Figure 2.

The Rtt107-Slx4 interaction is direct and is independent of Slx1, DNA damage, and DNA damage checkpoints. (A and B) Logarithmically growing cultures were treated with 0 or 0.03% MMS for 1 h. Extracts from yeast strains expressing the indicated epitope-tagged proteins were immunoprecipitated with IgG agarose. The immunoprecipitate was fractionated by SDS-PAGE, and immunoblots were probed with α-VSV to detect Rtt107-VSV and peroxidase-anti-peroxidase to detect Slx4-TAP. (C) Recombinant Rtt107, Slx1, and His6-Slx4 were coexpressed in E. coli, and the His6-Slx4 and associated proteins were purified and resolved on an SDS-polyacrylamide gel. The gel was stained with silver. Note that Rtt107 and His6-Slx4 have very similar mobility in this gel system. (D) Recombinant Slx4 and His6-RTT107-FLAG were coexpressed in E. coli and the His6-Rtt107-FLAG was purified. Purified proteins were fractionated on SDS-PAGE and stained with Coomassie blue. Note the slower mobility of the His6-Rtt107-FLAG compared with His6-Rtt107 and the faster mobility of Slx4 compared with His6-Slx4. (E) Truncated and full-length Rtt107-TAP were coexpressed with Slx4-FLAG. Rtt107-TAP and associated proteins were immunoprecipitated with rabbit IgG. Immunoprecipitates were fractionated by SDS-PAGE, and proteins were detected by probing the immunoblot with α-FLAG to detect Slx4-FLAG or rabbit IgG to detect Rtt107-TAP, Rtt107(1-511)-TAP, and Rtt107(512-1070)-TAP. The vector lane is a control strain in which no Rtt107-TAP is present.