Fig. 1.

(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)).