Extended Data Fig. 5: The assembly of Rev1 into the Polζ holocomplex in yeast cells depends upon Rev1 BRCT.
Polζ complexes were purified by α-Flag affinity purification of Flag-tagged Rev3 from protein extracts of yeast cells overexpressing individual subunits of the Rev1-Full-Polζ holocomplex and analyzed by SDS-PAGE and Coomassie staining. The complexes were eluted with prescission protease, and free GST and prescission protease were subsequently removed by incubation with Glutathione Sepharose. Lane 1, Rev1-Polζ holocomplex purified from cells expressing wild type Rev3/Rev7/Pol31/Pol32/Rev1 subunits. Rev1 copurifies with the Polζ holoenzyme. Lane 2, Polζ complex from cells as in lane 1, but lacking overexpression of the Pol32 subunit. Rev1 copurifies with the Polζ complex, despite the lack of Pol32. Lane 3, Rev1-Polζ holocomplex from cells as in lane 1, but overexpressing the rev1-1 mutant protein which harbors the G193R mutation within the BRCT domain. The rev1-1 mutant does not copurify with the Polζ complex. Expression of the rev1-1 protein was confirmed by Western blotting of total protein extract using affinity purified α-Rev1 antibodies. The gel was run once. The Protein identities and molecular weight sizes (kDa) are shown on the right and left, respectively.
