M5 gelred plus

Nick-closure assays were carried out as described previously (Zhang et al., 2017 (link)). Briefly, singly-nicked plasmid pBR322 was prepared by digestion with the nicking enzyme Nb. Bpu10I (Fermentas, United States) and purified. The nicked plasmid (0.3 μg) was incubated for 10 min at 25°C with various amounts of wild-type or mutant Cren7 in T4 ligase reaction buffer. After ligation with T4 DNA ligase (2 U; TransGen Biotech, China) for 2 min at 25°C, reaction was terminated with 50 mM EDTA and 0.5% SDS, and samples were deproteinized by treatment with protease K (2 mg/ml) for 2 h at 55°C. 20 μl of each sample (total volume of 30 μl) was then analyzed by electrophoresis in 1% agarose gel. The gels were imaged using BIORAD GELDOC2000 system after staining with M5 Gelred Plus (Mei5 Biotechnology, China).

Pulldown assays were conducted as described previously (Zhang et al., 2020 (link)). Briefly, 0.15 mg DNA-cellulose (Sigma-Aldrich), containing ∼750 ng dsDNA, was incubated for 30 min at room temperature with indicated amount of wild-type or mutant Cren7 and 500 ng pBR322 DNA in buffer A (10 mM HEPES, pH 7.6, 100 mM KCl, 2.5 mM MgCl₂ and 0.1 mg/mL BSA). After washing with buffer A (500 μl), the bound pBR322 DNA was eluted in buffer B (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 200 mM NaCl and 0.2% SDS), deproteinized by treatment at 55°C for 1 h with protease K (1 mg/ml) and resolved by 1% agarose gel electrophoresis. Gels were stained with M5 Gelred Plus (Mei5 Biotechnology, China), and imaged using a Gel imager (BIORAD GELDOC2000).