Figure 2.

Optimization of CMG Ubiquitylation in Yeast Cell Extracts

(A) TAP-SLD5 cells (YSS47) were synchronized in early S phase and used to prepare cell extracts as in Figure 1A. Recombinant ubiquitin was added at the indicated concentrations, before digestion of chromosomal DNA and isolation of the CMG helicase by immunoprecipitation of the TAP-tagged Sld5 subunit of GINS. The indicated proteins were then monitored by immunoblotting.

(B) In a similar experiment, wild-type ubiquitin (Ub) or the deubiquitylase inhibitor propargylated ubiquitin (Ub-Prg) were added to cell extracts at the indicated concentrations.

(C) In a similar experiment, 50 μM wild-type ubiquitin, 50 μM FLAG-tagged ubiquitin, and 3 mM ATP were added as indicated to an extract of S phase cells, together with 5 μM Ubi-Prg.

See also Figures S1 and S2.