Hel1, Hel2, Pep5 and Snt2 can efficiently ubiquitylate histone H4 <i>in vitro</i> in a reaction that is stimulated by Rad53.

<p>Recombinant and purified components were used to reconstitute the ubiquitylation of histone H4 <i>in vitro</i> exactly as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036295#pone.0036295-Singh2" target="_blank">[7]</a>. The reaction products were resolved on an 18% polyacrylamide gel and processed for Western blotting with H4 antibodies. Addition of commercially available recombinant yeast Uba1 (E1), human UbcH5A (E2; homolog of yeast Ubc4) and HIS6-tagged ubiquitin to recombinant human histone H4 with or without Rad53 did not result in appreciable histone modifications, apart from some monoubiquitylation (lanes 2 and 1 respectively). Only the addition of the E3 ligases Hel1 (H1), Hel2 (H2), Pep5 (P5) and Snt2 (S2) purified from yeast extracts via TAP epitope tags resulted in considerable high molecular weight histone modifications, which were further stimulated to varying degrees by the addition of recombinant Rad53 to the reaction mixture.