Cloning, expression and purification
ORC, Cdc6, Mcm2–7 – Cdt1, DDK, CDK, Sld2, Sld3 – Sld7, Cdc45, Dpb11, Pol ε, Pol ε exo-, Pol α, TopoI, Mcm10 and yeast histone octamer were purified based on a pre-established yeast histone octamer. protocols1,11,24,33,48,49,50,51.
Cloning, expression and purification of Mcm2–7 – Cdt1 mutants
The designed DNA fragments (Supplementary Table 1) were subcloned from pMA vectors (Supplementary Table 2) to pRS shuttle vectors (Supplementary Table 2), which were used to generate yeast strains (Supplementary Table 3) used to overexpress mutants. Mcm2–7 – Cdt1. The oMG25 DNA fragment was subcloned from pMG39 to pAM38 using MluI and XbaI restriction sites to obtain pMG69, which was integrated into the yJF21 yeast strain, thus generating the yAE164 strain that was used to overexpress the Mcm2 6A mutant. (Mcm2 V580A / P584A / K584A). / K587A / W589A / K633A). The oMG27 DNA fragment was subcloned from pMG43 to pJF4 using BsiWI and SphI restriction sites to obtain pMG53, followed by integration of pMG53 into the yAM20 strain, resulting in the yAE160 strain, which was used for overexpression. of the Mcm6 2E mutant (Mcm6 T423E R424E). The oMG28 DNA fragment was subcloned from plasmid pMG44 to pJF4 by BsiWI and SphI restriction sites, thus obtaining plasmid pMG54. Plasmid pMG54 was integrated into the yAM20 strain, resulting in the yAE161 strain that was used to overexpress the Mcm6 5E mutant (Mcm6 T408E / Q409E / L410E / G411E / L412E). All Mcm2–7 – Cdt1 mutants were essentially purified as wild type50.
Cloning, expression and purification of GINS
A block of genes encoding a double-stranded tag and the first three codons of Psf3 were amplified and cloned into pFJD5 using unrestricted cloning techniques. A list of used primers and gene blocks is included in Supplementary Table 1. BL21 (DE3) -CodonPlus-RIL (Agilent) cells were transformed with GINS expression plasmid (pJL003). Transforming colonies were inoculated into a 250 ml LB culture containing kanamycin (50 µg ml-1) and chloramphenicol 35 µg ml-1), which was grown overnight at 37 ° C with stirring at 200 rpm. The next morning, the culture was diluted 100 times in 6 × 1 l of LB with kanamycin (100 µg ml-1) and chloramphenicol (35 µg ml-1). The cultures were allowed to grow at 37 ° C until an optical density at 600 nm (DO600 nm) of 0.5 was reached; 0.5 mM β-d-1-thiogalactopyranoside (IPTG) isopropyl was added to induce expression, and the cells were allowed to stir for 3 h. Cells were collected by centrifugation at 4,000 rpm for 20 min on a JS.4.2 rotor (Beckman). For lysis, the cell pellets were resuspended in 120 ml of lysis buffer (100 mM Tris-HCl pH 8.0, 10% glycerol, 0.02% NP-40, 1 mM EDTA, 200 mM NaCl, Roche protease inhibitor tablets and 1 mM dithio). ) + 0.7 mM phenylmethylsulfonyl fluoride (PMSF). The lysate was sonicated for 120 s (5 s on, 5 s off) at 40% in a Sonics Vibra-Cell sonicator. The insoluble material was removed by centrifugation at 20,000 rpm for 30 min. a JS.25.50 rotor (Beckman). The supernatant was loaded by gravity into a 1 ml Strep-TactinXT (IBA) column. The resin was washed extensively with wash buffer (100 mM Tris-HCl pH 8.0, 10% glycerol, 1 mM DTT and 1 mM EDTA). GINS were eluted by the addition of 6 ml of 1 × BXT buffer (IBA) supplemented with 10% glycerol and 1 mM DTT. Fractions containing GINS were pooled and dialyzed overnight in gel filtration buffer (25 mM HEPES-KOH pH 7.6, 10% glycerol, 0.02% NP-40, 200 mM potassium acetate and 1 mM DTT). The sample was concentrated and loaded onto a HiLoad 16/600 Super dex 200 balanced in the same buffer. Fractions containing GINS were pooled, aliquoted, and rapidly frozen in liquid N2. About 22 mg of GINS were purified from a 6 liter culture.
Cloning, expression and purification of MH
The codon-optimized expression sequence for MH containing an HRV 3C protease cleavage site followed by a double-stranded tag was synthesized and cloned into pET302 using GeneWiz Synthesis (pJL004). Express T7 (NEB) cells were transformed with pJL004. Transforming colonies were inoculated into a 250 ml LB culture with ampicillin (100 µg ml-1), which was grown overnight at 37 ° C with stirring at 200 rpm. The next morning, the culture was diluted 100 times in 6 × 1 l of LB with ampicillin (100 µg ml-1). The cultures were allowed to grow at 37 ° C until a DO600 nm of 0.5 was reached; 0.5 mM IPTG was added to induce expression and the cells were allowed to stir for 3 h. Cells were collected by centrifugation at 4,000 rpm for 20 min on a JS.4.2 rotor (Beckman). For lysis, the cell pellets were resuspended in 80 ml of lysis buffer (20 mM Tris-HCl pH 8.5, 10% glycerol 0.5 mM EDTA, 500 mM KCl, inhibitory tablets). Roche protease and 2 mM tris (2-carboxyethyl) phosphine (TCEP) + 0.7 mM PMSF. The lysate was sonicated for 120 s (5 s on, 5 s off) at 40% in a Sonics Vibra-Cell sonicator. The insoluble material was removed by centrifugation at 20,000 rpm for 30 min on a JS.25.50 rotor (Beckman). The supernatant was loaded by gravity into a 5 ml Strep-TactinXT (IBA) column. The resin was washed thoroughly with lysis buffer. MH was eluted by the addition of 12 ml of 1 × BXT (IBA) supplemented with 10% glycerol and 1 mM DTT. The MH-containing fractions were pooled and loaded onto a HiLoad 16/600 Superdex 75 equilibrated in gel filtration buffer (20 mM Tris-HCl pH 8.5, 10% glycerol 0.5 mM EDTA, 100 mM KCl and 0.5 mM TCEP). Fractions containing MH were pooled, aliquoted, and quickly frozen in liquid N2. About 36 mg of MH was purified from a 6-liter culture.
DNA templates
The native ARs1 The origin of replication flanked by the Widom 601 and 603 sites or flanked by MH was amplified by PCR and purified as described above24. The 6 × ARs1 the array (pSSH005) was mounted by inserting an array of 6 ARs1 origins with a 40 bp space flanked by MH sites using the NEBuilder HiFi mount. The 6 × ARs1 The source matrix was amplified from pSSH005 by the first oSSH038 and concentrated by ethanol precipitation. A list of primers and DNA used is included in Supplementary Table 1.
Preparation and purification of DNA of chromatinized origin
Soluble yeast nucleosomes were reconstituted from octamers and DNA by salt gradient dialysis in several steps of 2 to 0.2 M NaCl as described above24. Following nucleosome folding, a final dialysis step was performed on the loading buffer (25 mM HEPES-KOH pH 7.6, 80 mM KCl, 100 mM sodium acetate, 0.5 mM TCEP) and loaded into a Superose 6 Increase 3.2 / 300 in the same balanced column. tampon. Fractions they contain ARs1 DNA from 2 nucleosomes bound together was collected, concentrated, and stored at 4 ° C. Reconstitution conditions were optimized by small-scale titration and nucleosomes were tested with 6% native PAGE.
Preparation and purification of MH-capped DNA
168 bp short origins flanked by MH
MH conjugation with source substrates was performed in 50 mM Tris-HCl pH 8.0, 1 mM EDTA and 0.5 mM 2-mercaptoethanol supplemented with 100 µM S-adenosylmethionine (NEB). The reaction was carried out overnight at 30 ° C, with a molar ratio of MH: DNA of 10: 1. After conjugation, the reactions were centrifuged at 14,680 rpm for 5 min and loaded onto a 1 ml RESOURCE-Q column equilibrated in DNA buffer (50 mM Tris-HCl pH 8, 0 and 5 mM 2). -mercaptoethanol). MH-conjugated DNA was eluted in a linear gradient of B-DNA buffer (50 mM Tris-HCl pH 8.0, 5 mM 2-mercaptoethanol, and 2 M NaCl) in 24 column volumes. Fractions containing MH-conjugated DNA were pooled, concentrated, and stored at -80 ° C. The conjugations were checked with 6% native PAGE.
Matrix flanked by 6 × ARS1 MH
MH conjugation with source substrates was performed in 25 mM Tris-HCl pH 7.5, 10 mM magnesium acetate, 50 mM potassium acetate and 1 mg ml-1 BSA supplemented with 150 µM S-adenosylmethionine (NEB). The reaction was carried out at 32 ° C for 1 h and then overnight at 4 ° C, with a 20: 1 molar ratio of MH: DNA. After conjugation, the reactions were centrifuged at 14,680 rpm for 5 min and loaded onto a Superose 6 Increase 10/300 column equilibrated in matrix buffer (25 mM HEPES-KOH pH 7.5, 200 mM NaCl and 1 mM DTT). Fractions containing MH-conjugated matrix DNA were pooled, concentrated, and stored at 4 ° C. The conjugations were checked with 6% native PAGE.
616 bp ARs1 circles
The 616 bp ARs1 the circles were assembled and prepared as described above1 with the following modifications. The dephosphorylation step was performed using quickCIP, instead of Antarctic phosphatase, for 30 min at 37 ° C followed by inactivation of the enzyme at 80 ° C for 2 min. After the binding step, the DNA was concentrated as described and incubated with T5 exonuclease (NEB; 37 ° C for 1 h) to remove unbound DNA. Ethanol precipitation, agarose electrophoresis, and electroelution were omitted; instead, phenol / chloroform / isoamyl alcohol was extracted, followed by ethanol precipitation using sodium acetate (pH 5.1) and the neutral carrier GeneElute Linear Polymer (LPA, MERCK).
In vitro CMG assembly on chromatized short sources
ARs1 DNA of flanked nucleosome origin (20 nM) was incubated with 52 nM ORC, 52 nM Cdc6 and 110 nM Mcm2–7 – Cdt1 for 30 min at 24 ° C in loading buffer (25 mM HEPES-KOH pH 7.6, 100 mM potassium glutamate, 10 mM magnesium acetate, 0.02% NP-40 and 0.5 mM TCEP) + 5 mM ATP. The reaction was supplemented with 80 nM DDK and incubation was continued for a further 10 minutes at 24 ° C. 1 × for 30 min at 24 ° C. The beads were washed three times with 100 µl of wash buffer (HEPES-KOH 25 mM pH 7.6, potassium glutamate 105 mM, magnesium acetate 5 mM, …