Figure 4.

CDK5 is not required for early DNA DSB signalling or DNA DSB repair. (A) Expression of dominant-negative CDK5 sensitises to PARP inhibitor. CAL51 cells transfected with empty vector (pcDNA3.1empty), CDK5 expression vector (pCDK5) or kinase-dead D145N CDK5 expression vector, and 24 h following transfection plated for clonogenic assay with KU0058948. (B) CDK5 is not required for ATM and ATR activation. CAL51 cells transfected 72 h earlier with siRNA where treated with 10-Gy irradiation, 50 J/m² ultraviolet light or not treated. Lystates where made 1 h following treatment, and a western blot was probed with antibodies against ATM Ser1981 autophosphorylation site and CHK1 Ser317 ATM/ATR phosphorylation site, with total ATM, CHK1, TP53 and a β-Tubulin loading control. (C) CDK5 does not affect homologous recombination by gene conversion. Gene conversion was measured using an adapted single-copy, chromosomally integrated HR reporter construct in a 293 human embryonic kidney cell line (Tutt et al, 2001). Silencing of BRCA1 acted as a positive control for reduced gene conversion. Details of this assay are outlined in Supplementary Figure 4. (D) CDK5 silencing does not affect DNA DSB end joining. FIGE assay in CAL51 cells transfected with siCDK5 or siCON. Time points represent minutes after 30 Gy irradiation and non-irradiated control (NIR).