Figure 1.

CDK activity regulates end resection downstream of RIF1 and MAD2L2. a) Analysis of RIF1 foci formation upon inhibition of CDK activity. HeLa cells were irradiated with 5Gy, fixed and stained for Cyclin A and RIF1 after 2h of recovery. 2 different thresholds of cyclin A levels were used to distinguish between G1/early S-phase (low cyclin A), S-phase (intermediate cyclin A), late S/G2 (high cyclin A). CDK inhibitor RO-3306 (10µM) was added to the medium 15 min before cell irradiation. The bracket highlights a subpopulation of cells in late S/G2 with a high number of foci, **** indicates p-value ≤ 0.0001. Statistical analysis is included in Material and Methods. b) Representative images of the experiment in a). c) Inhibition of CDK activity greatly reduced end-resection in MAD2L2 depleted U2OS cells. Cells were treated with 10µM RO-3306 or 20µM roscovitine for 15 minutes prior to adding neocarzinostatin (NCS) at 250ng/ml for 1 hour and western blotting of RPA phosphorylation (Ser-4/8) in whole cell extracts was used as a measure of end-resection. d) U2OS cells were treated with RO-3306 (10µM) or roscovitine (25µM) and irradiated with 5Gy. After 3 h, to allow for end-resection to take place, cells were pre-extracted to remove all chromatin-unbound RPA, fixed and stained for RPA foci. For each condition > 100 cells were quantified. Corresponding immunoblot shows the knockdown of MAD2L2 achieved.