Extended Data Figure 5. The rate of total DNA synthesis does not slow before dissolution.

(A–C) To further test whether replication stalls or slows prior to dissolution, p[lacOx12] was pre-incubated with LacR and replicated in Xenopus egg extracts. Termination was then induced by addition of IPTG after 5 minutes. Simultaneously, [α-³²P]dATP was added to specifically radiolabel DNA synthesized following IPTG addition (A). Radiolabelled DNA was then separated on a native agarose gel and total signal was measured by autoradiography (B). Total signal was quantified, normalized to peak signal, and graphed alongside the rate of dissolution, which was also measured in the same experiment (C). This approach gives a highly sensitive measure of DNA synthesis without manipulation of DNA samples. DNA synthesis should occur primarily within the lacO array (see Extended Data Fig. 1). Upon IPTG addition, there was an approximately linear increase in signal, which plateaued by 5.83 min. Importantly, dissolution was 65% complete by 5.83 min. Therefore, the large majority of dissolution occurs without stalling of DNA synthesis.

(D–E) Experimental repeats of (B–C)

(F) The experiments shown in (C–E) were graphed together with mean±s.d. Synthesis data was normalized so that for each experiment, synthesis at 1 min was assigned a value of 84.4%, since this was the average value from (C–D), where synthesis was allowed to plateau. Given the rate of replication fork progression in these egg extracts (260 bp/minute³²) and the size of the array (365 bp), forks should require, on average, 0.7 minutes to converge if no stalling occurs (365 bp ÷ 2 ÷ 260 bp/min = 0.7 min). The time required for dissolution was not appreciably longer than this (dissolution was 50% complete by 0.67 min after IPTG addition, (F)), consistent with a lack of stalling.

(G–H) The experiment shown in (B–C) was repeated using p[lacOx16]. Synthesis was approximately linear until 6.17 minutes, at which point 81% of molecules had dissolved, further demonstrating that the majority of dissolution occurs without stalling of DNA synthesis.