Extended Data Fig. 6: Characterization of stalled and uncoupled replication forks.
From: Replication fork uncoupling causes nascent strand degradation and fork reversal
(A) Cartoon depicting the strategy to test the role of helicase stalling during NSD. If helicase stalling triggers NSD then NSD should be increased in the absence of IPTG. If uncoupling triggers NSD then addition of IPTG should increase NSD. (B) Depiction of the DNA structures generated by Fig. 4A, prior to restriction digest. (C) Samples from (e) were separated on an agarose gel and visualized by autoradiography. θ* species arise from uncoupling. θ- species arise from the low level of uncoupling that occurs even in the presence of LacR, which is not a complete block to helicase progression13. (D) Depiction of different models for degradation of leading and lagging strands during NSD observed in Fig. 4 and the expected effect on degradation of LWS and RWS depicted in Fig. 4A. In (i) both leading and lagging strand are degraded simultaneously and synchronously, which should result in disappearance of RWS before LWS. In (ii) lagging strands only are degraded synchronously, which should result in persistence of ~50% of RWS and LWS until the leading strand are degraded by nuclease activity that initiated at lagging strands of the opposite fork. This would result in biphasic kinetics of degradation. In (iii) lagging strands only are degraded asynchronously, which should result in loss of RWS and LWS at the same rate. (E) Uncropped gel of Fig. 4B lanes 1-4 shown after a longer exposure so that the weaker rightward strands (RWS) could be visualized. (F) Quantification of LWS and RWS from (b). RWS are degraded before LWS, with no evidence of biphasic kinetics, indicating that both strands are degraded simultaneously, as in (a,i). Mean ± S.D., n = 3 independent experiments. (G) NSD was induced as in Fig. 4A in the presence or absence of DNA2-i. (H) Samples from (g) were separated on a native agarose gel and visualized by autoradiography. (I) Quantification of RI signal from (h). Mean ± S.D., n = 3 independent experiments. (J) Quantification of reversed forks from Fig. 4E expressed as a percentage of total signal at T = 0. Mean ± S.D., n = 3 independent experiments.
