Figure 3. PCNT is required for ATR dependent DNA damage signalling.

(a) G2-M checkpoint arrest was observed 2 h post UV treatment in control but not in ATR- or PCNT-Seckel LCLs. The mitotic index was examined 2 hours after treatment with 5 J m⁻² UV. Average of 3 experiments, error bars, s.d. Controls:- heterozygote relatives PCNT^E220X/+ and PCNT^S629fs/+ ; and an unrelated wild type (WT) control. UNT, untreated; UV, UV-C treated. Mitotic index is significantly increased in PCNT-Seckel cells compared with Wild-Type cells after UV treatment (p<0.001, E220X; p<0.01, S629fs; p<0.05, C1190fs) (b) ATR- and PCNT-Seckel LCL cells have increased hydroxyurea-induced nuclear fragmentation. Nuclear fragmentation was examined 24 h after treatment with 5mM hydroxyurea (HU). Percentage of cells displaying nuclear fragmentation (NF) is shown (p<0.001 WT versus PCNT-Seckel E220X) (c) ATR- and PCNT-Seckel LCL cells exhibit elevated levels of supernumerary centrosomes in mitosis. Percentage of mitotic (phosphohistoneH3 (Ser10) positive cells) with > 2 γ-tubulin stained foci (centrosomes) determined following 24 h incubation with nocodazole (p<0.05 WT versus E220X) (d) γH2AX foci formation is normal in PCNT-Seckel LCLs. Phosphorylation of the histone H2AX (termed γH2AX) by the PI3K-kinases is one of the earliest detectable responses to DNA damage, and is ATR-signalling specific after hydroxyurea treatment. Percentage of γH2AX foci was determined 2 hours after treatment with 5mM hydroxyurea. (e) ATR- and PCNT-Seckel cells show significantly reduced 53BP1 foci formation after treatment with 5mM hydroxyurea for 2 hrs, reflecting impairment in ATR/Chk1 dependent signalling. Immunofluorescent staining with anti-BrdU confirmed that all LCLs had equivalent S-phase populations (range: 20-23%) prior to exposure to HU (p<0.005 E220X; and p<0.001, S629fs versus WT after HU). (f) G2-M checkpoint arrest after Ionizing Radiation (IR) is normal in PCNT-Seckel and ATR-Seckel LCLs but not in Ataxia Telangiectasia mutated (ATM) LCLs.