Figure 7: Intrinsic E3 activity and nucleosome recognition each contribute to the rate of Histone H2A ubiquitination.

(a) Nucleosome ubiquitination catalysed by PCGF4 and PCGF5 at the indicated time points. (b) Identities and positions of residues important for nucleosome ubiquitination. RING1B K97 and R98, as well as PCGF4 K62 and R64 (shown at top) were shown previously to be functionally important³⁷. The residues in PCGF5 at positions equivalent to K62 and R64 are E62 and N64. There is an additional charged residue E67 that contributes to the acidic surface patch of PCGF5. (c,d) Mutants of PCGF4 and PCGF5 evaluated in a quantitative nucleosome ubiquitination assay. Incorporation of Alexa Fluor 488-tagged ubiquitin into H2A K119ub1 was quantified as described in Methods. Reactions were run in three independent experiments and data presented as mean value±s.e.m. (c) Wild-type complex (P4) is compared with the most active chimeric mutant shown in Fig. 5 (P4 SM), a mutant exchanging basic patch residues for those residues present in PCGF5 (P4 BP≡PCGF4 K62E/R64N), and a combination of P4 SM and P4 BP (P4 SM+BP). For the fastest reaction (P4 SM), product disappears at later time points because of conversion to a second product that appears to be H2Aub2 (see Supplementary Fig. 9a). (d) H2A K119ub1 formation catalysed by PCGF5 mutant complexes analogous to the PCGF4 variants shown in c. In the case of the P5 BP mutant, the flanking acidic residue E67 was also changed to the corresponding PCGF4 amino acid (E62K/N64R/E67L) to fully switch the charged patch from acidic to basic.