Figure 7.

Replication Fork Dynamics at Different DPCs in Xenopus Egg Extracts

Plasmids modified with MH^Lead, MH^Lag, clk-SA^Lead, and clk-SA^Lag were replicated in egg extracts in the presence of LacI to ensure that a single fork encounters the DPC (Duxin et al., 2014). At the indicated time points, samples were digested with Nb.BsmI and analyzed on a denaturing polyacrylamide gel. The upper schematics depict the nascent leading-strand products liberated by Nb.BsmI digest. After replication, plasmids containing clk-SA^Lead and clk-SA^Lag were also digested with AatII and FspI (bottom radiograph) that cleave on either side of the DPC and allows to monitor the nascent leading- and lagging-strand extensions past the DPC. Note that ∼50% of the plasmid contained crosslinked SA (Figure S7).