Nature research paper: MCM complexes are barriers that restrict cohesin-mediated loop extrusion
was incubated with SFP-Synthase and LD655-CoA at a 1:3:6 molar ratio for 2 h at 30 °C in buffer B + 100 mM KGlu, 10 mM MgCl. Labelled protein was further purified on a Superdex 200 increase 10/300 gel filtration column equilibrated in buffer B + 100 mM potassium acetate . Protein-containing fractions were pooled, concentrated with a MWCO 50000 Amicon Ultra Centrifugal Filter unit and stored in aliquots at −80 °C.
To produce the DNA substrate for single-molecule imaging, pMSuperCos-ARS1 was isolated from DH5α using a Plasmid Maxi Kit . One hundred micrograms of plasmid was digested with 100 U NotI-HF and XbaI for 7 h at 37 °C. The resulting 21,202 bp ARS1-DNA fragment was separated from the SuperCos1 backbone on a 10–40 % sucrose gradient. DNA handles were prepared by annealing oligonucleotides MS_200/201 MS202/203 in equimolar amounts in 30 mM HEPES, pH 7.
DNA was introduced to the flow cell at 5 pM in blocking buffer and incubated for 15 min in the absence of buffer flow to allow binding to the slide surface. To doubly tether DNA, the flow lane was flushed with 100 µM oligonucleotide MS_204 in blocking buffer at 100 µl per min.Helicase loading was achieved by introducing 0.25 nM ORC, 4 nM Cdc6 and 10 nM Cdt1–MCM
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