Extended Data Fig. 4: Reduced DNA synthesis and excess DNA unwinding in the presence of Pol ε polymerase mutants.

a, Total relative DNA synthesis in reactions of Fig. 1e were measured using ImageJ and plotted over time. b, Standard DNA replication reactions were carried out in the presence of wild-type or the indicated Pol ε variants (60 nM). 45 minutes after origin activation reactions were stopped and replication products analyzed by alkaline (left) or native (right) agarose gel-electrophoresis and autoradiography. Template: pARS1. c, Model for formation of θ and U* replication intermediates during plasmid replication in vitro. In normal DNA replication, the origin is initially unwound upon CMG activation (top left), followed shortly thereafter by the commencement of DNA synthesis and the coupling of leading strand synthesis to DNA unwinding by CMG (top center). Compensatory positive supercoils formed in the template during unwinding and fork progression are removed by Top1 and/or Top2. After deproteinization, the resulting θ structure is maintained (top right). In contrast, under conditions that slow-down DNA synthesis after origin unwinding the CMG helicase progresses along the template (bottom left) in advance of DNA synthesis; compensatory positive supercoils generated during DNA unwinding are removed by Top1 and/or Top2, and the unwound single-stranded DNA is stabilized by RPA binding (bottom center). Upon deproteinization, unwound complementary DNA strands reanneal, causing compensatory negative supercoils and thus resulting in a partially replicated, negatively supercoiled replication intermediate, U* (bottom right). Data for graph in panel a and uncropped gel images for b are available as source data.

Source data