Detection of programmed DSBs at IES boundaries during autogamy.

(A) LMPCR detection of DSBs at MAC or IES ends during autogamy of 51ΔA cells subjected to RNAi against ICL7 (control) or KU80c (same samples as in Figure 4). A Sanger DNA sequencing ladder provides size markers. On the diagram, the LMPCR linker is drawn as grey boxes and Paramecium DNA as black lines, with a black dot representing the 3′ end generated by Pgm-dependent cleavage. In the KU80c RNAi, the LMPCR signals at 51G4404 MAC ends are likely due to background DNA breaks generated at the MAC G⁵¹ locus during DNA extraction. This background is not detected for IESs of the A⁵¹ gene, because this locus is absent from the old MAC. (B) TdT tailing of free 3′OH ends during autogamy of 51ΔA cells subjected to RNAi against ICL7 (control) or KU80c (same samples as in (A)). On the diagram, the potentially resected 5′ end is represented by a dotted line.