a, Model for ICL repair by the FA pathway. Upon convergence of two replication forks at the crosslink, the CMG helicase is unloaded from the DNA to enable the approach of one replication fork to the −1 position. Ubiquitylation of FANCD2 promotes the recruitment of the XPF-ERCC1-SLX4 (XES) complex to the ICL, which enables nucleolytic incisions that unhook the crosslink. This step could be preceded by fork reversal of one of the stalled replication forks24. Incisions generate a broken strand and a strand with an adduct; the latter is bypassed by TLS whereas the broken strand is repaired by homologous recombination. In mammalian cells, it has been shown that a single fork can pass over the ICL without unhooking25; this ‘traverse’ gives rise to a structure that resembles the one generated after fork convergence and CMG unloading and could follow the same steps subsequently. b, The indicated plasmids were replicated in Xenopus egg extracts and reaction samples were analysed by western blot with FANCD2 antibody. Two independent experiments were performed. c, Western blot of FANCD2, showing a titration of Xenopus egg extracts compared to mock and FANCD2-depleted extracts. Two independent experiments were performed. d, The indicated plasmids were replicated in mock or in FANCD2-depleted extracts in the presence of [α-32P]dCTP. Repair products were digested by AflIII, separated on a sequencing gel alongside a ladder derived from extension primer S, and visualized by autoradiography. The white arrow denotes the −1 product, which is 2 nt larger in pICL-Pt owing to the position of the ICL. Three independent experiments were performed. e, The indicated plasmids were replicated in mock or FANCD2-depleted extracts and repair intermediates were digested with NotI, labelled at the 3′-end, and resolved by denaturing PAGE. Quantification of repair based on the intensity of the 44-nt product is shown in Fig. 1g. f, The independent experimental duplicate of Fig. 1g. g, The independent experimental triplicate of Fig. 1g, but using only pICL-AA NAT . h, Plasmid pICL-AA NAT was replicated in FANCD2-depleted extract, or FANCD2-depleted extract supplemented with a recombinant FANCI–FANCD2 complex (ID). Reaction samples were resolved by native agarose gel and visualized by autoradiography. RRIs, open circle (OC) and supercoiled (SC) products are indicated. The stalled repair product (grey arrow) is indicated. Two independent experiments were performed. i, The indicated plasmids were replicated in Xenopus egg extracts in the presence or in the absence of p97i and the intermediates were resolved by native agarose gel electrophoresis. The stalled repair products (grey arrow) are indicated. Seven independent experiments were performed. j, The indicated plasmids were replicated in Xenopus egg extracts in the presence or in the absence of p97i, and repair intermediates were digested with NotI, labelled at the 3′-end, and resolved by denaturing PAGE. The increase in intensity of the 44-nt band (white arrow) over time indicates ongoing replication and repair. A higher mobility band, probably generated from end-joining activity in some extracts, is indicated with an asterisk. k, Quantification of repair based on the intensity of the 44-nt product on the gel shown in j, as indicated in the Supplementary Methods. l, Quantified independent experimental duplicate of j. m, Quantified independent experimental triplicate of j.