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. 2017 Mar 9;168(6):990–999.e7. doi: 10.1016/j.cell.2017.02.020

Figure 1.

Figure 1

Injection of DNA Resembling an Excised IES Is Sufficient to Trigger the iesRNA Pathway and Lead to Positive Feedback-Mediated DNA Excision

(A) The current model for IES excision. An IES (red) with the TA repeats that mark its boundaries is represented in panel 1. The excisase PiggyMac cleaves at the ends leaving 4 nt 5′ overhangs centered on the TA (panel 2). The 5′ most nucleotide is resected (panel 2) and the broken DNA ends are annealed at the TA, then ligated. The missing nucleotide is filled in during the annealing-ligation process (green nucleotides) and a single TA dinucleotide is left in the genome (blue, panel 3).

(B) A cryptic IES oligonucleotide corresponding to crypIESa with its 4 nt 5′ overhangs and 5′ phosphate. The gel represents the product of PCR performed with primers flanking the targeted region. PCR was performed after injected cells had been left to expand vegetatively for four divisions, and the PCR reactions were set up with ten cells each. Lane 1 is uninjected, and lane 2 is successfully injected as is seen from the presence of a lower band representing amplicon with a 27 bp deletion (arrowhead). Sequencing of the band demonstrates the precise excision of the crypIESa, with one TA removed and one retained.

(C) Left: representative gels demonstrating excision of mating type IES (injection control) and crypIESb following co-injection of mating type IES-matching small RNAs (do not require Dcl5) and crypIESb DNA in control (lanes 1–4) versus Dcl5-silenced (lanes 5–8) cells. Arrowheads indicate bands corresponding to excision products. Right: co-injection of control RNA (mating type) and cryptic IES DNA was repeated to quantify the relative excision efficiencies in unsilenced (n = 13) and Dcl5-silenced (n = 19) cells. Charts are shown quantifying the relative efficiencies of excision.

(D) Cryptic IES DNA with 3′ overhangs does not induce excision. DNA oligonucleotides for crypIESa (3′ overhangs) and crypIESb (control, 5′ overhangs) are shown. PCR products corresponding to excised cryptic IES are only found for crypIESb after coinjection of both oligonucleotides. This experiment was repeated with successful co-injection of crypIESa (3′ overhangs) and crypIESb (control, 5′ overhangs) in 12 cells, none of which exhibited excision of crypIESa.

See also Figure S1.