Figure 2.
The C-terminal region of FANCD2 is responsible for histone binding and nucleosome assembly. (A) Schematic representations of full-length cFANCD2, and the cFANCD2(1-1389), cFANCD2(1-1267), cFANCD2(1-1167), cFANCD2(669-1439), and cFANCD2(953-1439) deletion mutants. The cFANCD2 domains, solenoid 1, helical domain 1, solenoid 2, helical domain 2, solenoid 3, and solenoid 4, are denoted as S1, HD1, S2, HD2, S3, and S4, respectively (Joo et al, 2011). The FANCD2 C-terminal acidic region is coloured red. The amino-acid sequences of the C-terminal regions of the Homo sapiens, Mus musculus, Gallus gallus, Xenopus laevis, Danio rerio, and Drosophila melanogaster FANCD2 proteins are aligned. The highly conserved residues are coloured red. The mutated residues in cFANCD2(R1336A/K1346A) and cFANCD2(D1350A/E1365A/E1382A) are indicated by orange and purple arrowheads, respectively. (B) The H3/H4 beads were incubated with extracts of HEK293T cells, producing either GFP-tagged cFANCD2, cFANCD2(1-1389), cFANCD2(1-1267), cFANCD2(1-1167), cFANCD2(669-1439), cFANCD2(953-1439), cFANCD2(R305W), cFANCD2(K563R), cFANCD2(D1350A/E1365A/E1382A), or cFANCD2(R1336A/K1346A). Proteins bound to the beads were detected by western blotting with an anti-chFANCD2 (polyclonal) antibody. The bottom panel indicates negative control experiments with beads lacking histones. (C) Nucleosomes were reconstituted on the relaxed plasmid DNA by cFANCD2 (lanes 3–7), cFANCD2(1-1389) (lanes 8–12), cFANCD2(1-1267) (lanes 13–17), cFANCD2(1-1167) (lanes 18–22), and cFANCD2(953-1439) (lanes 23–27) in the presence of wheat germ topoisomerase I. After deproteinization, the topoisomers were separated by agarose gel electrophoresis with ethidium bromide staining. The cFANCD2 concentrations were 0, 0.45, 0.90, and 1.8 μM. Highly supercoiled and relaxed DNAs are denoted as ‘sc’ and ‘relaxed’, respectively. (D) Graphic representation of nucleosome-assembly activities of the cFANCD2 mutants shown in C. Representative images are shown in C. The supercoiled DNA fractions were generated by nucleosome assembly in the presence of cFANCD2, and the intensities of the bands indicated by the arrows in C were quantitated by an LAS-4000 Image Analyser (GE Healthcare). Means of three independent experiments are shown with s.d.’s. (E) Nucleosomes were reconstituted on the relaxed plasmid DNA by cFANCD2 (lanes 3–7), cFANCD2(R1336A/K1346A) (lanes 8–12), and cFANCD2(D1350A/E1365A/E1382A) (lanes 13–17) in the presence of wheat germ topoisomerase I. The cFANCD2 concentrations were 0, 0.45, 0.90, and 1.8 μM. Highly supercoiled and relaxed DNAs are denoted as ‘sc’ and ‘relaxed’, respectively. (F) Graphical representation of the nucleosome-assembly activities of the cFANCD2 mutants shown in E. Representative images are shown in E. The supercoiled DNA fractions were generated by nucleosome assembly in the presence of cFANCD2, and the intensities of the bands indicated by the arrows in E were quantitated by an LAS-4000 Image Analyser (GE Healthcare). Means of three independent experiments are shown with s.d.’s.