Xenopus Egg Extract Replication

Xenopus egg extracts were prepared from Xenopus laevis wild type males and females 2–5 years of age, as approved by the Harvard Medical School Institutional Animal Care and use Committee (IACUC) and as described previously^{46 (link)}. For DNA replication, 1 volume of ‘licensing mix’ was prepared by adding plasmid DNA to High Speed Supernatant (HSS) of egg cytoplasm to a final concentration of 7.5–15 ng/µl. Licensing mix was incubated for 30 minutes at room temperature, leading to the formation of pre-replication complexes (pre-RCs). Next, licensing mix was supplemented with 0.1 volumes of Cyclin A to a final volume of 576 nM and incubated a further 10 minutes at room temperature, as previously described^{45 (link)}. Cyclin A treatment was performed to achieve highly synchronous DNA replication (Extended Data Fig. 10). Finally, 1.9 volumes of nucleoplasmic extract (NPE) was added to initiate Cdk2-dependent replication at pre-RCs. In all Figures, “0 minutes” represents the time 30 seconds after NPE addition. To radiolabel DNA, NPE was supplemented with [α-^{32 (link)}P]dATP. Reactions were stopped with 10 volumes Stop Solution (0.5% SDS, 25 mM EDTA, 50 mM Tris-HCl pH 7.5). DNA in Stop Solution was treated with RNase A (190 ng/µl final concentration) then Proteinase K (909 ng/µl final concentration) before either direct analysis by gel electrophoresis or purification of DNA as described previously^{40 (link)}. For UbVS experiments, UbVS (Boston Biochem) was added to final concentration of 20 µM, to HSS 5 minutes prior to addition of plasmid DNA (HSS) and to NPE 5 minutes prior to addition of HSS, with or without 120 µM Ubiquitin (Boston Biochem). Unless otherwise stated in the figure legend, all experiments were performed at least twice and a representative result is shown. Replicate samples were collected from independently assembled replication reactions, and therefore represent biological replicates.