Figure S1. Replication Fork Reversal Observed During ICL Repair. ... (B) Predicted action of XPF and SNM1A on a converged fork structure. If both leading ...
Cell Reports, Volume 23
Supplemental Information
Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading Ravindra Amunugama, Smaranda Willcox, R. Alex Wu, Ummi B. Abdullah, Afaf H. ElSagheer, Tom Brown, Peter J. McHugh, Jack D. Griffith, and Johannes C. Walter
Figure S1
C pICL
A
B XPF SNM1A
Undamaged plasmid
RPA
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Non-productive incisions Figure-8
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Figure S1. Replication Fork Reversal Observed During ICL Repair. Related to Figure 1. (A) Model depicting ICL unhooking by XPF and SNM1A, as described in (Abdulla et al. 2017). (B) Predicted action of XPF and SNM1A on a converged fork structure. If both leading strands advance to the -1 position, there would be no duplex DNA surrounding the ICL, which may preclude XPFERCC1 activity (not shown). If one leading strand remains arrested at the -20 position (Räschle et al., 2008), XPF in conjunction with SNM1A would probably incise both forks on the leading strand template, as shown, leading to breakage of both sister chromatids and a non-productive repair outcome. (C) Schematic representation of pICL repair intermediates. Structures arising from breakage are indicated in grey boxes. See text for details. (D) Quantification of all DNA structures from the time point at 45 minutes in the experiment shown in Figure 1C. Circular molecules are likely contaminated plasmids lacking an ICL or pICLs that have undergone repair. Sigma structures are molecules resulting from breakage or very early incisions. Linear species are structures resulting from breakage or incision and subsequent processing. Uninterpretable structures possibly contain a mixture of pre-incision structures and molecules that have undergone incisions. Therefore, for the quantification of pre-incision species shown in Figures 1C, 2D, 2E the uninterpretable structures are not considered. (E) Quantification of pre-incision structures and the uninterpretable molecules from the same time point shown in (D). (F) Quantification of the reannealed duplex region in plasmids containing a reversed fork. The median length is indicated with a black bar. Contour lengths of the reannealed duplex (See cartoon) were measured using ImageJ.
In extract
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Figure S2. Fork Convergence and CMG Unloading are Required for Fork Reversal During ICL Repair. Related to Figure 2 and Figure 3. (A) Top panel: pICL or pICLLacO was replicated using NPE containing [α-32P] dATP and vehicle, p97i, or LacR, and analyzed by agarose gel electrophoresis. SC, supercoiled; OC, open circular. Red arrowhead, slow Figure 8; Blue arrowhead, fast Figure 8; green arrowhead, reversed fork. See panel B for an explanation of these species. The structure of the species labeled “theta” is shown in Figure 2A. Bottom panel: Nascent strand analysis of pICL or pICLLacO repair intermediates by denaturing polyacrylamide gel electrophoresis (PAGE) in mock-treated, p97i-treated, or LacR-treated extracts. pICL repair intermediates were digested with the restriction enzyme AflIII, whereas pICL LacO repair intermediates were digested with AflIII and EcoRI. Schematic representation of leading strand species generated after digestion of pICL or pICL LacO repair intermediates with the restriction enzyme AflIII is also illustrated. For clarity, only the rightward fork is shown. (B) Proposed structure of slow Figure 8, fast Figure 8, and intermediate mobility (reversed fork) species shown in Figure 3A. When forks first converge on an ICL, a Figure 8 species is generated that migrates slowly in the gel (i). We propose that when DNA is extracted before CMG has dissociated, the ssDNA protected by the stalled CMGs re-anneals, leading to catenation of the sister chromatids and retardation of their mobility (ii). Consistent with this interpretation, the slow Figure 8 persists when extracts are treated with p97i (panel A; (Semlow et al., 2016; Fullbright et al., 2016)), we observed catenated dimers via EM in the presence of p97i (Figure 2Dii), and treatment of the slow Figure 8 with topoisomerase II converted it to a fast Figure 8 (panel C). When CMG unloads as part of the ICL repair reaction in egg extract, parental strands reanneal, and any resulting catenation is relaxed by endogenous topoisomerase II (iv), so that the structure migrates as a fast Figure 8, even after deproteinization (v). After leading strands advance to the -1 position (vi), the parental strands cannot reanneal, so there is no catenation upon protein extraction, again resulting in a fast mobility Figure 8 (vii). Finally, one of the forks undergoes reversal (viii-ix), generating the species with intermediate mobility, which we show below is a reversed fork. (C) To examine the effect of topology on the mobility of the Figure 8 structures shown in Figure 3A, an undamaged plasmid (pCTRL) or pICL was replicated in the presence of vehicle or p97i, and samples
were extracted at the indicated times. DNA was either treated with buffer or 0.1U/µL Topoisomerase II (Topogen) in a 10 µL reaction at 37°C for 1 hour. Samples were deproteinized and re-extracted before analysis by agarose gel electrophoresis. This analysis showed that the slow Figure 8 was converted to the fast Figure 8 by topoisomerase II (lanes 5-7 and 11-13, and panel B, ii-iii). SC, supercoiled; OC, open circular. Similar results were seen in one other independent experiment. (D) To address whether the structures with intermediate mobility in Figure 3A represent reversed forks we treated DNA with the bacterial Holliday junction resolvase RuvC. pICL was replicated in NPE, and at the indicated times, DNA was recovered, digested with RuvC, and analyzed by agarose gel electrophoresis. RuvC largely digested these intermediate-mobility structures (compare green arrowhead in lanes 4 and 8), whereas it did not affect slow Figure 8s (compare lanes 1-2 and 5-6). The digestion of fast Figure 8s by RuvC might be due to small amounts of spontaneous branch migration after DNA extraction, which would yield Holliday junctions cleavable by RuvC. SC, supercoiled; OC, open circular. Similar results were seen in two other independent experiments. (E) The indicated model DNA substrates were radiolabeled, digested with RuvC, analyzed by native PAGE, and subjected to autoradiography. The gel shows that under the conditions used in panel (D), RuvC specifically digests reversed forks. (F) To further examine the structure of the intermediate mobility species, pICL was replicated in NPE for 70 min. DNA was recovered, deproteinized, and separated by agarose gel electrophoresis. The reversed forks (red box) were excised from the gel after staining with SYBR gold. (G) The DNA extracted from the gel in panel (F) was subjected to EM image. ~ 83% of the intact molecules observed contained a reversed fork, although there were broken molecules in this preparation, probably resulting from gel extraction (inset). We conclude that the intermediate species detected on native agarose gels consist largely of reversed forks. (H) Nascent strand analysis of pICL repair intermediates by denaturing PAGE in extract supplemented with buffer, araCTP, or aphidicholin fifteen minutes after initiation of replication. pICL repair intermediates were digested with the restriction enzyme AflIII and analyzed as in panel (A). For clarity only the rightward fork is shown.
Figure S3 k Δ FA N C I-F A N C D 2
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Figure S3. FANCD2 Depletion Does Not Cause a Reversal Defect. Related to Figure 4. (A) Mock-depleted NPE and FANCD2-depleted NPE were blotted for FANCD2. A non-specific band served as a loading control. (B) pICL was replicated in Mock-depleted and FANCD2-depleted egg extracts in the presence of [32Pa]dATP, and products were analyzed by native agarose gel electrophoresis. SC, supercoiled; OC, open circular. (C) EM image of pICL repair intermediates in a FANCI-FANCD2 depleted reaction at 150 min. Brown arrowhead, sigma structures resulting from fork breakage. (D) DNA Substrates used in the Nuclease Assay Shown in Figure 4D. The DNA sequences of oligonucleotides used to generate each substrate are indicated in the Supplemental Experimental Procedures, according to the names shown in blue. The 3’-radioabel is indicated with a red asterisk. (E) An AP-pICL plasmid was replicated in NPE containing [α-32P] dATP and repair intermediates were separated on a native agarose gel and visualized by autoradiography. SC, supercoiled; OC, open circular. (F) EM images of AP-ICL repair intermediates at the indicated time points.
Figure S4
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Pol
ICL
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Fork Reversal
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Second End Capture and Branch Migration
Branch migration and Resolution
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Figure S4. New Model for Incision-Dependent ICL Repair. Related to Discussion. Upon fork convergence, the CMG complex is unloaded (i-ii), generating an X-shaped structure (ii) that is not a suitable structure for XPF incision. One of the two converged forks then undergoes reversal (iii), placing the ICL into the context of a single fork, which is cleaved by the SLX4-XPF-ERCC1 complex on the dsDNA side of the lesion (iv). The incision by XPF creates an entry point for a second nuclease (probably SNM1A) that digests in the 5ʹ to 3ʹ direction past the ICL (v), unhooking the crosslink. In the primary pathway observed in extracts, the reversed leading strand is restored to its original location (vi), allowing its extension by translesion DNA polymerases past the remaining mono-adduct (vii). Finally, the double stranded break is repaired by homologous recombination (HR, viii-x). In a potentially distinct pathway (for which we have no direct evidence), there is no restoration of the leading strand to its original location, and the incised template strand is extended during TLS (vi’). The resulting one-ended dsDNA end undergoes strand invasion (vii’), branch migration, and resolution (viii’). Ligation of the reversed leading strand and NER would complete repair (ix’). Such an alternative mechanism might contribute to ICLinduced sister chromatid exchanges (SCE) seen in hyper-recombinogenic cells such as chicken DT40 cells (Legerski, 2010; Thompson and Hinz, 2009). In cells, a single fork usually collides with ICLs, leading to replication fork traverse (top left; (Huang et al., 2013)). We speculate that this creates a structure equivalent to the one formed after fork convergence and CMG unloading, followed by fork reversal and the subsequent steps depicted for the fork convergence pathway.
Supplemental Experimental Procedures Xenopus egg extracts and DNA replication Xenopus egg extracts were prepared essentially as described (Lebofsky et al., 2009; Walter et al., 1998). For replication, a plasmid containing a site-specific cisplatin interstrand (pICL) (Räschle et al., 2008; Knipscheer et al., 2009), or pICL with an array of 48 Lac operator sequences (pICL LacO) (Zhang et al., 2015) was licensed for 30 minutes at room temperature in high-speed supernatant (HSS) of egg cytoplasm at a final concentration of 7.5 ng/ µL and 0.375 ng/ µL, respectively. Next, two volumes of nucleoplasmic extract (NPE) was added to initiate replication. For radioactive labeling of nascent DNA, reactions were supplemented with [α-32P] dATP. For replication analysis, at the indicated time-points, 1 µL of reaction mixture was added into five volumes of replication stop buffer I (80 mM Tris– HCl (pH 8.0), 8 mM EDTA, 5 % SDS, 0.13 % phosphoric acid, 10 % ficoll and 0.1 % bromophenol blue). Reaction intermediates were analyzed after proteinase-K treatment and electrophoresis on a 0.8% agarose gel. For quantification, gels were dried and exposed to a phosphoimager. For analysis of replication and repair intermediates, at the indicated time-points 3-6 µL of the reaction mixture was diluted into ten volumes of stop buffer II (80 mM Tris– HCl (pH 8.0), 25 mM EDTA and 0.5 % SDS). After RNaseA treatment samples were deproteinized with Proteinase-K, and purified by phenol-chloroform extraction and ethanol precipitation. The purified DNA pellet was resuspended in 3-6 µL of 10 mM Tris-HCl (pH 8.0) for further analysis. To block CMG unloading p97 inhibitor (NMS 873) (Magnaghi et al., 2013) was incubated with NPE at a final concentration of 200 µM. LacR treatment was as described previously (Zhang et al., 2015).
Incision Assay The incision assay was performed as described previously (Klein Douwel et al., 2014).
Nascent Strand Analysis For nascent strand analysis, purified pICL intermediates were digested with AflIII and pICLLacO intermediates were digested with AflIII and EcoRI, followed by addition of 0.5 volumes of denaturing PAGE Gel Loading Buffer II (Life Technologies). Radiolabeled nascent strands were separated on a 7% denaturing polyacrylamide gel, transferred to filter paper, dried, and visualized using a phosphorimager.
Sequencing ladders were generated with primer S (5ʹ-CATGTTTTACTAGCCAGATTTTTCCTCCTCT CCTG-3ʹ) using the Cycle Sequencing Kit (USB, Cleveland).
Lagging Strand Resection Analysis Purified pICLLacO repair intermediates were digested with BsaI and analyzed by 7% denaturing polyacrylamide gel electrophoresis. Sequencing ladders were generated with primer R (5ʹCGGTATCATTGCAGCACTGGGGCCA-3ʹ).
Crosslinking of DNA and sample preparation for electron microscopy analysis pICL was licensed at 15 ng/ µL final concentration in HSS, and replication was initiated as described above. At the indicated times after NPE addition, a 20 µL of reaction mixture was added into four volumes of replication stop buffer III (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 6.7 mM MgCl2 and 1% SDS). The high [Mg2+] was added to inhibit branch migration before crosslinking (Panyutin and Hsieh, 1994). Samples were then transferred to a 96–well plate and supplemented with Trimethylpsoralen (TMP, Sigma; 10 µg/ mL final concentration). The plate was incubated on a freezer pack for 5 min in the dark and irradiated with a hand-held UV lamp at 365 nM for 15min. TMP addition and crosslinking were repeated twice and DNA was purified as described previously. After cross-linking, roughly half of all T-A dinucleotides were cross-linked as determined by restriction enzyme analysis, indicating a very high density of TMP cross-linking (data not shown). Use of glycogen was avoided during the ethanol precipitation step as it interferes with EM imaging. DNA was incubated with 1µg E. coli SSB protein in 20 mM HEPES (pH 7.4), 100 mM NaCl, in a 20-50 µL volume, on ice for 15 minutes, followed by fixation of DNA-protein complexes with 0.3% glutaraldehyde for 5 min at room temperature. The sample was then passed over a 2-ml column of 6% agarose beads (ABT inc, Burgos Spain) previously equilibrated with TE buffer (10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA-NaOH), to eliminate unwanted salts and unbound proteins. Eluted fractions enriched for DNA-protein complexes were collected and mounted on grids as described below for EM analysis. For EM analysis of gel extracted DNA, samples were crosslinked as above and separated by electrophoresis on a 0.8% agarose gel. Gel was stained with SYBR gold
(Thermofisher) and the indicated band was excised, and DNA was extracted using Tini spin columns (Enzymax). Eluted DNA was then incubated with SSB as above and processed for EM. In a previous analysis of ICL repair by EM, we did not report the presence of reversed forks (Räschle et al., 2008). In that experiment, the DNA was not cross-linked or supplemented with SSB before EM. However, re-examination of the electron micrographs showed indirect evidence of fork reversal: at early time points (30 minutes), we detected almost exclusively Figure 8 structures (Räschle et al., 2008), whereas at later time points, these were replaced by late theta structures that likely represented reversed forks. Due to the absence of SSB, the reversed strand was not visible, and we therefore overlooked fork reversal in our original analysis. Thus, although structures consistent with fork reversal appear to be detected without psoralen cross-linking (see also agarose gel electrophoresis analysis), we included this step in all EM experiments to minimize any branch migration. We also included SSB to detect the reversed fork.
Surface spreading DNA with cytochrome C for EM Samples were prepared as described (Thresher and Griffith, 1992). Briefly, SSB-containing DNA was mixed with a buffer containing 250 mM ammonium acetate in a total volume of 48.2 µL. Just before spreading, 1.8 µL of 200 µg/mL cytochrome C solution was added, mixed briefly, and the entire volume spotted immediately on a piece of parafilm. After a 5-10 min incubation, the surface of the drop was touched lightly by a copper grid previously coated with parlodion film. The grid was then dehydrated for 30 sec in 75% ethanol and 30 sec in 90% ethanol. Following quick air-drying, the grid was placed in a high vacuum Denton evaporator and a thin layer of platinum (80%): palladium (20%) evaporated on the sample at an angle of 8 degrees at 2x10-6 torr. For Figure S3F, rotary shadowing was performed with a Leica ACE600 evaporator and 100% platinum. Finally, the grid was covered with a thin layer of carbon to aid in stabilizing the parlodion film. Samples were examined in an FEI T12 transmission electron microscope (TEM) equipped with a Gatan 2k SC200 CCD camera at 40 kV or a JEOL 1200EX TEM equipped with a 2k CCD camera (Advanced Microscopy Techniques).
Antibodies and Immunodepletion The polyclonal antibodies against FANCD2 (Knipscheer et al., 2009), FANCI (Duxin et al., 2014), and depletion of FANCI-FANCD2 (Semlow et al., 2016) were described previously.
Generation of Radiolabeled DNA substrates for the Nuclease Assay For 3’-end radiolabeling, 50 pmoles of DNA oligonucleotide was incubated with 1 unit of terminal deoxytransferase (TdT) (NEB) in the presence of 3.3 pmoles [α-32P]dATP in 25 µl reaction volume for 1 hour at 37°C. Then, the reaction mixture was loaded in a Bio-Gel P-6 spin column (Bio-Rad), centrifuged at 1,000xg for 4 minutes and diluted with nuclease-free water to a concentration of 500 nM. 10 µl of 500 nM radiolabeled oligonucleotide was annealed with unlabeled oligonucleotide(s) in 50 µl reaction volume by boiling at 95°C for 5 minutes and gradually cooled to below 30°C in the presence of annealing buffer (10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA). The quality of every batch of radiolabeled DNA substrates generated was analyzed using 10% non-denaturing polyacrylamide (PAGE) gel. See Figure S3D for structures used in the nuclease assay.
Code
Sequence of DNA oligonucleotides
6
5’-AAGGGGAGAAGAGAAAGAAAGG-3’
12
5’-CTGCCATGTCACTATGGTTAGCCGT-3’
13
5’-AGTAACGTGACAGCGTTGGCTAAGT-3’
CL-1
5’-ATAAATATTTTTTATTAATAATAGATCACCTTTCTTTCTCTTCTCCCCTT-3’ 3'P-TATTTATAAAAAATAATTATTATCTAGTCCTTCCTTCCTCTCCTCCCCTT-5'Bio
X
5’-GGTACAGTGATACCAATCGGCATACGGCTAACCATAGTGACATGG-3’ 3'-TTGCACTGTCGCAACCGATTCATACTTAGCCAACGCTGTCACGTT-5‘
Sequence of DNA oligonucleotides used to generate the DNA substrates for this study. Red lines represent a triazole interstrand crosslink; non-complementary regions are underlined; ‘P’ stands for 3’-phosphate; ‘Bio’ stands for 5’-biotin. Substrates annealed as indicated in Figure S3D.
Nuclease Assay 10 nM DNA substrates were pre-incubated in the presence or absence of 80 nM RPA (92.8 ng; purified human RPA obtained as described in (Abdulla et al. 2017)) on ice for 10 minutes in nuclease buffer (25 mM HEPES pH 8.0, 40 mM NaCl, 10% glycerol, 0.5 mM ß-mercaptoethanol, 0.1 mg/ml BSA and 0.4 mM
MnCl2), followed by the addition of 40 nM (62 ng) XPF-ERCC1 (Abdulla et al. 2017) and incubation at 30oC for 1 hour. The total reaction volume was 10 µL. The reaction was quenched by adding 3 µL stop solution (95 % formamide/5 % EDTA) and heating at 95oC for 5 minutes. Samples were loaded onto 10 % denaturing (7M urea) PAGE (19:1 Acylamide/Bis) gels containing 1x TBE and run for 1 hour at 525 V. Gels were fixed in fixing solution (40% methanol/ 20% Acetic Acid/5% glycerol) for 1 hour and dried in a Gel Dryer at 50oC for 4 hours or 80oC for 2 hours. Reaction products were visualized by phosphorimager.
RuvC Digestion The sequence of oligonucleotides 1-4 used to synthesize the model reversed fork (RF) structure is identical to the junction substrates X4 used previously (Van Gool et al, 1998). Briefly, Oligonucleotide 1 was 5’-end labeled with [γ-32P]ATP and T4 polynucleotide kinase (NEB), purified using a Bio-spin 6 column (Biorad) and annealed with a 5-fold molar excess of oligonucleotides 2, 3, and 4 in SSC buffer. Annealing was performed in a thermal cycler after heating to 95°C for 5min and gradually cooling to 25°C. For the production of 5’- flap substrate oligonucleotides 1, 2, and 3 (residues 1-25) were used. The resected, reversed fork (Res-RF) was produced by annealing oligonucleotides 1, 2, 3 and 4 (residues 25-50). All substrates were gel purified after separation via 8% native PAGE. For the analysis of pICL repair intermediates, reactions were stopped in stop buffer III and extracted as described above. Purified repair intermediates or the model substrates were treated with 25 nM RuvC (Abcam) or buffer in a 10 µL reaction containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 100 µg/ mL BSA and 1 mM DTT at 37°C for 30 min. Model substrates were deproteinized with 1 mg/ mL Proteinase-K and 0.5% SDS, and analyzed by 8% native PAGE followed by autoradiography. pICL repair intermediates were deproteinized, extracted with phenol-chloroform, ethanol precipitated, and analyzed on a 0.8% agarose gel electrophoresis.
Supplemental References Duxin, J.P., Dewar, J.M., Yardimci, H., and Walter, J.C. (2014). Repair of a DNA-protein crosslink by replication-coupled proteolysis. Cell 159, 346–357. Legerski, R.J. (2010). Repair of DNA interstrand cross-links during S phase of the mammalian cell cycle. Environ. Mol. Mutagen. 51, 540–551. Magnaghi, P., D’Alessio, R., Valsasina, B., Avanzi, N., Rizzi, S., Asa, D., Gasparri, F., Cozzi, L., Cucchi, U., Orrenius, C., et al. (2013). Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat. Chem. Biol. 9, 548–556. Panyutin, I.G., and Hsieh, P. (1994). The kinetics of spontaneous DNA branch migration. Proc. Natl. Acad. Sci. USA 91, 2021–2025. Thresher, R., and Griffith, J. (1992). Electron microscopic visualization of DNA and DNA-protein complexes as adjunct to biochemical studies. Methods Enzymol. 211, 481–490. van Gool, A.J., Shah, R., Mézard, C., and West, S.C. (1998). Functional interactions between the holliday junction resolvase and the branch migration motor of Escherichia coli. EMBO J. 17, 1838– 1845.
