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Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

Figure 8

Analysis of 5′- and 3′-end processing at double-strand breaks in LIG4-silenced cells.

A. LMPCR detection of 4-base and 3-base 5′ overhangs on the broken MAC ends generated at the left boundary of IES #5 = sm19-576 (66 bp) during autogamy of strain 51ΔA submitted to control or LIG4 RNAi. LMPCR products were separated on high resolution polyacrylamide denaturing gels to visualize the doublet of bands. The position of linker ligation to the free 5′ end was confirmed by gel purification of LMPCR products and sequencing using primer sm19-4 specific for the left flanking MAC sequences (the chromatograms show the sequence of the top strand). The structure of the ligation products is diagrammed at the bottom, with the linker represented in purple. Arrows indicate the nucleotides that are ligated to the linker on 4-base or 3-base 5′ extensions. B. Nucleotide addition to broken MAC 3′ ends is impaired in Ligase IV-depleted cells. Terminal transferase-mediated poly(C) tailing of the MAC left 3′ end of IES #6 (51G4404) was performed as indicated in [11]. Poly(C)-tailed products were amplified using primers 51G18 and I, then a nested PCR was performed with 51G13 and I before electrophoresis on a 3% Nusieve agarose gel. The position of size standards (in bp) is indicated: PCR products are expected around 122 bp, with some variability due to the length of the poly(C) tail added by the terminal transferase. To determine the position of poly(C) addition, gel-purified PCR products were sequenced using 51G13 (from the MAC left flanking sequences) as a sequencing primer. The chromatograms show the sequence of the top strand and the structure of the broken MAC ends identified in each sample is displayed at the bottom.

Figure 8