Figure 3

UV cross-linking experiments confirm SUMO-1 DNA interaction. (A) 100 ng of GST or GST-SUMO-1 were exposed in the absence or presence of 200 ng or 500 ng of dsDNA (69-mer) to UV (312 nm, 200 W) for 10 min. After 12.5% SDS-PAGE and western-blotting protein complexes were detected using a GST antibody and compared to non-exposed samples ('control'). (B) 100 ng of SUMO-1 were exposed in the absence or presence of 400 ng or 1 μg of dsDNA (27-mer) to UV (312 nm, 200 W) for 10 min. After 12.5% SDS-PAGE and western-blotting protein complexes were detected using a SUMO-1 antibody and compared to non-exposed samples ('control'). (C) The GST-SUMO-1 + DNA complex is sensitive to DNase treatment. Experiments as in A using 200 ng of dsDNA were post cross-linking and prior SDS-PAGE incubated with or without DNaseI (2 h, 37°C). (D) The high molecular weight bands formed by GST-SUMO-1 in the presence of DNA can also be shown to contain DNA using a fluorescent dsDNA. (E) Sequence independence of SUMO-1 DNA interaction is demonstrated using two different dsDNAs and detected after 10% SDS-PAGE.