Figure 5. Rescue of homologous recombination in Brca1^Δ11/Δ11 cells by 53BP1 deletion correlates with increased phosphorylation of RPA and is dependent on ATM and CtIP.

(A) Western blot analysis showing Kap1 and RPA phosphorylation in WT and 53BP1^-/- cells in response to 30Gy ionizing radiation. (B) RPA and Kap1 phosphorylation after 30Gy ionizing radiation in Brca1- and Lig4-deficient cells. Knockout Brca1^ϕ/ϕ and Lig4^ϕ/ϕ cells were prepared by sorting B cells homozygous for the conditional alleles after infection with pMX-Cre-GFP retrovirus. (C) Western blot showing Kap1 and RPA phosphorylation in Brca1^Δ11/Δ11 53BP1^-/- cells after 30Gy ionizing radiation with and without ATM inhibitor (KU55993). ATMi (5 μM) was added 2 hours prior to irradiation. (D) Western blot analysis showing ionizing radiation-induced RPA phosphorylation in Brca1^Δ11/Δ11 53BP1^-/- MEFs after CtIP shRNA. Protein lysates were prepared from B cells infected with either vector (-) or CtIP shRNA (CTIP), with and without 30 Gy ionizing radiation. (E) Quantification of radial chromosome structures in metaphases from Brca1^Δ11/Δ11 53BP1^-/- cells treated with ATM inhibitor (5 μM) and / or PARP inhibitor (1 μM). ATMi was added at the start of the B cell culture, PARPi at 24 hours, and metaphases were made at 48 hours (n ≥ 240 metaphases; mean +/- s.d. shown from two experiments). (F) CFSE dilution analysis showing proliferation of B cells cultured with ATM inhibitor and/or PARP inhibitor for 96 hours. (G) Quantification of Rad51 foci in Brca1^Δ11/Δ11 53BP1^-/- cells. Rad51 foci were induced with 5Gy of ionizing radiation and cells fixed for Rad51 staining 2 hours later. Cells were either untreated, or pre-treated for 2hrs with 5μM ATM inhibitor. See also Fig. S3.