Survival of the Replication Checkpoint Deficient Cells Requires MUS81-RAD52 Function
Figure 2
Loss of CHK1 function leads to MUS81-dependent DSBs production independently from RAD51 regulation.
(A) Analysis of CHK1 phosphorylation in GM01604 cells transfected with control siRNAs (siCtrl) or siATR, siTIPIN and siRAD9 and treated with 2 mM HU for 6 h. Lysates were subjected to SDS-PAGE and immunoblotted for pS345CHK1 and CHK1. (B) Evaluation of MUS81-dependent DSBs formation by neutral Comet assay in cells in which CHK1 function was chemically inhibited. GM01604 cells were transfected with control siRNAs (siCtrl) or siMUS81. Forty-eight hours thereafter, cells were treated or not with CHK1 inhibitor (UCN-01) for 1 h and with 2 mM HU for 6 h and then subjected to Comet assay. Data are presented as mean tail moment and are means of three independent experiments. Error bars represent standard errors. Where not depicted, standard errors were <15% of the mean. In the panel, representative images are shown. (C) Western blotting in GM01604 cells transfected with control siRNAs (siCtrl) or siRAD51 and/or siMUS81. Depletion of proteins was verified 48 h after transfection using the relevant antibodies. Tubulin was used as loading control. (D) Analysis of DSBs formation in the absence of RAD51. Cells in which RAD51 and/or MUS81 was down-regulated were treated or not with UCN-01 for 1 h and with 2 mM HU for 6 h, then cells were subjected to neutral Comet assay. Cells treated with UCN-01 were used as positive control. Graph shows data presented as mean tail moment +/− SE from three independent experiments. Error bars represent standard errors. Where not depicted, standard errors were <15% of the mean. (E) Experimental scheme for genetic knock-down and rescue experiments. GM01604 cells were transfected with siRNA oligos targeting the UTR of RAD51 or GFP (siCtrl). RAD51-depleted cells were nucleofected to express RNAi-resistant wild-type or phosphorylation mutant form of RAD51 (RAD51-T309A). (F) Depletion of RAD51 and expression of the ectopic wild-type or RAD51-T309A were verified by immunoblotting 48 h thereafter using the anti-RAD51 antibody. Tubulin was used as loading control. (G) Analysis of DSBs formation in cells with impaired RAD51 function. GM01604 cells were transfected with control siRNAs (siCtrl) or siMUS81 and/or siRAD51. Forty-eight hours thereafter, cells were transfected with the RAD51-T309A plasmid (see Text S1). Then cells were treated or not with UCN-01 for 1 h and exposed to 2 mM HU for 6 h before being subjected to neutral Comet assay. Sample treated with UCN-01 was used as positive control. Data are presented as mean tail moment and are means of three independent experiments. Error bars represent standard errors. Where not depicted, standard errors were <15% of the mean.