The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

Tan, Jinzhi; Vonrhein, Clemens; Smart, Oliver S.; Bricogne, Gerard; Bollati, Michela; Kusov, Yuri; Hansen, Guido; Mesters, Jeroen R.; Schmidt, Christian L.; Hilgenfeld, Rolf

The zone-interference gel electrophoresis (ZIGE) device was adapted from Abrahams et al. [77] . ZIGE assays were performed using a horizontal 1% agarose gel system in TBE buffer (20 mM Tris, 50 mM boric acid, 0.1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.3). The protein was incubated at room temperature for 30 min with different concentrations of oligodeoxynucleotides, such as (dG) 10 and bcl-2 promoter region (59-GGGCGCGGGAG-GAATTGGGCGGG-39), or oligoribonucleotides (59-UGGGGG-GAGGGAGGGAGGGA-39 and 59-UGGGGU-39). The samples were mixed with dimethylsulfoxide (DMSO; final concentration 10% (v/v)) and a trace of bromophenolblue (BPB). These proteinoligonucleotide samples were applied to the small slots. Oligonucleotide with the same concentration as in the small slots was also mixed with DMSO and BPB in 1xTBE buffer and applied to the long slots of the gel (total volume 100 ml). Electrophoresis was performed at 4uC for 1 h with a constant current of 100 mA. Staining was performed as outlined in [77] .