Alcohol-derived DNA crosslinks are repaired by two distinct mechanisms

Abstract

Acetaldehyde is a highly reactive, DNA-damaging metabolite that is produced upon alcohol consumption¹. Impaired detoxification of acetaldehyde is common in the Asian population, and is associated with alcohol-related cancers^1,2. Cells are protected against acetaldehyde-induced damage by DNA crosslink repair, which when impaired causes Fanconi anaemia (FA), a disease resulting in failure to produce blood cells and a predisposition to cancer^3,4. The combined inactivation of acetaldehyde detoxification and the FA pathway induces mutation, accelerates malignancies and causes the rapid attrition of blood stem cells^5-7. However, the nature of the DNA damage induced by acetaldehyde and how this is repaired remains a key question. Here we generate acetaldehyde-induced DNA interstrand crosslinks and determine their repair mechanism in Xenopus egg extracts. We find that two replication-coupled pathways repair these lesions. The first is the FA pathway, which operates using excision-analogous to the mechanism used to repair the interstrand crosslinks caused by the chemotherapeutic agent cisplatin. However, the repair of acetaldehyde-induced crosslinks results in increased mutation frequency and an altered mutational spectrum compared with the repair of cisplatin-induced crosslinks. The second repair mechanism requires replication fork convergence, but does not involve DNA incisions-instead the acetaldehyde crosslink itself is broken. The Y-family DNA polymerase REV1 completes repair of the crosslink, culminating in a distinct mutational spectrum. These results define the repair pathways of DNA interstrand crosslinks caused by an endogenous and alcohol-derived metabolite, and identify an excision-independent mechanism.

Extended Data Figure 1. Acetaldehyde ICLs are stable and repaired in Xenopus egg extract.

(a) Scheme for the synthesis of the precursor, 4-(R)-Aminopentane-1,2-diol. (b) Site-specific synthesis of an (R)-α-CH3-γ-OH-1,N2-Propano-2’-deoxygunosine (PdG) adduct in a DNA oligonucleotide. (c) Denaturing PAGE showing crosslink formation between dG and PdG, but not between dG and Inosine (Ino), which lacks an N2 amine. Two independent experiments. (d) Confirmation of AA-ICL formation by MALDI. The peak at 12,370.2 represents imine or pyrimidopurinone form. Two further peaks at 5979.41 and 6409.74 Da, equate to masses for the two parent oligos, consistent with the mass of the carbinolamine form after dissociation back to PdG and dG upon desorption/ionization conditions. Three independent experiments. (e) Stability of AA_NAT-ICL as a function of temperature and time, as determined by radiolabelling (α-³²P-dCTP) and resolution by denaturing PAGE. Error Bars represent S.E.M., from three independent experiments. (f) The AA_NAT-ICL is susceptible to hydrolysis with aqueous acid, whereas AA_RED-ICL is stable. Pre-purification crosslink reactions were incubated with or without formic acid and products were resolved by denaturing PAGE. Three independent experiments. (g) Scheme depicting the type and position of the DNA lesions used in this study. Duplex DNA with or without the indicated lesion was annealed into a backbone vector to generate a circular plasmid with lesion. (h) To determine the percentage of crosslinks the ICL-containing plasmids were digested with NotI, 3’ labelled by end filling with α-³²P-dCTP, and separated by denaturing PAGE. Crosslinked DNA (88 nt) will show slower mobility compared to non-crosslinked DNA (44 nt). The crosslink percentage was calculated by comparing the 88 nt with the 44 nt products. Two independent experiments. (i) ICL-AA_NAT and ICL-Pt are stable in Xenopus egg extract. Plasmids were incubated in a highspeed supernatant (HSS) extract. DNA was extracted and analyzed as described in (h). Three independent experiments. (j) Solution structures of a cisplatin ICL and a reduced form of an acetaldehyde ICL (pdb: 1DDP and 2HMD, cartoon representation generated in PyMol). (k) Indicated plasmids were replicated in Xenopus egg extract, repair intermediates were digested with NotI, 3’ labelled, and resolved by denaturing PAGE. Increase in intensity of the 44 nt band in time indicates ongoing replication and repair. A higher mobility band, probably generated from end-joining activity in some extracts is indicated (*). Independent experimental duplicate of Main Fig. 1e (l) Quantification of repair based on the intensity of 44 nt product on gel in (k), as described in methods (Supplementary Information Methods). Independent experimental duplicate of Main Fig 1f Additional replicates of this experiments are presented in Main Fig. 2a, Extended Data Figs. 2k-m, and Extended Data Fig. 4b.

Extended Data Figure 2. Acetaldehyde ICLs are repaired by Fanconi-dependent and independent mechanisms.

(a) Model for ICL repair by the FA pathway. Upon convergence of two replication forks at the crosslink, the CMG helicase is unloaded from the DNA to allow approach of one replication fork to the -1 position. Ubiquitylation of FANCD2 promotes the recruitment of the XPF-ERCC1-SLX4 (XES) complex to the ICL that enables nucleolytic incisions that unhook the crosslink. This step could be preceded by fork reversal of one of the stalled replication forks. Incisions generate a broken strand and a strand with an adduct, the latter is bypassed by TLS while the broken strand is repaired by homologous recombination. In mammalian cells, it has been shown that a single fork can pass over the ICL without unhooking, this ‘traverse’ gives rise to a structure that resembles the one generated after fork convergence and CMG unloading and could follow the same steps subsequently. (b) Indicated plasmids were replicated in Xenopus egg extract, reaction samples were analysed by western blot with FANCD2 antibody. Two independent experiments. (c) FANCD2 Western blot showing a titration of Xenopus egg extract compared to mock and FANCD2 depleted extract. Two independent experiments. (d) Indicated plasmids were replicated in Mock or FANCD2 depleted (ΔFANCD2) extract in the presence of α-³²P-dCTP. Repair products were digested by AflIII, separated on a sequencing gel alongside a ladder derived from extension primer S, and visualized by autoradiography. White arrow denotes –1 product that is 2 nt larger in pICL-Pt due to the position of the ICL. Three independent experiments. (e) Indicated plasmids were replicated in Mock or FANCD2 depleted (ΔFANCD2) extract, repair intermediates were digested with NotI, 3’ labelled, and resolved by denaturing PAGE. Quantification of repair based on the intensity of 44 nt product is shown in main Fig. 1g. (f) Independent experimental duplicate of Main Fig. 1g. (g) Independent experimental triplicate of Main Fig. 1g, but only using pICL-AA_NAT. (h) Plasmid pICL-AA_NAT was replicated in FANCD2 depleted extract, or FANCD2 depleted extract supplemented with recombinant FANCI-FANCD2 complex (ID). Reaction samples were resolved by native agarose gel, visualised by autoradiography. Replication/repair intermediates (RRI), open circle (OC), and supercoiled (SC) products are indicated. Stalled repair product (grey arrow) is indicated. Two independent experiments. (i) Indicated plasmids were replicated in Xenopus egg extract in presence or absence of p97i and the intermediates were resolved by native agarose gel electrophoresis. Stalled repair products (grey arrow) are indicated. Seven independent experiments. (j) The indicated plasmids were replicated in Xenopus egg extracts in presence or absence of p97i and repair intermediates were digested with NotI, 3’ labelled, and resolved by denaturing PAGE. Increase in intensity of the 44 nt band (white arrow) in time indicates ongoing replication and repair. A higher mobility band, probably generated from end-joining activity in some extracts is indicated (*). (k) Quantification of repair based on the intensity of 44 nt product on gel in (j) as indicated in methods (Supplemetary Information Methods). (l) Quantified independent experimental duplicate of (j). (m) Quantified independent experimental triplicate of (j).

Extended Data Figure 3. Reduced acetaldehyde ICLs are repaired by the Fanconi Pathway.

(a) MALDI data confirms identity and stability of the reduced ICL. Three independent experiments. (b) The indicated plasmids were replicated in Xenopus egg extracts in presence or absence of p97i and the repair intermediates were analysed using the NotI digestion assay. Increase in intensity of the 44 nt band indicates ongoing replication and repair. A band with higher mobility, probably generated from end joining activity in some extracts is indicated (*). Quantification of repair based on this gel is shown in Main Figure 1j. Three independent experiments. (c) Independent experimental duplicate of main figure 1j. (d) Independent experimental triplicate of main figure 1j.

Extended Data Figure 4. AA-ICL repair requires DNA replication and replication fork convergence.

(a) Indicated plasmids were replicated in Xenopus egg extracts in presence or absence of Geminin. Repair intermediates were digested with NotI, 3’ labelled, and resolved by denaturing PAGE. Quantification of repair based on the intensity of 44 nt product is shown in main Figure 2a. (b) Independent duplicate experiment of main Figure 2a. (c) As in (b). Independent triplicate experiment of main Fig. 2a but only representing pICL-AA_NAT (d) pICL-Pt-LacO was replicated in Xenopus egg extract containing α-³²P-dCTP, in presence or absence of LacR. The repair intermediates were digested by AflIII and EcoRI, separated on a sequencing gel and visualized by autoradiography. Two independent experiments. (e) The indicated plasmids were replicated in extract in presence or absence of LacR. Repair products were digested with NotI, 3’ labelled, and resolved by denaturing PAGE. (f) Quantification of repair based on the intensity of 44 nt product on gel in (e), as described in methods. (g) Independent experimental duplicate of (f). (h) Independent experimental triplicate (f).

Extended Data Figure 5. The alternate route of AA-ICL repair does not involve DNA excision.

(a) NEIL3 Western blot showing a titration of Xenopus egg extract compared to NEIL3 depleted extract and NEIL3 depleted extract supplemented with recombinant wildtype NEIL3 (WT) or catalytically inactive NEIL3 (MUT). Three independent experiments. (b) Indicated plasmids were replicated in NEIL3-depleted extract (ANEIL3) containing α-³²P-dCTP, supplemented with wild-type (WT) or catalytically inactive (MUT) NEIL3. Replication intermediates were resolved by native agarose gel electrophoresis and visualised by autoradiography. Three independent experiments. (c) Clonogenic survival of WT, NEIL3, FANCL or NEIL3/FANCL deficient human HAP1 cells after 2 h exposure to acetaldehyde. Three independent experiments. Measure of centre represents the average values, error bars were calculated as S.E.M. (d) The LD50 for the survival of WT and deficient HAP1 cells was calculated by regression analysis of the curves presented in (c). Measure of centre represents the LD50, error bars were calculated as standard error. Three independent experiment. (e) Quantification of the arm fragments resulting from APE1 treatment (AP sites) from the gel in Main Figure 2e. (f) Quantification of the APE1 arms as in (e), from an independent duplicate experiment without p97i addition. (g) Quantification of the APE1 arms as in (e), from an independent triplicate experiment without p97i addition. As a positive control we used a plasmid containing an abasic site-induced interstrand crosslink (pICL-AP) that is also repaired via the glycosylase NEIL3. (h) Quantification of the HincII arm fragments from the gel in Main Figure 2f. (i) Quantification of the HincII arms as in (h), from an independent duplicate experiment. (j) Quantification of the HincII arms as in (h), from an independent triplicate experiment. (k) Schematic representation of the formation of DNA adducts by unhooking incisions during ICL repair (left). These adducts are not removed during ICL repair in Xenopus egg extracts and can therefore be visualized. Plasmids were replicated in Xenopus egg extracts in the presence or absence of p97i. Late reaction samples were digested with AflIII and AseI and separated on a sequencing gel. Adducts on either the top or bottom strand (white arrow heads) were detected by strand-specific Southern blotting. Three independent experiments.

Extended Data Figure 6. Both routes of AA-ICL repair are mediated by REV1 and REV7.

(a) Indicated plasmids were replicated in Mock or REV1 depleted (ΔREV1) extracts containing α-³²P-dCTP, in the presence or absence of p97i. Reaction samples were digested by either AflIII or AflIII and BamHI, separated on a sequencing gel alongside a ladder derived from extension of primer S, and visualized by autoradiography. White arrows denote 0 products, dark grey arrows indicate -1 products, and light grey arrows indicate 0/-1 products (not separated). Two independent experiments. (b) Western blot detection of REV7 in REV1 (ΔREV1) or Mock depleted Xenopus egg extract. Two independent experiments. (c) Western blot detection of REV7 and REV1 in REV7 (ΔREV7) or Mock depleted Xenopus egg extract. Two independent experiments. (d) Indicated plasmids were replicated in mock or REV7 depleted (ΔREV7) extracts containing α-³²P-dCTP. Reaction samples were digested by either AflIII or AflIII and BamHI, separated on a sequencing gel and visualized by autoradiography. Two independent experiments. (e) Indicated plasmids were replicated in mock or REV1 depleted (ΔREV1) extracts containing α-³²P-dCTP. Reaction samples were digested by AflIII and BamHI, separated on a denaturing PAGE gel alongside a ladder derived from extension primer S, and visualized by autoradiography. (*) Indicate 121 nt background fragment caused by a second BamHI restriction site in the leftward fork. Two independent experiments.

Extended Data Figure 7. Mutagenic outcome of acetaldehyde crosslink repair.

(a) Frequency of nucleotide misincorporation in a 15 bp region flanking the lesions present in the indicated plasmids. Co-plotted are the mutation frequencies for the same plasmid that has not been replicated in Xenopus egg extract (NR) and for control vector, pCon. Strand specificity is lost because sample preparation involves PCR amplification, therefore, only the top sequence is indicated below the graphs. Related to Fig. 4. (b) Distribution and frequency of nucleotide misincorporations in a 15 bp region flanking the lesions present in the indicated plasmids. Independent duplicate sequencing experiment. The height of the bars represents the mutation frequency minus baseline mutations found in pCon. (c) Frequency of nucleotide misincorporations in a 15 bp region flanking the lesions present in the indicated plasmids (data from the same duplicate sequencing experiment as in b). Co-plotted is the mutation frequency for pCon.

Fig. 1. AA-ICL repair by Fanconi-dependent and independent mechanisms.

(a) Reaction scheme of AAnat-ICL formation. Two acetaldehydes react with deoxyguanine (dG) generating N2-deoxypropanoguanine (PdG), this reacts with a 5’-CpG guanine on the opposite strand. The AAnat-ICL exists in a three-state equilibrium. (b) Replication intermediates generated during ICL repair. (c) Plasmids were replicated in Xenopus egg extract, reaction products were resolved by native agarose gel and visualized by autoradiography. Figure 8 structures (F8), later replication/repair intermediates (RRI), open circle/supercoiled products (OC/SC, grey arrow) are indicated. Six independent experiments. (d) Scheme for the NotI ICL repair assay. Wavy lines: synthesized during repair. (e) Plasmids were replicated in extract, repair intermediates were isolated, NotI-digested, and resolved by denaturing PAGE. Accumulation of the 44 nt product (open arrow) indicates ongoing replication and repair. (*) Product probably generated from end-joining activity in some extracts. Ten independent experiments. (f) Quantification of repair based on the gels in (e), as described in methods (Supplementary Information Methods). Ten independent experiments. (g) Quantification of repair in mock or FANCD2-depleted extract. Based on gel in Extended Data Fig. 2e. Three independent experiments. (h) Scheme of the reduction of ICL-AAnat to ICL-AA_RED. (i) Plasmids were replicated in extract and products were resolved by native agarose gel. Three independent experiments. (j) Quantification of repair with or without p97i. Based on gel in Extended Data Fig. 3b. Three independent experiments.

Fig. 2. The alternate AA-ICL repair route requires DNA replication and fork convergence but no DNA excision.

(a) Quantification of repair with or without Geminin. Based on gel in Extended Data Fig. 4a. Three independent experiments. (b) Scheme of AA-ICL LacO plasmids. (c) Plasmids were replicated in extract with or without LacR. Repair intermediates were digested and separated on a sequencing gel. Grey arrows: –1 product, open arrows: –20 product. Two independent experiments. (d) Scheme of the assays used to detect base excision (left) and nucleotide excision (right) pathways in (e) and (f), respectively. DNA fragments formed after HincII/APE1 (left box) or HincII digestion (top and right box). (e) Plasmids were replicated in extract with p97i. Repair intermediates were digested and separated by native agarose gel. Red arrows: Arms from APE1 incisions. Quantification in Extended Data Fig. 5e, replicates in Extended Data Fig. 5f, g. Four independent experiments. (f) Plasmids were replicated in extract with or without p97i. Repair intermediates were digested and separated by native agarose gel. White arrows: Arms from backbone incisions. Quantification in Extended Data Fig 5h, replicates in Extended Data Fig. 5i, j. Six independent experiments.

Fig. 3. AA-ICL repair is mediated by REV1.

(a) Scheme of products detected on the sequencing gel in (c). (b) REV1 Western blot for mock and REV1-depleted extract compared to a titration of undepleted extract. Nine independent experiments. (c) Plasmids were replicated in mock or REV1-depleted (ΔREV1) extracts, repair intermediates were digested and separated on a sequencing gel alongside a sequencing ladder. White arrows: 0 products, dark grey arrows: –1 products, light grey arrows: 0/-1 products. Three independent experiments. (d) Scheme of the primer extension assay. Late repair products are digested with AflIII and BamHI and used as template for primer extension from labelled TOP or BOT (bottom) primers. (e) Primer extension products were separated by denaturing PAGE. Four independent experiments.

Fig. 4. AA-ICL repair causes point mutations.

(a) Scheme of the positions of DNA lesions and adducts (red) generated during repair. The FA pathway generates an adduct on the top or bottom strand, the second pathway for AA-ICL repair creates an adduct on one or both strands. The PdG monoadduct is present on the bottom strand. Mutations are generated during translesion synthesis (blue N’s and arrow). (b) Distribution and frequency of nucleotide mis-incorporation in a 15 bp region flanking the lesions. Strand specificity is lost because sample preparation involves PCR amplification, therefore, only the top sequence is indicated below the graphs. Mutation frequency is minus baseline mutations found in pCon.