Amersham typhoon

To prepare the substrate, the oligonucleotide (5'-(dT)₄₀GTTTTCCCAGTCACGACG-TTGTAAAACGACGGCCAGTGCC-3') containing a 40 nt region complementary to the M13mp18(+) strand and a 40 nt oligo-dT at the 5′ end was labeled at the 3′ terminus with [α-³²P] dCTP (Perkin Elmer) and annealed to the single-stranded DNA M13mp18 (Huang et al., 2020 (link)). 0.1 nM (in molecules) DNA substrates were mixed with 5 µg recombinant MCM8/9 complex as well as its mutants as indicated within each 15 µl volume reaction in the helicase buffer (25 mM HEPES, pH 7.5, 1 mM magnesium acetate, 25 mM sodium acetate, pH 5.2, 4 mM ATP, 0.1 mg/ml BSA, 1 mM DTT). 2.5 µg HROB was used as an activator. To avoid re-annealing, the reaction was supplemented with a 100-fold unlabeled oligonucleotide. The reactions were then incubated at 37 °C for 60 min and stopped by adding 1 µl of stop buffer (0.4% SDS, 30 mM EDTA, and 6% glycerol) and 1 µl of proteinase K (20 mg/ml, Sigma) into the reaction for another 10 min incubation at 37 °C. The products were separated by 15% polyacrylamide gel electrophoresis in 1x TBE buffer and analyzed by the Amersham typhoon (Cytiva).

Each cyclization reaction contained the following components: 1 μL 10X T4 DNA ligase reaction buffer (New England BioLabs), 2 μL water, 1 μL 10X ligation buffer additives (400 μg/mL UltraPure BSA, 100 mM KCl, 0.1% NP-40), 2 μL 80 μM Cas9 (or protein-purification size exclusion buffer), 2 μL 100 μM sgRNA (or RNA storage buffer), 1 μL 25 nM cyclization substrate, 1 μL T4 DNA ligase (400,000 units/mL, New England BioLabs) (or ligase storage buffer). All reaction components were incubated together at 20°C for 15 minutes prior to reaction initiation except for the ligase, which was incubated separately. Reactions were initiated by combining the ligase with the remainder of the components, allowed to proceed at 20°C for 30 minutes, then quenched with 2.5 μL 5X native quench solution. Samples were then incubated at 55°C for 15 minutes, resolved on an analytical native PAGE gel (8% acrylamide:bis-acrylamide 37.5:1, 0.5X TBE) at 4°C, and imaged for fluorescein on an Amersham Typhoon (Cytiva). Monomolecular cyclization efficiency (MCE) for a given lane is defined as (band volume of circular monomers)/(sum of all band volumes). Bimolecular ligation efficiency (BLE) is defined as (sum of band volumes of all linear/circular n-mers, for n≥2)/(sum of all band volumes). The non-specific degradation products indicated in Extended Data Fig. 6a were not included in the analysis.