FIGURE 3.

H2Aub does not affect Dot1L MTase activity on mononucleosomes. A, surface rendition of mononucleosome structure shown in axial and radial views (Protein Data Bank code 1KX5). DNA is colored black, and histones H3, H4, H2A, and H2B are colored green, lavender, yellow, and orange, respectively. The Dot1 methylation site (H3K79) is colored dark blue. Positions that when ubiquitylated strongly stimulate Dot1L activity are colored red. Located at the opposite side of the nucleosome axial face from H2BK120, H2AK119 is colored brown. B, DTT titration of Dot1L MTase activity on unmodified mononucleosomes (unmod nucl) as quantitated by scintillation counting of incorporated ³H. C, Dot1L MTase activity was tested on human unmodified and ssUb mononucleosomes as indicated. A representative experiment is shown. Three-fourths of each reaction was separated by SDS-PAGE and Coomassie Blue-stained (upper panel), and activity was assessed by fluorography (middle panel). Activity was also assessed by scintillation counting of incorporated ³H in one-fourth of each reaction (lower panel). Error bars indicate the S.D. of multiple scintillation counts of the same reaction.