Figure 5.

UV-mediated crosslinks between core-type DNA oligomer and the carboxy-terminal domains of Int. C65 was mixed with a 5′-³²P-labeled, 13-bp, core-type DNA fragment (300 pmols each) in 50 μl of buffer B (see Fig. 4) plus 10% glycerol. The mixture was incubated 10 min at room temperature and 20 min on ice and then layered on a Petri dish on ice where it was irradiated with UV light (254 nm) for 20 min. An aliquot of the UV-crosslinked complex was subjected to limited proteolysis with 100 ng of endoproteinase Arg-C (rat submaxillary gland, HPLC-purified), at 25°C for 1 h and analyzed by 15% SDS/PAGE (13) followed by autoradiography. (Left) Coomassie blue staining of the gel before and after proteolysis. Lanes A and B contain purified recombinant C65 and C170 proteins, respectively, as markers. (Right) The autoradiogram of the UV-crosslinked products before and after limited proteolysis with Arg-C.