(A) Schematic representation of lesion bypass in ICL repair (12). (B) Purified FANCI-FANCD2^WT and FANCI-FANCD2^K562R stained with Coomassie blue. (C) Reciprocal co-immunoprecipitation of xlFANCI and xlFANCD2 from Xenopus egg extract. Input (I) and supernatant (S) (0.2 μl extract), or precipitated proteins (P, from 1 μl extract) were blotted for FANCI and FANCD2. PI: pre-Immune serum. (D) Replication-dependent binding of FANCI-FANCD2 to damaged chromatin. Crosslinked sperm chromatin was replicated in undepleted extracts supplemented with FANCI-FANCD2^WT or FANCI-FANCD2^K562R (310 nM). Chromatin-bound fractions (from 2 μl extract) or total extract (0.2 μl), were analyzed by Western blotting with anti-strep-tag (to visualize recombinant FANCD2), anti-FLAG-tag (recombinant FANCI), and anti-RCC1 (loading control) antibodies. Where indicated, replication was inhibited with Geminin. Note that only ubiquitylated FANCD2 binds chromatin, while both ubiquitylated and unubiquitylated FANCI bind (see also (14)).

FANCD2 and its ubiquitylation are required for ICL repair. (A) Sequence surrounding the ICL and relevant restriction sites of pICL. (B) pICL (2.3 ng/μl) was replicated in mock-depleted extract, FANCD2-depleted extract (ΔFANCD2) or ΔFANCD2 extract supplemented with 375 nM FANCI-FANCD2^WT or FANCI-FANCD2^K562R, and repair efficiency was plotted. For primary data and calculation of repair efficiency, see Fig. S5A.

Insertion of a nucleotide across from the damaged base is compromised in FANCD2-depleted extracts (A) Schematic representation of leading strand intermediates generated after pICL digestion with AflIII. (B) Samples from the reactions described in Fig. 2B were digested with AflIII, separated on a sequencing gel alongside a ladder derived from extension of primer S (S, in panel A) on pControl, and visualized via autoradiography. Nascent strands generated by the rightward (red) and leftward (green) replication forks are indicated to the right and illustrated in (A). −1 and extension products observed in (B) were quantified and graphed in (C) and (D), respectively.

ICL-proximal incisions are compromised in FANCD2-depleted extracts. (A) Schematic representation of predicted fragments generated by HincII digestion of pICL, before and after dual incisions and lesion bypass. Parental strands in black, nascent strands in grey. (B) pICL (2.5 ng/μl) was replicated in mock-depleted extract, FANCD2-depleted extract (ΔFANCD2) alone or supplemented with 386 nM FANCI-FANCD2^WT. Samples were digested with HincII and separated on a denaturing agarose gel. Both parental and nascent strands are detected by Southern blotting. Unreplicated pControl and pICL were optionally digested with the indicated enzymes and used as size markers for arm/linear species and X-shaped molecules, respectively (lanes 1–3). Note the small amount of linear products in lane 3 (3% of the total), which represents contaminating non-crosslinked plasmids. X-structures and linear species observed in (B) were quantified and graphed in (C) and (D), respectively.