Preparation of DNA Substrates for Biochemical Assays

The Φx174 viral (+) strand ssDNA and replicative form I dsDNA were purchased from New England BioLabs. Oligonucleotides were gel-purified as previously described (29 (link)). To prepare 5′-end-labeled ssDNA for Benzonase protection assays, 80-mer Oligo 1 was incubated with polynucleotide kinase (New England Biolabs) and [γ-P³²-ATP] (PerkinElmer) for 5′-end-labeling. The unincorporated nucleotide was removed using a Spin 6 column (Bio-Rad). For oligonucleotide-based DNA binding, the DNA substrates Oligos 2–4 and Oligos 5–10 were derived from previous researches (30 (link),31 (link)). The sequences of DNA substrates used in our DNA mobility shift and Benzonase protection assays are summarized in Supplementary Table S1, and the composition of annealed DNA substrates for oligonucleotide-based DNA binding is summarized in Supplementary Table S2. For tethering particle motion analysis, a gapped DNA substrate (151 bp dsDNA handle + (dT)135 nt ssDNA + 19 bp dsDNA handle) was prepared by annealing the PCR products as described in previous reports (32 (link),33 (link)).