Extended Data Fig. 3: Characterization of replication products.
From: Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks
a, Standard replication reactions with pARS1 were carried out in the absence or presence of Top1 or Top2, as indicated. Reactions were stopped 60 minutes after origin activation, and replication products analyzed by native agarose gel-electrophoresis and autoradiography after linearization with the unique cutter Nde I, which cuts near the origin (fully replicated DNA molecules will resolve into linear monomers after linearization, whereas replication products containing unreplicated regions will resolve into double Y-shaped intermediates. Representative gel is shown on the left. The bar diagram depicts the average dissolution efficiency (fraction of linear full-length molecules per reaction) and s.d. of three independent experiments. b, Replication products from experiment in Fig. 1b were analyzed by alkaline agarose gel-electrophoresis and autoradiography. c, Pulse-chase experiment demonstrating that the ERI is a precursor of the LRI. Replisomes were formed on chromatin templates in the presence of α-32P-dCTP and stalled by omission of topoisomerase from the reaction (lane 1). After 15 minutes, Top1 was added to the reaction to release the stalled replisomes and intermediates chased by simultaneous addition of excess cold dCTP. At the indicated time points (lanes 2 and 3) replication products were isolated and analyzed by native (left) or denaturing agarose gel-electrophoresis and autoradiography. d, Standard replication reactions carried out in the presence of either Top1, Top2, or both. Replication products were analyzed by native agarose gel-electrophoresis and autoradiography. Uncropped gel images and data for graph in panel a are available as source data.
