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Figure 4


Fig. 4. HU-induced ATR foci and chromatin retention is decreased both in NBS1-deficient and NBS1 phospho-mutant cells. (A-R) NBS-ILB1 (NBS1), NBS-ILB1 (vector only) and NBS-ILB1 (S278A/S343A) cells were synchronized and treated with 2 mM HU for 3 hours. To visualize detergent-resistant ATR foci, cells were extracted with detergent before fixation. (D-F) In NBS cells restored with wild-type NBS1, ATR foci (green) were still visible after detergent extraction (D) and ATR foci colocalized with HU-induced RPA32 foci (red) (F). (G-R) Decreased retention of ATR (J,P) and a loss of colocalization with RPA (L,R) was observed in NBS1-deficient and in NBS1 phospho-mutant cells. (S) ATR retention in chromatin fractions of synchronized NBS-ILB1 (NBS1) and NBS-ILB1 (S278A/S343A) cells was examined by immunoblotting with anti-ATR antibodies. NBS cells expressing NBS1 retained ATR in the chromatin fractions, whereas NBS cells expressing a phospho-mutant form of NBS1 (S278A/S343A) did not retain ATR in the chromatin. Orc2-chromatin binding was not altered following DNA damage and was used as a loading control.





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