Extended Data Fig. 1 |. DNA replication and ICL repair in Xenopus egg extracts.

a, Schematic of pICL replication in the nucleus-free Xenopus egg extract system⁴⁸. Incubation of the plasmid in HSS supports the recruitment of inactive MCM2-7 double hexamers (red hexamers; “Licensing”). Addition of NPE activates replication initiation, including the assembly of active CMG helicases (green hexamers), and elongation of nascent strands (red lines) leads to convergence of forks at the ICL.

b, Intermediates generated during replication-coupled repair of a cisplatin-ICL. Top, progression through the incision-dependent Fanconi anemia repair pathway generates distinct intermediates resulting from fork convergence, CMG unloading, leading strand approach to the ICL, fork reversal, incisions, and repair of the double strand break by homologous recombination. Bottom, deproteinization of the DNA intermediates depicted along the top yields DNA structures that travel with characteristic mobilities during native agarose gel electrophoresis. These structures are indicated along the side of the gel and with colored dots and/or arrowheads in Fig. 1b and other figures. The Slow Figure 8 arises upon fork convergence on the ICL (Fig. 1b, purple dot). Conversion of Slow to Fast Figure 8s results from CMG unloading and an accompanying change in plasmid topology (Fig. 1b, orange dot)⁷. Next, a species of intermediate mobility appears (Fig. 1b, green arrowhead), which represents reversed forks, as shown by electron microscopy⁷. Following XPF-dependent unhooking of the reversed structure^7,49, double-strand DNA break repair generates joining products that barely enter the gel⁵⁰ (Fig, 1b, Well product). Some of these species are resolved into monomeric, supercoiled plasmids that represent the final, fully repaired product (Fig. 1b, SC) that is sensitive to SapI digestion.

c, Intermediates generated during replication-coupled repair of an AP-ICL (pICL^AP). Top, progression through the NEIL3 repair pathway generates intermediates resulting from fork convergence, NEIL3-dependent N-glycosyl bond cleavage, nascent strand ligation, decatenation, and translesion synthesis (TLS). Bottom, deproteinization of the DNA intermediates depicted along the top yields DNA structures that travel with characteristic mobilities during native gel electrophoresis. These structures are indicated along the side of the gel and with colored dots in Fig. 3a and other figures.