In vitro gel shift assays

Recombinant Yan^A86D and Yan^V105R (amino acids 1–499) were prepared by TEV protease cleavage of GST fusion proteins purified from the BL21 codon plus Escherichia coli cells (see the Supplemental Material for details). Yan protein in the eluate was assessed for quality and concentration by SDS-PAGE followed by ImageJ quantification of the intensity of Yan products. Eluates were stored for up to 5 d at 4°C.

Double-stranded probes were end-labeled with T4 polynucleotide kinase (New England Biolabs) and γ³²PATP (Perkin Elmer) for 1 h at 37°C prior to annealing. All oligonucleotide sequences are listed in the Supplemental Material.

For competition assays, ∼30 pmol of Yan^A86D was incubated with 50× cold probe for 20 min on ice in 25-µL reactions (0.5 μg of poly-dI-dC, 5 mg of BSA, 25% glycerol in 13 mM HEPES at pH 7.9, 40 mM KCl, 0.7 mM EDTA, 0.3 mM DTT, 1 pmol of labeled probe). Cold competitor (100×) was added, and reactions were incubated an additional 20 min on ice. Cooperative binding was assessed by incubating equimolar amounts of Yan^V105R and Yan^A86D for 20 min on ice in the presence of 1 pmol of labeled probe. Samples were resolved on 10% polyacrylamide gels (competition assay) or on 4%–20% gradient gels (cooperative binding) to capture both bound and free probe run in 0.5× Tris-borate-EDTA (TBE) buffer for 45 min at 120 V.