Krypton

Co-sedimentation assays were performed with LMM titrated by WYR in quintuplicate. Components were added to a final buffer of 215 mM NaF, 20 mM Na-P, 0.5 mM TCEP in a total volume of 60 µL. Twenty microliters were removed and labelled Pre-Spin (PS). Solutions were incubated overnight at 4 °C and then centrifuged at 15,000 rcf at 4 °C for 10 min. Twenty microliters of supernatant (S) were separated without disturbing the pellet. The remaining 20 µL of solution were included in the pellet (P) fraction and accounted for in the calculations, as described in the next section. Sedimentation of LMM with Insulin (5.8 kDa, 51 aa) was used as a control, performed in triplicate, to determine non-specific binding/sedimentation.
Samples were combined with 5 µL of 5× Sample Buffer (50% glycerol; 300 mM Tris HCl, pH 6.8; 10% SDS; 0.05% Bromo Blue, 125 mM DTT) and boiled for 10 min. The samples were loaded into 15 well 15% SDS PAGE minigels and run at 170 V. Gels were fixed in 40% Ethanol, 10% Acetic acid with one exchange at 20 min. They were rocked in this solution overnight. Krypton (Thermo Fisher) was used to stain the gels for 2.5 h and destained in accordance to Krypton protocol prior to viewing. Gels were viewed by Gel Doc (Bio-Rad) and the .tiff files were exported for analysis.

2 pmole ORC, 3 pmole Cdc6 and 6 pmole Mcm2-7/Cdt1 were sequentially added to the 40 μl reaction solution containing 1 pmole of bead-coupled 1.3 Kbps ARS1 DNA in helicase loading buffer (25 mM HEPES-KOH (pH7.6), 12.5 mM magnesium acetate, 0.1 mM zinc acetate, 300 mM potassium glutamate, 20 μM creatine phosphate, 0.02% NP40, 10% glycerol, 3 mM ATP, 1 mM dithiothreitol and 2 μg creatine kinase). The reaction mix was incubated at 25°C at 1200 rpm for 30 min in a thermomixer (Eppendorf). Beads were washed three times with Buffer H containing 0.3 M potassium glutamate and DNA bound proteins were eluted from the beads using DNase I. Eluted proteins were separated by SDS-PAGE and stained with a fluorescent protein stain (Krypton, Thermo Scientific). For high salt wash experiments, Buffer H containing 0.5 M NaCl was used at the second wash step. In ATPγS experiments, 6 mM ATPγS was used instead of ATP.