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. 2020 Aug 7;9:e58223. doi: 10.7554/eLife.58223

Figure 2. Foldback inversion GCRs are mediated by a ssDNA hairpin intermediate.

Proposed mechanism underlying foldback inversion formation based on the inversion junction sequences recovered. 5’ to 3’ resection from a DSB or other initiating form of DNA damage exposes a ssDNA region predicted to form a ssDNA hairpin. Appropriately positioned DSBs can lead to properly paired hairpin stems, whereas other DSBs will lead to 3’ flaps that require processing before extension by DNA polymerases. Extension of the hairpin leads to the inversion junction sequences observed.

Figure 2—source data 1. Predicted ssDNA hairpin structures for observed foldback inversions.
The structures of the key predicted ssDNA hairpin intermediates observed (panels A to BK are sorted by the chromosomal position of the predicted hairpin). The chrV coordinates for each predicted hairpin correspond to the coordinates reported for the foldback inversion-containing GCRs.

Figure 2.

Figure 2—figure supplement 1. Inversion junction formed by two sequential ssDNA hairpin intermediates.

Figure 2—figure supplement 1.

(A) The inversion sequence junction from PGSP1995 (center line), which was present in a GCR from a sae2Δ mutant, shows an unusual feature relative to other inversion junction sequences recovered as it contains a five base TGAGG sequence that does not align to either inverted sequence from chrV. For most inversion junction sequences, the two alignments to chrV overlap, and the overlap indicates the sequences involved in the ssDNA hairpin stem. Thus, this inversion junction sequence must involve a different mechanism of formation. (B) A mechanism of formation of the PGSP1995 inversion junction that is consistent with the junction sequence identified involves two sequential ssDNA hairpins. After resection from a DSB (or other initiating damage), the first hairpin forms by annealing the stem sequences (highlighted in orange) to generate a chrV:27,444_27,465 hairpin with a 10 nt loop and 6 bp stem. DNA synthesis after formation of the first hairpin adds the unaligned TGAGG sequence and bases required for forming the stem of a second hairpin. Dissociation of the first hairpin and formation of a second hairpin (eight nt loop, 4 bp stem) using the newly synthesized bases generates an intermediate that after extension generates an inversion junction sequence identical to the one shown in panel A.
Figure 2—figure supplement 2. Inversion junction formed by two sequential ssDNA hairpin intermediates.

Figure 2—figure supplement 2.

(A) The inversion sequence junction from PGSP4918 (center line), shows an unusual feature relative to other inversion junction sequences recovered as it contains a six base TGATAA sequence that does not align to either inverted sequence from chrV. For most inversion junction sequences, the two alignments to chrV overlap, and the overlap indicates the sequences involved in the ssDNA hairpin stem. Thus, this inversion junction sequence must involve a different mechanism of formation. (B) A mechanism of formation of the PGSP4918 inversion junction that is consistent with the junction sequence identified involves two sequential ssDNA hairpins.