p30 inhibits DSB HR DNA repair. (A) Schematic representation of the in vivo HR assay with DR-GFP HR reporter vector (see “In vivo HR and NHEJ DNA-repair assay”). (B-F) HeLa cells were cotransfected with DR-GFP vector either with the control vector or with the pI-SceI and with the vector expressing p30 and p30T232A (B); p30, p30-9RA, and p30-9RA-TA (C); p30-9RA and p30-9RA-delC660 (D); p30 and HTLV-2 p28 (E); or with p30, HTLV-2 p28, and HTLV-2 p28-P40T (F). Forty-eight hours later, the expression of GFP was assessed by FACS analysis. The relative percentage of GFP-expressing cells was represented by histograms corresponding to the average of 5 independent experiments. Western blots show p30, p30T232A, p30-9RA, p30-9RA-TA, p28, and p28-P40T expression in the assay. (G) HTLV-I molecular clones ACH and ACHΔp30 were transfected into Jurkat T cells to evaluate the effect of p30 on HR DNA repair when expressed in the context of the whole virus. Results demonstrated an increase of 13.3% of HR when p30 was ablated from an HTLV-I molecular clone. Data are derived from 2 independent transfection experiments and the standard deviation value for ACHΔp30 was 3. (H) p30 delays the response to DNA damage. HeLa cells infected with mock or p30-expressing lentivirus were γ-irradiated (2 Gy) and collected at 0, 2, 4, 6, and 8 hours after irradiation. Shown are Western blot analyses of pS957-SMC1, pThr68-CHK2, and p-H2AX checkpoints involved in the response to DNA damage.