Figure 1. Dissection of E3 ligase functions in UV-mediated DNA damage repair. (A) Quantitative analysis of H2A-ubiquitylation levels. Immunoblots (as in B and Fig. S1, A and B) were probed with histone H2A antibody. The intensities of H2A and H2A-ubiquitin bands were quantified by the ImageJ software. The graphs illustrate the relative H2A ubiquitylation calculated as (H2A ubiquitin)/(H2A + H2A ubiquitin), normalized to Ponceau staining intensity after knockdown of the respective proteins (H2A ubiquitin/H2A). Values are normalized to the value from nonirradiated cells and are given as mean ± SEM (n = 4). (B) Monoubiquitylation of histone H2A at lysine 119 after UV irradiation is mainly catalyzed by RING1B. Chromatin association assays of control and RING1B knockdown HEK293T cells after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The specificity of the H2A-ubiquitin antibody was verified (Fig. S1 C). (C) Epistatic relationship of xpc-1 and spat-3. Wild-type nematodes (N2) or spat-3 mutants (VC31) were fed with either control or xpc-1 RNAi–producing bacteria. The relative viability was analyzed after UV irradiation (200 J/m2). Values are given as mean ± SEM (n = 3). (D) Impact of BMI-1 on RING1B-mediated H2A ubiquitylation after UV irradiation. Chromatin association assays of UV-irradiated HEK293T cells treated with siRNAs (control, BMI-1). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. Relative intensities of H2A ubiquitin/H2A and RING1B abundance after BMI-1 depletion were measured. Values are given as mean ± SEM (n = 4). (E) Epistatic relationship of RING1B and BMI-1 in response to UV irradiation. Relative colony formation potential of control or RING1B knockdown cell lines treated with siRNA was analyzed at different UV doses. Control cells were transfected with either control siRNA (control) or BMI-1 siRNA (BMI-1). RING1B knockdown cell lines were transfected with either control siRNA (RING1B) or BMI-1 siRNA (RING1B + BMI-1). Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM (n = 9).
Figure 1.

Dissection of E3 ligase functions in UV-mediated DNA damage repair. (A) Quantitative analysis of H2A-ubiquitylation levels. Immunoblots (as in B and Fig. S1, A and B) were probed with histone H2A antibody. The intensities of H2A and H2A-ubiquitin bands were quantified by the ImageJ software. The graphs illustrate the relative H2A ubiquitylation calculated as (H2A ubiquitin)/(H2A + H2A ubiquitin), normalized to Ponceau staining intensity after knockdown of the respective proteins (H2A ubiquitin/H2A). Values are normalized to the value from nonirradiated cells and are given as mean ± SEM (n = 4). (B) Monoubiquitylation of histone H2A at lysine 119 after UV irradiation is mainly catalyzed by RING1B. Chromatin association assays of control and RING1B knockdown HEK293T cells after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The specificity of the H2A-ubiquitin antibody was verified (Fig. S1 C). (C) Epistatic relationship of xpc-1 and spat-3. Wild-type nematodes (N2) or spat-3 mutants (VC31) were fed with either control or xpc-1 RNAi–producing bacteria. The relative viability was analyzed after UV irradiation (200 J/m2). Values are given as mean ± SEM (n = 3). (D) Impact of BMI-1 on RING1B-mediated H2A ubiquitylation after UV irradiation. Chromatin association assays of UV-irradiated HEK293T cells treated with siRNAs (control, BMI-1). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. Relative intensities of H2A ubiquitin/H2A and RING1B abundance after BMI-1 depletion were measured. Values are given as mean ± SEM (n = 4). (E) Epistatic relationship of RING1B and BMI-1 in response to UV irradiation. Relative colony formation potential of control or RING1B knockdown cell lines treated with siRNA was analyzed at different UV doses. Control cells were transfected with either control siRNA (control) or BMI-1 siRNA (BMI-1). RING1B knockdown cell lines were transfected with either control siRNA (RING1B) or BMI-1 siRNA (RING1B + BMI-1). Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM (n = 9).

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