Loss of <i>T</i>. <i>brucei</i> RNase H1 leads to increased levels of nuclear damage in replicating cells and in the VSG expression site.

A. Percentage of WT and Tbrh1-/- cells (n >200 in each of three replicates) that have detectable nuclear anti-γ-H2A signal. Data shown as mean +/- SD; *** P B. Distribution of intra-nuclear γ-H2A signal in WT and Tbrh1-/- cells (n >200 in each of three replicates). Data shown as mean +/- SD; ** P C. Percentage of cell cycle stages in WT and Tbrh1-/- cells with nuclear anti-γ-H2A signal; cell cycle stages were determined by DAPI staining and fluorescent imaging followed by counting the number and shape of nuclear (N) and kinetoplast (K) structures in individual cells: 1N1K, 1N1elongated K (1N1eK), 1N2K and 2N2K (n ≥50 for each cell cycle stage in three replicates). Data shown and analysed as in C; **** P D. Super-resolution structure-illumination microscopy imaging of anti-γ-H2A signal and co-localisation with DAPI in representative replicating (1N1eK and 1N2K; see below) Tbrh1-/- cells; only in the merge of anti-γ-H2A (magenta) and DAPI (cyan) images is colour provided (further examples of mutant and WT cells are provided in S5 Fig). E. Localisation of γ-H2A by ChiP-seq in WT and Tbrh1-/- cells. ChiP-seq signal is shown mapped to BES1 and BES3, which are represented as in Fig 2. Magenta and green tracks show normalised ratios of read-depth fold-change (1–3 fold) in IP samples relative to input in WT and Tbrh1-/- mutants, respectively.