Extended Data Fig. 9: Cisplatin sensitivity assay in DT40 cells and ICL repair in Xenopus egg extracts.

a, Clonogenic survival assay assessing cisplatin sensitivity of DT40 FANCD2^−/− cells complemented with either FANCD2^WT (WT), FANCD2^K563R (ubiquitination deficient), or KR helix variants (KKEE, EERR, EEEE). Results are based on 2 independent clones of each mutant and 6 replicates per clone, with mean and standard deviation plotted. P-values enclosed in brackets are for samples compared to WT (ns = not significant, **** P < 0.000001; see Supplementary Table 2 for all p values). The arginine mutants (KKEE and KKAA) fully rescue cisplatin sensitivity but the lysine mutants (EERR and AARR) do not (see also Fig. 6a). This is consistent with the lysine mutants having a stronger effect on stalling (Fig. 5c). However, since the EEEE and AAAA mutants show high sensitivity to cisplatin (compare EEEE to EERR and KKEE), it is likely that all four residues contribute to ss-dsDNA junction binding. Interestingly, cell lines expressing alanine substitutions were more sensitive to cisplatin than glutamic acid substitutions. We hypothesize that the glutamic acid substitutions do not bind DNA efficiently in cells and therefore act as nulls (like D2^−/−) whereas the alanine substitutions act as dominant negatives (like FANCD2^K563R) that bind DNA but are non-functional. For the colony survival assay, three cell dilutions (1:1, 1:10, and 1:100) were plated in duplicate for each cisplatin concentration. This resulted in a maximum of six replicates per concentration. Additionally, two clones were tested for each mutant. This brought the theoretical maximum number of observations to N = 12 for each cisplatin concentration, including the untreated control. However, some conditions were excluded due to limitations in counting at very high or very low colony numbers. For example, the WT DT40 at dilution 1:1 (untreated) was too confluent for accurate counting, and no colonies were found in the replicates of DT40 K563R at a 10 µM cisplatin for cell dilution of 1:100. Consequently, the final number of observations for these examples is N = 8. Statistical testing was performed by the R package CFAssay using two way analysis of the cell survival data by fitting a linear quadratic model with maximum likelihood (all values reported in Supplementary Table 2). b, ICL repair in Xenopus egg extracts. Mock-depleted (Mock), and FANCD2-depleted (ΔD2) NPE complemented with wild-type FANCD2 (ΔD2 + D2^WT) or mutant FANCD2 (ΔD2 + D2^EEEE), were analyzed by western blot using α-FANCD2 antibody (top left blot). These extracts were used to replicate pICL. Absolute ICL repair efficiency was calculated and plotted (right panel). Dotted line indicates SapI fragments from contaminating uncrosslinked plasmid present in pICL preparations. Total extract samples were collected at indicated timepoints and analyzed by western blot using α-FANCD2 (bottom left blot). Note that the basic KR helix residues tested in the chicken system (KKRR) are KRRR in xlFANCD2. Representative of 2 independent experiments (n = 2). c, Repeat of experiment shown in panel b and Fig. 6b including all FANCD2 mutants (ΔD2 + D2^EEEE, ΔD2 + D2^EERR and ΔD2 + D2^KREE). Note that the 30-minute timepoint of ΔD2 + D2^KREE is not shown due to poor digestion of this sample; we can assume that no repair has taken place at 30 min since this is the case at 60 min. Representative of 2 independent experiments (n = 2). For gel source data see Supplementary Fig. 1.