Extended Data Fig. 7 |. ICL repair by NEIL3 requires CMG association with chromatin.

a, Analysis of proteins associated with pICL^AP during replication with NEIL3-depleted extract supplemented with rNEIL3^WT or catalytically inactive rNEIL3^K60A, p97i, and Culi. At the indicated times after replication initiation, chromatin was recovered and blotted for the indicated proteins. Consistent with NEIL3 dissociating rapidly after unhooking, rNEIL3^K60A was recovered more efficiently with pICL^AP than rNEIL3^WT.

b, pICL^AP was replicated with NEIL3-depleted extract supplemented with rNEIL3^WT or rNEIL3^K60A, p97i, and Culi, as indicated, and analyzed as in Fig. 1b.

c, The extracts used in Fig. 3c were blotted for TRAIP. Non-specifically detected proteins are marked with asterisks.

d, If NEIL3 activity is coupled to ubiquitylated CMG, NEIL3 should only function before CMG has been unloaded. To test this prediction, we inhibited all unhooking events by depleting egg extracts of NEIL3 (to block the NEIL3 pathway) and FANCD2 (to block the FA pathway). At a late time point (90 min), we added back rNEIL3 to extract where CMG had been allowed to unload (-p97i), or extract where CMG unloading was prevented (+p97i). Our model predicts that rNEIL3 should unhook the ICL only in the latter setting. Top, schematic illustrating late addition of rNEIL3 to NEIL3- and FANCD2-depleted egg extracts in the absence (left) or presence (right) of p97i. Bottom, replication of pICL^AP in mock-, NEIL3-, or NEIL3- and FANCD2-depleted extracts in the presence of [α-³²P]dATP. Extracts were supplemented with p97i as indicated and rNEIL3 was added at 90 min where indicated (black arrowheads). Replication intermediates were resolved and visualized as in Fig. 1b. Depletion of NEIL3 and FANCD2 blocked all unhooking of the AP-ICL, resulting in an accumulation of reversed forks (lane 9, green arrowhead). Addition of rNEIL3 at 90 min. in the absence of p97i (after CMG unloading) failed to induce unhooking, based on the persistence of the reversed forks (lanes 21-24). In contrast, when CMG unloading was prevented with p97i (lanes 25-30; note the persistence of Slow Figure 8 intermediates), late rNEIL3 addition led to efficient ICL unhooking, as seen from the rapid conversion of Slow Figure 8s to open circular and supercoiled species (lanes 34-36). This gel image was compressed vertically to fit the page.

e, To confirm the presence or absence CMG at the AP-ICL, DNA was recovered from the reactions described in d and subjected to nascent strand analysis as in Extended Data Fig. 3a. Top, extension products and nascent strands of the leftward fork. Bottom, nascent strands of the rightward fork. Black arrowheads, rNEIL3 addition. Depletion of NEIL3 and FANCD2 did not affect loss of the CMG footprint at −20 and caused persistence of nascent DNA strands at −1 (lanes 13-24), indicative of failure to unhook the ICL. Late addition of NEIL3 failed to stimulate further nascent strand extension (lanes 21-24), indicating that unhooking did not occur. Treatment with p97i caused persistence of the CMG footprint at −20 (lanes 25-30), consistent with retention of CMG at the ICL, and late addition of NEIL3 stimulated formation of full-length nascent strand extension products (lanes 34-36), indicative of efficient unhooking. Taken together, the data in d and e strongly suggest that NEIL3 activity is coupled to the presence of CMG at the site of the ICL, although we cannot rule out that NEIL3 activity is suppressed by downstream events, such as fork reversal, that depend on CMG unloading.