Radiolabeled DNA-protein Binding Assay

DNA fragments (see S3 Table) containing oriC_Mtb [33 (link)] and rrnAPL [19 (link)] were amplified by PCR and products were gel-purified using QIAquick column (Qiagen). 250 ng of gel-purified dsDNA or 3x FLAG ssDNA oligo (see S3 Table) were labeled with T4 polynucleotide kinase (NEB) and [γ-³²P]-ATP, and unincorporated [γ-³²P]-ATP was removed using Illustra ProbeQuant G-50 microcolumns (GE Healthcare). 20,000 cpm of labeled probe, 10 μg BSA, and the indicated amounts of DciA_Mtb or DciA_Mtb^W113A were mixed with buffer (50mM Tris-HCl pH7, 150mM NaCl, 1mM EDTA, 1mM DTT) in a total volume of 12μl and incubated for 20 minutes at room temperature. Samples underwent native electrophoresis in 4–20% nondenaturing TBE polyacrylamide gels (Invitrogen). The gels were dried and exposed to film for detection by autoradiography.