FIG. 5.

DSBs at IES boundaries generate 3- and 4-base overhangs. (A and B) Primer extension and sequencing analysis of the linker ligation products obtained for IESs 51A6649 (A) and 51G4404 (B). Genomic DNA from autogamous 51 new at t = 10 h (Fig. 1B) was ligated to linkers I′/(GTAG)J′ (IES ends) or I′/(CTAC)J′ (mac ends) for 51A6649 and to I′/(ATAC)J′ (IES ends) or I′/(GTAT)J′ (mac ends) for 51G4404. The positions of all primers are shown on the diagrams, with an asterisk marking those used for primer extension and sequencing. IES sequences (capital letters) are represented by thick lines, and flanking macronuclear sequences (lowercase letters) are represented by thin lines. All ³³P-labeled primer extension products were analyzed on sequencing gels and revealed by autoradiography. Sequencing chromatograms (for 51A6649) or sequencing gels (for 51G4404) are shown for the left boundary of each IES. The sequence of the strand complementary to the one on which ligation has taken place is displayed above each panel, with the major sequence in bold and the linker underlined. Product U corresponds to linker ligation on the nucleotide 5′ to the TA, while L corresponds to ligation on the T residue (each circled L represents the unexpected major ligation product observed for the macronucleus-destined ends). (C) Ligation reactions leading to products U and L, with the linker in gray and Paramecium genomic DNA ends in white. (D) LMPCR detection of free 5′PO₄ groups on the IES side of the TA, at the left boundary of IES 51G4404. The Asp718-precleaved control fragment was mixed with DNA from vegetative cells, and genomic DNA samples were as in Fig. 3. The experimental strategy was as for Fig. 3A, except that linker I/J and primers hybridizing within macronuclear flanking sequences were used. An autoradiogram of the sequencing gel used for the detection of primer extension products is shown, with the positions of size markers (from a sequencing ladder) indicated on the left. In this experiment, bands U and L were labeled following the same rule as in Fig. 3B and correspond to the same positions as linker ligation products U and L, respectively, detected at macronucleus-destined ends in panel B. Their observed sizes are consistent with the estimation of the distance separating the 5′ end of the primer from the IES boundary (indicated on the diagram), incremented by the 25 bp from linker primer I.