The DDB1-CUL4ADDB2 ubiquitin ligase is deficient in xeroderma pigmentosum group E and targets histone H2A at UV-damaged DNA sites -- Kapetanaki et al. 103 (8): 2588 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Kapetanaki et al. 10.1073/pnas.0511160103.

Supporting Figures

Files in this Data Supplement:

Supporting Figure 6
Supporting Figure 7
Supporting Figure 8

Supporting Figure 6

Fig. 6. Subcellular distribution of the RBX1-CUL4A-based ubiquitin ligase (DDB1-CUL4A^DDB2) E3 components after UV irradiation of normal cells. The control lymphoblastoid cells GM01953 were UV-irradiated with 40 J/m² and collected either immediately after treatment or after the indicated recovery periods. The cell fractions were obtained by the same method used for preparation of native chromatin in Figs. 3 and 5 (see Materials and Methods), except that the chromatin-bound proteins were released by sonication in the same volume of buffer used for extraction of nuclear soluble proteins (0.3 M fraction). The fractions (20 mg) were subjected to immunoblot analysis with the indicated antibodies.

Supporting Figure 7

Fig. 7. Recovery of uH2A in response to UV irradiation in xeroderma pigmentosum group E (XP-E) and control cells. The lymphoblastoid cells from control (GM01953) and XP-E patients (XP25PV) were UV-irradiated with 40 J/m² and cells were collected at the indicated times. The samples were prepared as described in Fig. 4 and analyzed by immunoblotting using antibodies against histone H2A, monoubiquitinated histone H2A (uH2A), and actin (loading control).

Supporting Figure 8

Fig. 8. Coimmunoprecipitation of partially or completely digested native chromatin with UV-damaged DNA-binding protein (UV-DDB) antibodies. The chromatin-insoluble fraction from normal GM01953 cells, collected 45 min after UV treatment, was digested with micrococcal nuclease (MNase) at 37°C for the indicated times. The solubilized chromatin fractions obtained after MNase treatment for 2.5, 5 (polynucleosomes), and 25 (mononucleosomes) min, were immunoprecipitated with DDB1-IgY and DDB2 antibodies. The levels of DDB2, uH2A, and H2A in coprecipitates were immunodetected with the use of the appropriate antibodies. Immunoprecipitations with DDB1 protein revealed interactions between the E3 ligase and uH2A, which were not detectable in the sample enriched in mononucleosomes.