Wu RA , Semlow DR , Kamimae-Lanning AN , Kochenova OV , Chistol G , Hodskinson MR , Amunugama R , Sparks JL , Wang M , Deng L , Mimoso CA , Low E , Patel KJ , Walter JC .

F31 GM122277 NIGMS NIH HHS , R01 HL098316 NHLBI NIH HHS , K99 GM129422 NIGMS NIH HHS , F31 GM122277 National Institute of General Medical Sciences, K99 GM129422 National Institute of General Medical Sciences, R01 HL098316 National Heart, Lung, and Blood Institute, Howard Hughes Medical Institute , MC_U105178811 Medical Research Council , R00 GM129422 NIGMS NIH HHS

	Extended Data Fig. 2 |. Recombinant TRAIP supports CMG unloading at cisplatin-ICLsa, NPE immunodepleted of TRAIP was loaded alongside a dilution series of mock-depleted NPE and blotted for TRAIP. A relative loading amount of 100 corresponds to 2 µl of NPE. Non-specifically detected proteins are marked with asterisks.b, Bacterially-expressed rTRAIPWT, rTRAIPR18C, His6-SUMO-rTRAIPWT, and His6-SUMO-rTRAIPΔPIP (comprising residues 1-455) were partially purified, resolved by SDS-PAGE, and visualized with Coomassie Brilliant Blue staining. Note that His6-SUMO-rTRAIP is obscured by co-migrating, contaminating proteins. Bacterially-expressed rTRAIP was used for all subsequent experiments, unless otherwise indicated.c, rTRAIPWT and rTRAIPR18C were expressed in Sf9 insect cells and purified using an N-terminal 3×FLAG tag. The tag was then cleaved using 3C protease. The recombinant proteins, along with a buffer sample containing 3C protease only, were resolved by SDS-PAGE and visualized with Coomassie Brilliant Blue staining.d, Mock- and TRAIP-depleted extracts supplemented with rTRAIPWT or rTRAIPR18C used in Fig. 1b were analyzed by immunoblotting for TRAIP. The absence of the non-specific bands seen in a may be due to shorter incubation with the TRAIP antibody. The concentration of added recombinant TRAIP relative to endogenous TRAIP fluctuates among experiments (e.g. compare panel d and Extended Data Fig. 3b). We ascribe this difference to variations in non-specific removal of endogenous TRAIP from extracts during the mock-depletion procedure, and possibly also in the delivery of recombinant TRAIP into extract.e, pICLPt was replicated in mock- or TRAIP-depleted extracts supplemented with [α-32P]dATP and Sf9-expressed rTRAIPWT, rTRAIPR18C, or 3C protease alone and analyzed as in Fig. 1b.f, Extracts used in the replication reaction shown in e were analyzed as in d.g, pICLPt was replicated in the indicated egg extracts with [α-32P]dATP and analyzed as in Fig. 1b.h, Extracts used in the replication reaction shown in g were analyzed as in d. Note that deleting the C-terminal PIP box disrupts the epitope for the TRAIP antibody used for immunoblotting. Therefore, to assess the activity of TRAIPΔPIP in ICL repair relative to TRAIPWT, His6-SUMO-tagged proteins were added back to TRAIP-depleted extract and assayed in g. The relative amounts of His6-SUMO-TRAIPWT and His6-SUMO-TRAIPΔPIP were compared by detecting the histidine tag. By blotting the same extracts for TRAIP, a comparison of the relative concentrations of His6-SUMO-TRAIPΔPIP and endogenous TRAIP was made.i, Mock- and TRAIP-depleted extracts used in the replication reactions shown in Fig. 1c and d were analyzed as in d.
	Extended Data Fig. 3 |. TRAIP, but not FANCM, ATR, or BRCA1, is required for CMG unloading at cisplatin-ICLsa, Left, schematic of nascent strands generated at ICLs. When forks converge on an ICL, nascent strands stall ~20 nt from the ICL on either side of the lesion due to the footprint of CMG (green hexamer). AflIII cuts 144 nt to the left and 534 nt to the right of the ICL, generating characteristic products for the leftward and rightward leading strands upon fork convergence, CMG unloading, and leading strand extension. Right, nascent strand analysis of pICLPt replication in the indicated extracts. After replication with [α-32P]dATP, nascent strands were extracted, digested with AflIII, and resolved on a denaturing polyacrylamide gel alongside a sequencing ladder and visualized by autoradiography. As seen previously2,51, when replication forks converged on the ICL in mock-depleted egg extracts, leading strands initially stalled 20-40 nucleotides (nt) from the lesion (lane 1) and then advanced to the −1 position (lanes 2-6), which depends on CMG dissociation19,51. In contrast, in TRAIP-depleted egg extracts, the −20 footprint persisted for three hours (lanes 7-12). This effect was rescued with rTRAIPWT but not rTRAIPR18C (lanes 13-24).b, Extracts used in the replication reaction shown in a were blotted for TRAIP.c, Mock- or FANCM-depleted extracts were blotted for FANCM. A non-specifically detected protein is marked with an asterisk.d, Nascent strand analysis of pICLPt replicating in mock- or FANCM-depleted extracts was performed as in a. The CMG footprint disappeared at the ICL in FANCM-depleted egg extract, consistent with FANCM not being required for CMG unloading at ICLs.e, pICLPt was replicated in the absence or presence of ATR inhibitor ETP-46464 (ATRi), and nascent strand analysis was performed as in a. ATR inhibitor was added to the reaction 2.5 min after initiation. The CMG footprint disappeared at the ICL with or without ATR inhibitor, indicating that ATR signaling is not required for CMG unloading at ICLs.f, Extracts used in e were sampled at various time points and blotted for Xenopus CHK1 Serine-344 phosphorylation to verify ATR inhibition. MCM6 was detected as a loading control.g and h, Mock- and TRAIP-depleted extracts used in one of the replicate reactions quantified in Fig. 1e (g) and f (h) were blotted for TRAIP.i, We previously showed that the immunodepletion of BRCA1 from egg extracts inhibits CMG unloading at ICLs, but this defect could not be rescued with recombinant BRCA1-BARD1 complex6,19. To test whether TRAIP is co-depleted with BRCA1, NPE was immunodepleted of BRCA1 with BRCA1 antiserum, loaded alongside a dilution series of mock-depleted NPE, and blotted for BRCA1 and TRAIP. A relative loading amount of 100 corresponds to 2 µl of NPE. Non-specifically detected proteins are marked with asterisks. This analysis revealed that immunodepletion of BRCA1 also removed TRAIP from NPE. Notably, we also observed TRAIP co-depletion with antibodies against other proteins (data not shown), suggesting it interacts non-specifically with different antibodies.j, The extracts described in i were supplemented with pICLPt, [α-32P]dATP, and rTRAIP, as indicated, and analyzed as in Fig. 1b. rTRAIPWT suppressed the stabilization of the Slow Figure 8 species seen in BRCA1-depleted extract, consistent with the restoration of CMG unloading, and indicating that the unloading defect seen in BRCA1-depleted egg extracts is due primarily to the removal of TRAIP from the extract.k, The extracts used in j were blotted for TRAIP.l, To determine whether BRCA1 contributes to TRAIP-dependent CMG unloading, NPE was immunodepleted of TRAIP or TRAIP and BRCA1 using Protein A Sepharose-purified antibodies purified from antiserum. A dilution series of mock-depleted NPE was loaded alongside the depleted extracts, and extracts were blotted for BRCA1 and TRAIP. A relative loading amount of 100 corresponds to 2 µl of NPE.m, The extracts described in l were supplemented with pICLPt, [α-32P]dATP, and rTRAIP, as indicated, and analyzed as in Fig. 1b. rTRAIPWT suppressed the accumulation of Slow Figure 8s to a similar extent in TRAIP-depleted egg extracts whether or not BRCA1 was co-depleted (lanes 19-30), indicating that BRCA1 is not needed to support TRAIP function. BRCA1 depletion reproducibly resulted in a decrease in well product formation, suggesting a role for BRCA1 in recombination after a double-strand break is formed by ICL unhooking incisions.n, The extracts used in m were blotted for TRAIP.
	Extended Data Fig. 4 |. TRAIP and CRL2LRR1 promote distinct CMG unloading pathwaysa, pCTRL was replicated in NPE in the presence of Geminin or the CDC7 inhibitor PHA-767491 (CDC7i). Eight minutes after addition of NPE, the plasmid was recovered and the indicated proteins were analyzed by immunoblot.b, Cartoon depicting the effects of Geminin and CDC7i, and the step at which TRAIP is recruited to chromatin.c, Extracts used in the replication reaction shown in Fig. 2e were blotted for TRAIP.d, Top, upon termination of pCTRL replication, CMG (green) unloading depends on CRL2LRR1-mediated MCM7 ubiquitylation (purple)21,52, but it’s unknown whether unloading also requires TRAIP. Middle, CMGs that have converged at an ICL undergo TRAIP-dependent ubiquitylation (Fig. 1), but the involvement of CRL2LRR1 is unknown. Bottom, if two origins fire on a single plasmid, one pair of replication forks converges at the ICL and undergoes TRAIP-dependent CMG unloading whereas a second pair undergoes CRL2LRR1-dependent unloading. Because both pairs of CMGs should undergo ubiquitylation, in some experiments we include Culi to monitor only TRAIP-dependent ubiquitylation.e, To determine whether TRAIP is required for CMG unloading during replication termination, we analyzed proteins associated with plasmids 60 min after replication initiation in mock- or TRAIP-depleted extracts containing p97i or Culi, as indicated. Chromatin was recovered and blotted for the indicated proteins. CMG unloading from pCTRL was unaffected by TRAIP depletion (compare lanes 4 and 7). Consistent with this, in the presence of p97i, TRAIP was not required for MCM7 ubiquitylation on pCTRL (compare lanes 2 and 5). In contrast, in the absence of TRAIP, CMG unloading from pICLPt was inhibited compared to the mock-depleted control (compare lanes 10 and 13), consistent with Fig. 1c. Similarly, TRAIP was essential for efficient MCM7 ubiquitylation on pICLPt (compare lanes 8 and 11, note the greater level of unmodified MCM7 in lane 11). The partial CMG unloading (lane 13) and residual MCM7 ubiquitylation observed on pICLPt in the absence of TRAIP (lane 11) were likely the result of termination events that occurred elsewhere on the plasmid (as described in d, bottom). Consistent with this interpretation, the combination of TRAIP depletion and Culi abolished MCM7 ubiquitylation (lane 12).f, To determine whether CRL2LRR1 contributes to CMG unloading at ICLs, pICLPt was replicated in undepleted extract containing p97i or Culi and analyzed as in Fig. 1b. Culi had no significant effect on the accumulation of Fast Figure 8 structures, consistent with CRL2LRR1 being dispensable for CMG unloading at ICLs.g, Left, to assess the effect of LRR1 depletion on CMG unloading at ICLs, NPE was immunodepleted of LRR1, loaded alongside a dilution series of mock-depleted NPE, and blotted for LRR1 and CUL2. A relative loading amount of 100 corresponds to 2 µl of NPE. Non-specifically detected protein is marked with an asterisk. Right, pICLPt was replicated in mock- or LRR1-depleted egg extracts and analyzed as in Fig. 1b. The absence of LRR1 had no effect on the formation of Fast Figure 8 structures, supporting the idea that CRL2LRR1 is dispensable for CMG unloading at ICLs.h, Nascent strand analysis of pICLPt replicating in mock- or LRR1-depleted extracts was performed as in Extended Data Fig. 3a. The CMG footprint disappeared with normal kinetics at the ICL in LRR1-depleted egg extract, consistent with CRL2LRR1 not being required for CMG unloading at ICLs.
	Extended Data Fig. 5 |. TRAIP ubiquitylates numerous CMG subunits with heterotypically-linked chains upon fork convergence at an ICLa, pICL-lacOPt was incubated with LacR prior to replication in mock- or TRAIP-depleted extracts. After 1 hr of replication (to allow for termination of replication forks that do not converge on the ICL or lacO array as depicted in Fig. 2c), 0.3 mM NMS-873 was added and the extracts were incubated for 5 min to inhibit p97. 10 mM IPTG and Sf9-expressed rTRAIPWT were then added as indicated and the extract was incubated for 2 hr to disrupt LacR DNA binding and allow fork convergence and TRAIP-dependent ubiquitylation. The plasmid was recovered and blotted for the indicated proteins.b, The extracts described in a were blotted for TRAIP.c, pICL-lacOPt was incubated with LacR prior to replication in undepleted egg extracts. After 30 min of replication (to allow for termination of replication forks that do not converge on the ICL or lacO array as depicted in Fig. 2c), 0.3 mM NMS-873 was added and the extracts were incubated for 5 min to inhibit p97. 10 mM IPTG was then added as indicated to disrupt LacR DNA binding and the extract was incubated for 1 hr to allow fork convergence. The plasmid was recovered and blotted for the indicated proteins.d, Recombinant CMG was purified, resolved by SDS-PAGE, and visualized with SYPRO ruby staining.e, rTRAIP ubiquitin ligase activity. rTRAIPWT or rTRAIPR18C was combined with ubiquitin, E1, three E2s (UbcH5a/b/c), and ATP as indicated. Polyubiquitin chain synthesis (top gel) and TRAIP autoubiquitylation (bottom gel) were detected by immunoblotting the reactions with ubiquitin and TRAIP antibody, respectively. Notably, rTRAIPR18C was much more compromised in forming free polyubiquitin chains in this assay than it was in ubiquitylating rCMG (see Fig. 2d). The data suggest that the interaction between TRAIP and CMG can suppress, to a great extent, the profound ubiquitylation defect of the R18C mutation.f, pCTRL-lacO and pICL-lacOPt were replicated in undepleted extract as in c and recovered. Samples were treated with the indicated DUBs and blotted for the indicated proteins.g, pCTRL-lacO and pICL-lacOPt pre-bound with LacR were replicated in undepleted extract as in c. At the time of IPTG addition to release the LacR array and allow fork convergence, 100 µM recombinant ubiquitin (wild-type or various lysine-to-arginine mutants) was added to the extract (which contains ~8 µM endogenous ubiquitin) and incubated for 1 hr. The plasmid was recovered and blotted for the indicated proteins.h, Extracts used in g were blotted for ubiquitin. Some ubiquitin mutants contain a di-ubiquitin species (marked with asterisk). Whether this arises upon addition to extract is unclear.
	Extended Data Fig. 6 |. AP-ICL repair by NEIL3 requires TRAIPa, Analysis of chromatin-associated proteins during replication of pICL-lacOPt or pICL-lacOAP in undepleted extract in the presence and absence of Geminin, as indicated. At different times after replication initiation, chromatin was recovered and blotted for the indicated proteins.b, pICL-lacOPt and pICL-lacOAP were replicated in undepleted extract supplemented with Culi, Geminin, and p97i, as indicated, and analyzed as in Fig. 1b.c, The extracts used in the replication reaction shown in Fig. 3a were blotted for TRAIP.d, Extracts used in one of the reactions quantified in Fig. 3b were analyzed as in c.e and f, pICLAP was replicated in the extracts shown in Extended Data Fig. 2h (e) and Extended Data Fig. 2f (f) with [α-32P]dATP and analyzed as in Fig. 1b.
	Extended Data Fig. 7 |. ICL repair by NEIL3 requires CMG association with chromatina, Analysis of proteins associated with pICLAP during replication with NEIL3-depleted extract supplemented with rNEIL3WT or catalytically inactive rNEIL3K60A, 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, rNEIL3K60A was recovered more efficiently with pICLAP than rNEIL3WT.b, pICLAP was replicated with NEIL3-depleted extract supplemented with rNEIL3WT or rNEIL3K60A, 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 pICLAP 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.
	Extended Data Fig. 8 |. The zinc-finger domains of NEIL3 contribute to its recruitment to the replication forka, Left, to determine whether rNEIL3Δ291 is catalytically active, a model AP-ICL substrate comprising a synthetic 5′-radiolabeled 24mer oligonucleotide cross-linked to a ~3mer was mixed with rNEIL3Δ291 or rNEIL3WT. Cross-linked and unhooked species were resolved by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Asterisks indicate the 32P radiolabel. Note that the cross-linked species migrates as a doublet due to heterogeneity in the bottom strand following RNase digestion (see Methods for details). Right, quantification of unhooking. Equivalent results were obtained in three independent experiments, which show that rNEIL3Δ291 retains full glycosylase activity.b, Interaction of the NEIL3 NPL4-type zinc finger (NZF; residues 300 to 328) with ubiquitin. GST-NEIL3 NZF fusion protein (WT or TL,LV substituted) was immobilized on a biosensor tip and monoubiquitin binding was measured by biolayer interferometry (BLI). The ubiquitin binding response was corrected for non-specific binding to GST and plotted as a function of ubiquitin concentration. Error bars represent standard error of the mean from three independent experiments.c, Extracts used in Fig. 3e were blotted for NEIL3. Black arrowheads, NEIL3-specific bands. rNEIL3Δ291 is not efficiently detected by the NEIL3-specific primary antibody. Non-specifically detected proteins are marked with asterisks.d and e, To test whether the two GRF zinc fingers in NEIL3 interact with ssDNA, we expressed each individually and performed electrophoretic mobility shift assays. rMBP-NEIL3 GRF zinc finger fusion proteins (wild-type or substituted) were incubated with 5′-radiolabeled 25-mer ssDNA or dsDNA. Bound and unbound DNAs were resolved by native polyacrylamide gel electrophoresis and visualized by autoradiography. This analysis reveals that both GRF domains bind specifically to ssDNA.f, Analysis of proteins associated with pICLPt during replication in undepleted extract in the presence of p97i and Culi. Extracts were supplemented with wild-type or mutated rNEIL3. At different times, chromatin was recovered and blotted for the indicated proteins. The individual GRF substitutions modestly affected recovery of rNEIL3 upon pICL pull-down while combination of the substitutions or deletion of both GRF zinc fingers strongly reduced rNEIL3 recovery, indicating that interactions mediated by the GRF zinc fingers promote recruitment of NEIL3 to an ICL.g, pICLAP was replicated in mock- or NEIL3-depleted extracts supplemented with wild-type or mutated NEIL3 as indicated and analyzed as in Fig. 1b. Relative to rNEIL3WT, rNEIL3 with substitutions in either GRF zinc finger that abolish ssDNA binding (K500E and K546E) exhibited modest defects in pICLAP unhooking that were exacerbated when the substitutions were combined, indicating that interactions between the GRF zinc fingers and ssDNA contribute to ICL repair.h, Extracts used in the replication reactions shown in g, were blotted for NEIL3. Non-specifically detected proteins are marked with asterisks.i, Model for recruitment of NEIL3 to chromatin by zinc finger-mediated interactions. Upon replication fork convergence at an ICL, TRAIP-dependent CMG ubiquitylation recruits NEIL3 through direct interactions between NEIL3’s NZF domain and ubiquitin. Association of NEIL3 with chromatin is further enhanced by interactions between the tandem GRF zinc fingers and single stranded DNA, possibly on the lagging strand template.
	Extended Data Fig. 9 |. Knockout of the FA and NEIL3 pathways have additive effects on ICL sensitivity in mammalian cellsa, Immunoblot analysis of NEIL3 expression in wild-type, NEIL3, and FANCL/NEIL3 knockout HAP1 cell lines. Histone H3 was detected as a loading control. Non-specifically detected proteins are marked with asterisks.b, Schematic of FANCL CRISPR targeting. (i) Human FANCL exon 10, sgRNA binding sites, and homology arm targets, (ii) FANCL-Puro targeting construct with homology arms flanking exon 10, (iii) Targeted FANCL allele with integrated puromycin resistance cassette.c, Detection of the integrated puromycin resistance cassette in HAP1 cells by FANCL long-range PCR.d, Analysis of FANCD2 ubiquitylation in MMC-treated wild-type, FANCL, and FANCL/NEIL3 knockout HAP1 cell lines to confirm FANCL knockout. Vinculin was detected as a loading control. FANCL is the catalytic subunit of the FA core complex, which ubiquitylates FANCD2.e, Neil3 qRT-PCR confirming gene disruption in CH12 cell lines. ND, not detected.f, Analysis of FANCD2 ubiquitylation in mitomycin C-treated CH12 Fancb single and Neil3/Fancb double knockout cell lines to confirm Fancb knockout. Vinculin was detected as a loading control. A non-specifically detected protein is marked with an asterisk.g, Cell viability of wild-type, Fancb, Neil3, or Fancb/Neil3 knockout CH12 cells after exposure to trioxsalen and UV-A irradiation. Two independent clones were used for the single mutants and three independent clones were used for the double mutant. Error bars, standard error of the mean. We speculate that, relative to HAP1 cells, CH12 cells may be more reliant on the FA pathway to repair trioxsalen-induced damage due to lower expression levels of NEIL3.
	Extended Data Fig. 10 |. Model of TRAIP’s action in interphase and mitosisa, TRAIP (blue) travels with the replisome and associates with CMG, directly or indirectly (i-iv, top). In interphase, TRAIP is oriented such that its E2 (whose identity is unknown) points ahead of the replisome. As a result, TRAIP cannot ubiquitylate the CMG it is associated with, but it can ubiquitylate any protein that lies in the replisome’s path. TRAIP can thus ubiquitylate CMG in trans when two replisomes meet (i), and it can ubiquitylate DPCs that block the path of the replisome (ii). We speculate that TRAIP ubiquitylates any proteinaceous structure that causes extended stalling of the replisome. In mitosis, TRAIP undergoes a conformational change so that it can ubiquitylate the CMG with which it is associated in cis. As a result, in mitosis, stalled forks undergo TRAIP-dependent CMG ubiquitylation in the absence of fork convergence (iii) (Deng et al., in press). We propose that TRAIP does not ubiquitylate terminated CMGs in interphase because they rapidly move past each other29, precluding ubiquitylation by the forward-pointing TRAIP (iv, middle). However, upon mitotic entry, terminated CMGs undergo TRAIP-dependent ubiquitylation in cis (iv, bottom)(Deng et al., in press). Importantly, TRAIP-dependent CMG unloading in interphase egg extracts is not dependent on residual CDK1 activity (b and c), indicating that TRAIP is regulated differently in interphase and mitosis.b, In interphase egg extracts, TRAIP travels with DNA replication forks but ubiquitylates CMGs only when forks converge. In the presence of Cyclin B1-CDK1, TRAIP is activated in the absence of fork convergence (Deng et al., in press)(summarized in a). We therefore wanted to know whether TRAIP-dependent CMG unloading in interphase egg extract depends on residual CDK1 activity. Top, reaction scheme to determine whether the action of TRAIP at ICLs in interphase egg extract requires residual CDK1 activity. Replication of pICL-lacOPt with a pre-assembled LacR array was initiated by addition of NPE (−55 min). Forty-five min after initiation (−10 min), reactions were supplemented with buffer or the CDK1 inhibitor RO-3306 (CDK1i) and allowed to incubate for an additional 10 min; CDK1i was added late to avoid inhibition of replication initiation. The LacR array was then released with addition of IPTG to trigger fork convergence and ICL repair (0 min). In a control, buffer instead of IPTG was added to maintain the LacR array and thereby prevent fork convergence. At the indicated times after IPTG addition, samples were collected and analyzed as in Fig. 1b to look for evidence of CMG unloading and ICL processing. Bottom, experiment described in top scheme. Fork stalling at the boundaries of the LacR array lead to a theta structure (lanes 1, 7, 13, 19). Upon addition of IPTG, theta was converted to Slow Figure 8, Fast Figure 8 (co-migrating with theta), and well product with equal efficiency in the presence and absence of CDK1i (compare lanes 7-12 and 19-24). The data suggest that CDK1 is not required for CMG unloading or ICL repair.c, Left, reaction scheme of a control experiment to ensure that the addition of CDK1i in b blocked CDK1 activity. As recently described (Deng et al., in press), when replication forks are stalled at a LacR array, the subsequent addition of Cyclin B1-CDK1 promotes TRAIP-dependent CMG unloading and fork breakage, alternative end joining, and the formation of aberrant replication products that migrate in the well of an agarose gel. To verify that CDK1 was effectively inhibited in a, we used this CDK1-dependent well-product formation as an assay. To this end, replication of pCTRL-lacO with a pre-assembled LacR array was initiated (−55 min) by addition of NPE. Forty-five min after initiation (−10 min), reactions were supplemented with CDK1i or buffer and allowed to incubate for an additional 10 min. Cyclin B1-CDK1 was then added to activate fork breakage and well product formation (0 min). Right, experiment described in left scheme. As expected, addition of Cyclin B1-CDK1 in the absence of CDK1i led to the formation of well products (lanes 25-30) but not in the presence of CDK1i (lanes 31-36). Note that the experiments in b and c were performed in parallel; extracts were aliquoted for the various conditions described at 0 min. Therefore, the CDK1i effectively inhibited CDK1-dependent activation of TRAIP. We conclude that TRAIP activation at ICLs does not depend on residual CDK1 activity.
	Fig. 1 |. CMG unloading at ICLs requires the E3 ubiquitin ligase TRAIPa, Models of ICL repair by the FA (left branch) and NEIL3 (right branch) pathways. TLS, translesion DNA synthesis; AP, abasic site.b, pICLPt was replicated in egg extracts containing [α-32P]dATP. Replication intermediates were resolved by native agarose gel electrophoresis and visualized by autoradiography. Recombinant TRAIPWT, rTRAIPR18C, and p97 inhibitor NMS-873 (p97i) were added as indicated. OC, open circular; SC, supercoiled; pQnt, undamaged control plasmid; Purple dot, Slow Figure 8; Orange dot, Fast Figure 8; Green arrowhead, reversed fork (see Extended Data Fig. 1b for discussion). For TRAIP levels in extracts, see Extended Data Fig. 2d.c and d, Analysis of proteins associated with pICLPt during replication in the indicated extracts in the absence (c) or presence (d) of p97i and Cullin RING ubiquitin ligase inhibitor MLN4924 (Culi). Culi was added to eliminate ubiquitylation by CRL2LRR1 (see Extended Data Fig. 4d). For TRAIP levels in extracts, see Extended Data Fig. 2i. Residual MCM7 ubiquitylation in the presence of R18C was observed in eight independent experiments (e.g. Fig. 3c). Histone H3, recovery control.e, Left, pICLPt was replicated for 90 min in the indicated extracts, and the relative abundance of reversed forks, as determined by electron microscopy, was quantified. Values were normalized to the mock-depleted extract. Error bars, range from two independent experiments. Right, representative electron micrograph of a reversed fork (green arrowhead) from TRAIP-depleted extract supplemented with rTRAIPWT. For TRAIP levels in extracts, see Extended Data Fig. 3g.f, Error-free repair as measured by regeneration of a SapI restriction site coinciding with the ICL. The ~7% basal SapI cutting is due to contaminating undamaged plasmid8. Error bars, standard error of the mean from three independent experiments. For TRAIP levels in extracts, see Extended Data Fig. 3h.
	Fig. 2 |. TRAIP ubiquitylates CMGs that have converged at ICLsa, Left, experimental scheme. Plasmid containing an ICL flanked by 48 copies of the lac operator (lacO) (pICL-lacOPt) was incubated with Lac repressor (LacR) prior to replication in undepleted egg extract. After 30 min, IPTG addition dissociated LacR and allowed fork convergence. Right, pICL-lacOPt was recovered and blotted for the indicated proteins. Culi suppressed CRL2LRR1-dependent ubiquitylation.b, Chromatin-associated proteins during replication of pICLPt or pCTRL in undepleted extract in the presence or absence of the licensing inhibitor Geminin and p97i.c, Left, following CRL2LRR1-dependent unloading of CMGs that underwent termination on plasmids with multiple initiation events (see Extended Data Fig. 4d), release of the LacR array with IPTG leads to convergence of forks at the ICL, allowing analysis of TRAIP-specific ubiquitylation. Right, analysis of proteins associated with pICL-lacOPt during replication in the presence of p97i. MCM2 ubiquitylation was inferred from the increase in unmodified MCM2 upon USP2 treatment, which was used to remove all ubiquitin modifications. Asterisks, non-specifically detected proteins.d, Analysis of rCMG subunits incubated with rTRAIP in the presence of ubiquitin, E1, E2s UbcH5a/b/c, and ATP. MCM4 was present as full-length and degradation products. Asterisk, non-specifically detected protein.e, Nascent strand analysis of pDPC2×Lead replicated in the indicated extracts. Red dot, combined footprint of CMG and the DPC in the presence of TRAIP. For TRAIP levels in extracts, see Extended Data Fig. 4c.
	Fig. 3 |. TRAIP promotes NEIL3-dependent ICL repaira, pICLAP was replicated in the indicated extracts and analyzed as in Fig. 1b. Red dot, OC-OC catenane; Blue dot, OC-SC catenane. For TRAIP levels in extracts, see Extended Data Fig. 6c.b, Error-free repair of pICLAP was quantified as in Fig. 1f. Error bars, standard error of the mean from three independent experiments. For TRAIP levels in extracts, see Extended Data Fig. 6d.c and f, Analysis of proteins associated with pICLPt during replication in the indicated extracts (c) or undepleted extract (f) in the presence of p97i and Culi. Asterisk, non-specifically detected protein.d, Schematic of Xenopus NEIL3 proteins.e, pICLAP was replicated in the indicated extracts and analyzed as in Fig. 1b. ICL unhooking was estimated by quantifying OC and SC signal normalized to pQnt. For NEIL3 levels in extracts, see Extended Data Fig. 8c.
	Fig 4 |. MCM7 ubiquitin chain length influences ICL repair pathway choicea, Left, replication of pICLPt or pICLAP in undepleted extract supplemented with buffer, UbWT, or UbNoK was analyzed as in Fig. 1b. Right, Slow Figure 8 structures quantified as a percentage of total replication products. Note that UbWT and UbNoK delayed replication by ~20 minutes. Error bars, standard error of the mean from three independent experiments.b, Proteins associated with pICLPt during replication in undepleted extract in the presence of p97i and Culi and UbWT or UbNoK, as indicated. Black bar, longer ubiquitin chains sensitive to UbNoK addition.c, Model for hierarchical activation of the NEIL3 and FA pathways by TRAIP. Green, CMG helicase; purple, ubiquitin; orange, NEIL3.d, Clonogenic survival of wild-type, FANCL, NEIL3, or FANCL/NEIL3 CRISPR knockout HAP1 cells after exposure to cisplatin (top) or trioxsalen and UV-A irradiation (bottom). Error bars, standard error of the mean from at least three independent experiments.

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