RBX1 prompts degradation of EXO1 to limit the homologous recombination pathway of DNA double-strand break repair in G1 phase

摘要

High-level ubiquitination mediated suppression of EXO1 in G1-phase cells.  HeLa cells were synchronized to different phases of the cell cycle by double-blockage of thymidine, and the cell phase distributions were monitored by flow cytometry analysis. EXO1 protein was detected in the synchronized G1- and G2-phase HeLa cells by microscopic observation of immunofluorescent staining with anti-EXO1 antibody. Nuclei were stained with DAPI. The expression levels of EXO1, RBX1, Cullin1, and phosphorylated DNA-PKcs-S2056 were assessed by western blotting analysis. Among them, phosphorylation of DNA-PKcs, is indicative of cells in different phase of cell cycle. , Ubiquitination of EXO1 protein was detected respectively in the synchronized G1- and G2-phase cells. After HeLa were transfected with HA-Ub and SBP-Ub plasmid for 48 h, the immunoprecipitation (IP) product obtained with the EXO1 antibody and S-bead agarose, were subjected to western blotting analysis with the anti-flag antibody and anti-EXO1 antibody separately. In order to achieve the same content of EXO1 for immunoprecipitation, when HA-Ub and SBP-Ub transfected cells were synchronized in G1- and G2-phase using a double thymidine block, cell were pre-treated with proteasome inhibitor MG132 for 2 h. Hela were arrested at the G1/S boundary followed by a release into fresh media to allow cells to progress through the cell cycle. Cells were synchronized in G2 phase (7–9h) and G1 phase (12–20h) after TDR release. 10 μM MG132 was added in G1 and G2 cells for 2 h and EXO1 contents were detected. Neddylation of Cullin1 protein was detected separately in the synchronized G1- and G2-phase cells. The immunoprecipitation (IP) product obtained with the Cullin1 antibody was subjected to western blotting analysis with anti-NEDD8 antibody. The effect of neddylation on the stability of the EXO1 protein. Cells were pre-treated with the neddylation inhibitor MLN4924 or DMSO as a control for 8 h and then co-cultured with cycloheximide (CHX). EXO1 levels were assessed by western blotting analysis at the indicated times after CHX treatment