Ubiquitin vinyl sulfone

Preparation of Xenopus egg extracts was performed as described previously (Lebofsky et al., 2009 (link)). For DNA replication, plasmids were first incubated in a high-speed supernatant (HSS) of egg cytoplasm (final concentration 7.5 ng DNA/μL extract) for 20 minutes at 21°C, leading to the formation of pre-replication complexes (pre-RCs). Next, two volumes of nucleoplasmic egg extract (NPE) was added to one volume of HSS, initiating Cdk2-dependent replication at pre-RCs. For all figures, the 0 minute time point corresponds to NPE addition. For DNA labeling, reactions were supplemented with [α-³²P]dATP, which is incorporated into nascent strands during replication. For UbVS reactions, NPE was supplemented with 14 μM ubiquitin vinyl sulfone alone, or with 50 μM ubiquitin (both from Boston Biochem, Cambridge, MA, USA) prior to mixing with HSS. Reactions were stopped with 10 volumes Stop Solution A (0.5% SDS, 25 mM EDTA, 50 mM Tris-HCl pH 7.5) and replication intermediates were purified as described (Raschle et al., 2008 (link)). Replication and repair intermediates were separated by 0.8% native agarose gels and visualized using a phosphorimager to determine replication efficiency (Lebofsky et al., 2009 (link)). All experiments were performed at least twice, and a representative result is shown.