen, and stored at -80ºC. To elute the retained HIS tag and the Ulp1 protease of the 5ml HisTrap TM FF crude column, buffer D+200mM imidazole was injected into the column.
• HIS-Mcm5. Fundamentally like HIS-Mcm2 with several changes. All the buffers used for this protein also contained 0.05% IGEPAL (substitute of NPGO). The pellet was resuspended with a lysis buffer containing 1M NaCl instead of 0.7M NaCl, and an additional 1mM DTT. Furthermore, after the His Trap FF elution, 1mM EDTA, 1mM EGTA, 1mM PMSF, and 1mM DTT was added to the protein. Plus, in this case, the MonoQ TM 5/50 GL elution was performed with a 0.15-0.7M NaCl gradient in buffer F over 8CVs.
• HIS-Mcm5DC. The starting cultures were 1.5L in volume. Like HIS-Mcm5 with some changes at the end of the process. After the Ulp1 digestion, the protein was fractionated onto a Superdex TM 200 Increase 10/300 GL pre-equilibrated in buffer D+10% glycerol, and no MonoQ column was performed. Peak fractions were aliquoted, snap-frozen in liquid nitrogen, and stored at -80ºC.
• HIS-Mcm6. Essentially like HIS-Mcm2 with some changes. All the buffers used for this protein also had 1mM ATP, 5mM Mg(OAc)2, and 1mM EDTA. The lysis buffer also contained 2 tablets of protease inhibitor cocktail. Additionally, after the His Trap FF beads elution, 1mM DTT, 1mM PMSF, and 1mM EGTA were added to the protein. Plus, before applying the protein to the MonoQ TM 5/50 GL column a dilution of 1:2.7 with buffer E was made. The column was eluted with a step from 0.25 to 0.7M NaCl in buffer F.
• HIS-Mcm6DC. The starting cultures were 1.5L in volume. Exactly like HIS-Mcm6 but the digestion was made with 3C protease instead of Ulp1.
• HIS-Mcm6 C-WHD and HIS-Mcm6 C-WHD 1-1. A similar protocol to the purification of HIS-Mcm4 C-WHD was followed, except that the induction was carried out 5h at 30ºC. • Mcm7. We generally followed the protocol described by Davey et al., 2003 but with some differences. The buffers used are the same as the original protocol. The starting cultures were 2.8L in volume. For cell lysis, the pellet was resuspended with 45ml with a final concentration of 1MNaCl, 2mM DTT, 1mM PMSF, 1mM lysozyme, 50mM Tris-HCl pH 7.6. Cells were lysed by sonication and centrifuged like HIS-Mcm2. The supernatant was treated with 0.3mg/ml ammonium sulfate. After centrifugation for 30 min at 10000rpm at 4ºC, the pellet was resuspended in buffer A containing 0.25mg/ml ammonium sulfate and then centrifuged as above. This step was repeated using buffer A containing 0.2mg/ml ammonium sulfate. The resulting pellet was resuspended in 12ml of buffer A and dialyzed against buffer A for 1h. The protein was loaded onto a 5ml HisTrap TM Q FF column (GE Healthcare) pre-equilibrated in buffer A+70mM NaCl. The column was eluted with 50ml, 70mM-500mM NaCl gradient in buffer A. Peak fractions containing the protein were dialyzed overnight against buffer B+90mM NaCl. Mcm7 was applied to a 2ml HiTrap TM Heparin HP column (GE Healthcare) pre-equilibrated in buffer B+90mM NaCl. The column was eluted with a 20ml, 90mM-500mM NaCl gradient in buffer B. Peak fractions containing the protein were pooled. A 1:3 dilution was made with buffer A. Then, the sample was injected into a 1ml MonoQ TM 5/50 GL column pre-equilibrated in buffer A+100mM NaCl. Bound proteins were eluted with a 3ml, 100mM-1M NaCl gradient in buffer A. Peak fractions were then injected to a 24ml Superdex TM 200 Increase 10/300 pre-equilibrated in 0.3M Kacetate, 25mM HEPES pH 7.6, and 10% glycerol buffer. Peak fractions containing Mcm7 were pooled, aliquoted, snap-frozen in liquid nitrogen, and stored at -80ºC.
• HIS-Mcm7DC1. The starting cultures were 1.5L in volume. Essentially like HIS-Mcm2 with several changes at the end of the process. After the Ulp1 protease digestion, the protein was fractionated on a Superdex TM 200 Increase 10/300 pre-equilibrated in buffer F, and no MonoQ column was performed. Then, peak fractions were pooled, aliquoted, snap-frozen in liquid nitrogen, and stored at -80ºC.
• HIS-Mcm7DC2. Essentially like HIS-Mcm7DC1. Except that after the Superdex TM 200 Increase 10/300 column, peak fractions were pooled, a 1:7 dilution with buffer E was made, and the protein was concentrated onto a MonoQ TM 5/50 GL column. The MonoQ was eluted with a 0.1-0.5M NaCl linear gradient in buffer F over 20CVs. Finally, Mcm7DC2 was aliquoted, snap-frozen in liquid nitrogen, and stored at -80ºC.
• HIS-Cdt1 Flag. Like HIS-Mcm2 at the beginning of the process with some changes at the end. After the Superdex200 16/600 column, peak fractions were injected to a Superdex TM 200 Increase 10/300 column pre-equilibrated in buffer G. Peak fractions were pooled and concentrated with a 10K MWCO centricon. Protein was finally aliquoted, snap-frozen in liquid nitrogen, and stored at -80ºC.
• HIS-Cdt1 S272, HIS-Cdt1 S272 1-1, HIS-Cdt1 1-1, and HIS-Cdt1 2-1. The starting cultures were 0.75L in volume for the Cdt1 S272 and S272 1-1, and 1.5L for the other two proteins. Essentially like the HIS-Mcm2 purification protocol, but with several changes at the end of the process. After the Ulp1 protease digestion, the protein was applied to a Superdex TM 200 Increase 10/300 pre-equilibrated in buffer D. Then, fractions containing the protein were pooled and injected to a second Superdex TM 200 Increase 10/300 pre-equilibrated in buffer G. Peak fractions of the second gel filtration column were aliquoted, snap-frozen in liquid nitrogen, and stored at -80ºC.

Formation of Mcm2-7+Cdt1 complexes from individually purified subunits.
We followed the protocol described by Frigola et al., 2013. The only change was that the gel filtration buffer also contained 5mM Mg(OAc)2.