FIGURE 4.

ELG1 facilitates USP1-mediated PCNA deubiquitination. A, overexpression of ELG1 and USP1 decreased UV-induced PCNA monoubiquitination (PCNA-Ub). HEK293T cells were transected with FLAG-ELG1 or Myc-USP1 expression vectors. Cells were irradiated with 20 J/m² UV after 48 h and incubated further for 6 h. The soluble (S) and chromatin-bound (B) fractions were isolated and subjected to immunoblot analysis. Histone H3 and tubulin proteins were used as loading controls for chromatin-bound and soluble fractions, respectively. B, the increase in PCNA monoubiquitination following USP1 knockdown depends on ELG1. HEK293T cells were cotransfected with different combinations of control (Ctrl) or ELG1 siRNA with a Myc-USP1 expression vector as indicated. The chromatin-bound fractions were analyzed by immunoblotting. C, ELG1 knockdown did not affect FANCD2 monoubiquitination. The chromatin-bound fractions were prepared 72 h after transfection of HEK293T cells with the indicated siRNAs and then subjected to immunoblot analyses. D, the I-SceI-induced HR assay was performed as described previously (20) after knockdown of USP1 or ELG1. BLM depletion was used as a positive control. The inset shows the structure of the recombination substrate DR-green fluorescent protein and the location of the I-SceI recognition site. Error bars, S.D. of triplicate experiments. E, SupF mutation frequencies for UV-irradiated plasmids are indicated for control siRNA and ELG1 siRNA-treated cells. Error bars, S.D. of triplicate experiments. The number in the graph indicates probability (p value) calculated by Student's t tests.