Double strand breaks upstream of the active VSG are not typically lethal.

(A) Schematic showing the VSG2 BES on chromosome 6a with the individual modifications. I-SceI recognition site (SceR) and reporter genes incorporated to give VSG up [ 37 ]; 70int sce ; Pseudo sce and ESAG1 sce . Centre lines indicate the medians. Arrow, BES RNA Pol1 promoter; grey boxes, ESAGS; black boxes, 70 bp repeats; yellow box, VSG; black circles, telomere. RFP : PAC , Red Fluorescent Protein and Puromycin N–Actyltransferase ; black circles, telomere. (B) Clonogenic survival assay, cells were plated out into 96 –well plates in either non or I-SceI inducing conditions. Clones were counted after 7 days. Centre lines indicate the medians. (C) Proportion of survivors following a I-SceI break. Number of replicate plates are as follows: VSG up , 3 plates per condition; 70int sce , 9 plates per condition; Pseudo sce , 9 plates for the–Tet and 12 plates for the + Tet and ESAG1 sce , 6 plates per condition. (D) Cell in G2 were determined by DAPI staining and scored according to the position of the nucleus (n) and kinetoplast (k) following an induction of an I-SceI break. n = 2 (independent inductions,) 100 cell counted for each time point by two independent researchers. (E) Focal accumulation of γH2A as assessed by immunofluorescence assay following an induction of an I-SceI break. n = 2 (independent inductions,) 100 cell counted for each time point by two independent researchers. Error bars, SD. Individual biological clones were used for each cell line. VSG up served as a control [ 37 ].