Figure 5

Polθ-helicase is bound to DNA throughout the entire cell cycle. (A) Epimastigote cells overexpressing GFP-Polθ-helicase were fixed, permeabilized, incubated with mAbAC, which recognizes flagellum (red), and stained with DAPI (blue). N – nucleus, k – kinetoplast, f – flagellum and * – new flagellum. Bars represent 2 μM. (B) Epimastigote cells were treated with HU for 24 h, washed and maintained in culture for 6 h (S), 18 h (G2), and 24 h (G1). “Control” indicates cells that were not treated. Cells were stained with propidium iodide and analyzed according to their DNA content. Blue lines indicate the peak of the fluorescence intensity of G1/S cells (left line) and G2/M cells (right line). (C) The samples analyzed in (B) were subjected to cell fractionation. In this assay, cells were lysed, and after centrifugation, the supernatants were saved as soluble fraction 1 (SF1). Again, pellets were incubated with lysis buffer and centrifuged, and the supernatants were saved as soluble fraction 2 (SF2). Finally, pellets were treated with DNase to obtain DNA-bound proteins (DBPs) and then centrifuged; the supernatants were saved as DBPs. Samples were subjected to western blotting using anti-Polθ-helicase and anti-histone H3 as a control in DBP fractions.