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.

(A) 对经针对ICL7（对照组）或KU80c的RNA干扰（RNAi）处理的51ΔA细胞的自配生殖过程中，采用连接介导PCR（LMPCR）检测大核（MAC）或内部消除序列（IES）末端的DNA双链断裂（DSBs），所用样本与图4一致。以桑格DNA测序分子量梯作为尺寸标记。示意图中，LMPCR接头以灰色方框表示，草履虫DNA以黑色线条绘制，黑色圆点代表由Pgm依赖型切割产生的3'末端。在KU80c RNAi实验组中，51G4404大核末端的LMPCR信号可能源于DNA提取过程中在MAC G51位点产生的背景DNA断裂。该背景信号未在A51基因的IES中被检测到，原因是该位点不存在于旧大核中。
(B) 对经针对ICL7（对照组）或KU80c的RNA干扰（RNAi）处理的51ΔA细胞的自配生殖过程中，采用末端脱氧核苷酸转移酶（TdT）加尾法检测游离3'-羟基末端，所用样本与(A)一致。示意图中，可能经切除修饰的5'末端以虚线表示。