A reconstituted system reveals how activating and inhibitory interactions control DDK dependent assembly of the eukaryotic replicative helicase

During G1-phase of the cell-cycle the replicative MCM2–7 helicase becomes loaded onto DNA into pre-replicative complexes (pre-RCs), resulting in MCM2–7 double-hexamers on DNA. In S-phase, Dbf4-dependent kinase (DDK) and cyclin-dependent-kinase (CDK) direct with the help of a large number of helicase-activation factors the assembly of a Cdc45–MCM2–7–GINS (CMG) complex. However, in the absence of S-phase kinases complex assembly is inhibited, which is unexpected, as the MCM2–7 double-hexamer represents a very large interaction surface. Currently it is unclear what mechanisms restricts complex assembly and how DDK can overcome this inhibition to promote CMG-assembly. We developed an advanced reconstituted-system to study helicase activation in-solution and discovered that individual factors like Sld3 and Sld2 can bind directly to the pre-RC, while Cdc45 cannot. When Sld3 and Sld2 were incubated together with the pre-RC, we observed that competitive interactions restrict complex assembly. DDK stabilizes the Sld3/Sld2–pre-RC complex, but the complex is only short-lived, indicating an anti-cooperative mechanism. Yet, a Sld3/Cdc45–pre-RC can form in the presence of DDK and the addition of Sld2 enhances complex stability. Our results indicate that helicase activation is regulated by competitive and cooperative interactions, which restrict illegitimate complex formation and direct limiting helicase-activation factors into pre-initiation complexes.


A RECONSTITUTED SYSTEM REVEALS HOW ACTIVATING AND INHIBITORY INTERACTIONS CONTROL DDK DEPENDENT ASSEMBLY OF THE EUKARYOTIC REPLICATIVE HELICASE
M. Carmen Herrera, Silvia Tognetti, Alberto Riera, Juergen Zech, Pippa Clarke, Alejandra Fernández-Cid and Christian Speck

Oligonucleotides, plasmids and antibodies
Oligonucleotides, plasmids and antibodies used in this study are listed in Table S1, S2 and S3, respectively.

Cloning
The coding sequence of Sld2 and Dpb11 were amplified by PCR with primers (CS1144, CS1145 and CS1260, CS1261, respectively) that added NotI and EcoRI restriction sites. The PCR products were cloned into pGEX 6P1 vector.
The coding sequence of Sld7 was amplified by PCR with primers CS1653 and CS1654, adding NotI and BamHI restriction sites and cloned into pGEX-6P1 (GE Healthcare).
The coding sequence of Sld3 was amplified by PCR with primers CS785 and CS818, adding BamHI and SalI restriction sites and cloned into pESC-HIS (Agilent Technologies). This construct was then modified by insertion of an N-terminal MBP tag, amplified from pMALC2x with primes CS921 and CS922. The primers also coded for a PreScission protease site and a myc tag and BamHI restriction sites.
For yeast expression, the coding sequence of Cdc45 (missing the stop codon) was amplified by PCR with primers CS561 and CS572, adding NotI and SpeI restriction sites and cloned in pESC-HIS, pESC-LEU, pESC-TRP and pESC-URA vectors (Agilent Technologies). The stop codon was deleted in order to have a C-terminal FLAG tag.
The coding sequence of Cdc45 was amplified by PCR with primers CS591 and CS592 adding BamHI and NotI restriction sites and cloned in pGEX 6P1 vector (GE Healthcare) for bacterial expression.
MBP-Prescission-Dbf4 and Cdc7 were cloned into pESC-TRP (Stratagene). The dbf4 gene was amplified from genomic DNA of the S. cerevisiae strain S288C with the primers CS1052 and CS1053 via SmaI/NheI. The MBP-PreScission gene was amplified from pCS245 with primers CS921 and CS922 and cloned in before the dbf4 gene using a BamHI site. The cdc7 gene was amplified from genomic DNA of the S. cerevisiae strain S288C with the primers CS1058 and CS1059 and cloned via NotI/SpeI the vector. The resulting plasmid is pCS313.

Bacterial protein expression
The respective plasmids were transformed in BL21 Codon Plus RIL E. coli competent cells (Agilent). The cells were grown in Terrific Broth supplemented with appropriate antibiotics at 37°C to an OD600 of 1. Protein expression was then induced by addition of 0.5 mM IPTG for 5h at 16°C.

Yeast protein expression
Yeast strain AS499 (MATa. bar1Δ,pep4) was transformed. The yeasts were grown overnight in selective medium at 30°C. The proteins were expressed as described previously (3).

Sld3 purification
Cells were lysed in a freezer mill.

Sld2 purification
The cells were lysed by sonication in buffer A [50 mM PIPES (pH 6.5), 500 mM AS, 10 mM MgCl2, 5 mM DTT, and 1% Triton, 10% Glycerol, 2 mM ATP]. The fusion protein was bound to glutathione agarose (Sigma) in buffer A at 4 °C for 2 h, and then the protein was eluted by addition of PreScission Protease (GE Healthcare) for 2h at 4 °C. The eluate was diluted with 1 volume buffer A with no salt and bound to SP Sepharose (GE Healthcare). Sld2 was eluted with buffer B [50 mM PIPES (pH 6.5), 500 mM AS, 5 mM DTT, and 0.1% Triton, 10% Glycerol].

Cdc45 purification
The protein was purified as previously described (4). To remove the FLAG peptide the sample was incubated for 30 min with DEAE beads (GE Healthcare), the resin was extensively washed and the protein was eluted with purification buffer containing 500 mM KCl.

Sld7 purification
The cells were lysed by sonication in buffer C [50 mM HEPES-KOH (pH 7.5), 300 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 1% Triton, 10% Glycerol, 2 mM ATP]. Ammonium sulphate was added to a final concentration of 250 mM to the extract. Nucleic acids were precipitated from the cell extract by the addition of 30 µl 10 % polymin P/HCl (pH 6.5) per ml of extract. GST-Sld7 was precipitated by adding 0.4 g fine powdered ammonium sulphate per ml of extract. The fusion protein was bound to glutathione agarose (Sigma) in buffer C at 4 °C for 2 h, and then Sld7 was eluted by addition of PreScission Protease (GE Healthcare) for 2h at 4 °C. The eluate was concentrated using a centricon concentrator (Millipore).

DDK purification
Cells were lysed in a freezer mill. The resulting extract was incubated with amylose resin (NEB) pre-equilibrated with buffer DK [50 mM Hepes-KOH pH 7.5, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 0.02% NP40, 1 mM DTT] and supplemented with proteases and phosphatases inhibitors for 2 h at 4°C. The protein was eluted by cleaving the MBP tag via addition of PreScission protease for 2 h at 4°C. The eluate was incubated with Glutathione agarose for 2 h to remove the PreScission protease. The GST flow-through was collected and concentrated by centricon (Millipore).

Dpb11 purification
The cells were lysed by sonication in buffer D [50 mM PIPES (pH 6.5), 100 mM NaCl, 3 mM DTT, and 1% Triton, 10% Glycerol]. Ammonium sulphate was added to a final concentration of 250 mM to the extract. Nucleic acids were precipitated from the cell extract by the addition of 45 µl 10 % polymin P/HCl (pH 6.5) per ml cell extract. Protein from the supernatant was precipitated by adding 0.35 g fine powdered ammonium sulphate per ml of extract. The fusion protein was bound to glutathione agarose (Sigma) in buffer D at 4 °C for 2 h, and then Dpb11 was eluted by addition of PreScission Protease (GE Healthcare). The eluate was diluted with 1 volume buffer D with no salt and bound to SP Sepharose (GE Healthcare). Dpb11 was eluted with buffer E [50 mM PIPES (pH 6.5), 500 mM NaCl, 5 mM DTT, 10 mM MgCl2, 0.1% Triton, 10% Glycerol].

Competitor DNA
Two complementary oligonucleotides (CS1126 and CS 1127, table S1) were incubated in buffer M [100 mM Tris-HCl (pH 7.9), 100 mM NaCl, 1 mM DTT, 10 mM MgCl2] for 5 min at 95 °C and then slowly cooled down to RT over 30 min. The hybridized oligos were concentrated using Microcon (Millipore) to a concentration of 5 µM.

In vitro transcription and translation
To generate 35 S-Met-labelled proteins, the coding sequences of the genes were amplified using a sense primer and anti-sense primers, as suggested by the supplier's instruction (Promega). To compensate for different methionine content additional methionines were incorporated into the anti-sense primer. 400 ng of the resulting PCR product was used as template in the TNT-T7 Quick for PCR DNA system (Promega), combined with 20 μl of TNT Quick Master Mix and 20 μCi of 35 S-Met (Perkin-Elmer) in a total volume of 25 μl and incubated for 90 min at 30°C with mixing.
Immobilisation (Sld3): Sld3 (150 ng) was immobilized on anti-Sld3 318 beads (protein G) for 15 min at 24°C with mixing in 50 μl of Sld3 binding buffer followed by three washes and used for pull downs, together with IgG control beads.