Extended Data Fig. 1: Acetaldehyde ICLs are stable and are repaired in Xenopus egg extracts.

a, Scheme for the synthesis of the precursor, 4-(R)-aminopentane-1,2-diol. b, Site-specific synthesis of a 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 were performed. d, Confirmation of AA-ICL formation by MALDI MS. The peak at m/z 12,370.2 represents the imine or pyrimidopurinone form. Two further peaks at m/z 5,979.41 and 6,409.74 equate to masses for the two parent oligonucleotides, consistent with the mass of the carbinolamine form after dissociation back to PdG and dG under the desorption/ionization conditions. Three independent experiments were performed. 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, AA_NAT-ICL is susceptible to hydrolysis in 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 were performed. g, Scheme depicting the type and position of the DNA lesions used in this study. Duplex DNA with or without the indicated lesions was annealed into a backbone vector to generate circular plasmids with or without damage. h, To determine the percentage of crosslinks, the ICL-containing plasmids were digested with NotI, labelled at the 3′-end by end filling with [α-³²P]dCTP, and separated by denaturing PAGE. Crosslinked DNA (88 nt) shows slower mobility compared with non-crosslinked DNA (44 nt). The percentage of crosslinks was calculated by comparing the 88-nt product with the 44-nt products. Two independent experiments were performed. i, AA_NAT-ICL and Pt-ICL are stable in Xenopus egg extracts. Plasmids were incubated in a high-speed supernatant (HSS) extract. DNA was extracted and analysed as described in h. Three independent experiments were performed. j, Solution structures of a cisplatin ICL and a reduced form of an acetaldehyde ICL (PDB: 1DDP²² and 2HMD²³, cartoon representation generated in PyMOL). k, The indicated plasmids were replicated in Xenopus egg extracts and repair intermediates were digested with NotI, labelled at the 3′-end, and resolved by denaturing PAGE. The increase in intensity of the 44-nt band over time indicates ongoing replication and repair. A higher mobility band, probably generated from end-joining activity in some extracts, is indicated by an asterisk. This gel is the independent experimental duplicate of that in Fig. 1e. l, Quantification of repair based on the intensity of the 44-nt product on the gel in k, as described in the Supplementary Methods. This graph is the independent experimental duplicate of that in Fig. 1f. Additional replicates of these experiments are presented in Fig. 2a, Extended Data Figs. 2k–m, 4b.