Figure 5. An AP-ICL is repaired by an incision-independent pathway that does not require FANCI-D2 or CMG unloading.

(A) Mechanism of AP-ICL formation.

(B) Cartoon of an AP-ICL containing plasmid (pICL^AP), in which the SapI restriction site coincides with the ICL.

(C) pCtrl, pICL^Pt, pICL^AP, or pICL^Pso was replicated in egg extracts and analyzed as in Figure 1C. White arrowhead, homologous recombination intermediate that accumulates for pICL^AP.

(D) pICL^AP was replicated in mock-depleted egg extract, FANCI-D2-depleted extract (ΔI-D2), or ΔI-D2 extract supplemented with xFANCI-D2 (ΔI-D2+xI-D2), and analyzed as in Figure 1C. White arrowhead, Figure 8 structures that persist in the absence of FANCI-D2. The fraction Figure 8 indicates the proportion of Figure 8 structures relative to total species at 300 min.

(E) Schematic illustration of nascent leading strands liberated by digestion of pICL^AP with AflIII.

(F) pICL^Pt and pICL^AP were replicated with [α-³²P]dATP in the presence or absence of NMS-873. The nascent strands were purified and digested with AflIII before separation on a denaturing polyacrylamide gel. Two portions of the autoradiograph are shown with different contrasts for optimal display. White arrowhead, leftward -1 products. Black arrowhead, rightward -1 products.