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. 2016 Nov 10;12(11):e1006410. doi: 10.1371/journal.pgen.1006410

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© 2016 Hartlerode et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 6 — (A) Homologous template switching during non-canonical LTGC termination in a XRCC4^fl/fl clone. Upper panel: Cartoon depicts the HR-mediated template switch between the displaced nascent strand product of LTGC (black) and identical rabbit β-globin intron sequences within the episomal I-SceI expression vector. Lower panel: Sequence of the homologous template switch complex breakpoint. Orange: intron sequences copied from the I-SceI expression vector. Brown: additional sequence copied from the episomal I-SceI expression vector. Red: single N-addition at second breakpoint. Blue: second end of the DSB, resected 9 bp prior to end joining with the twice-displaced nascent strand. (B) Microhomology-mediated complex breakpoint formation during non-canonical LTGC termination in a XRCC4^Δ/Δ clone. Southern blotting analysis of this clone is presented in Fig 3B. Upper panel: Cartoon shows map of the complex breakpoint, which involved rearrangement of the second (non-invading) DNA end. Red: 21nt insertion. Blue arrows: duplicated 36bp sequence from second end of DSB (located 3579-3614bp from the I-SceI site). Orange arrow: Inverted 110bp sequence adjacent to duplicated sequence (located 3469-3578bp from the I-SceI site). The MMEJ breakpoint within the second DNA end is located 3579-3582bp downstream of the I-SceI site. Blue sequences (including correctly oriented blue arrow) to the right of MH breakpoint are unrearranged ROSA26 locus. With the exception of the inverted 110 bp sequence, a segment of the second DNA end ~3.5kb adjacent to the I-SceI site was deleted during the rearrangement. Lower panel: Sequence of the MH-mediated complex breakpoint. Black: LTGC product (gene conversion tract length was 1249bp). Red: 21nt insertion. First blue underlined: inverted 36bp repeat. Orange: 110bp inversion. Second blue underlined: correctly oriented 36bp repeat, contiguous with unrearranged ROSA26 sequence. Bold underlined blue: 4bp MH breakpoint. Hypothetical model of this complex breakpoint is presented in Fig 7.