Secondary structure forming sequences drive SD-MMEJ repair of DNA double-strand breaks

Abstract

Alternative end-joining (alt-EJ) repair of DNA double-strand breaks is associated with deletions, chromosome translocations, and genome instability. Alt-EJ frequently uses annealing of microhomologous sequences to tether broken ends. When accessible pre-existing microhomologies do not exist, we have postulated that new microhomologies can be created via limited DNA synthesis at secondary-structure forming sequences. This model, called synthesis-dependent microhomology-mediated end joining (SD-MMEJ), predicts that differences between DNA sequences near double-strand breaks should alter repair outcomes in predictable ways. To test this hypothesis, we injected plasmids with sequence variations flanking an I-SceI endonuclease recognition site into I-SceI expressing Drosophila embryos and used Illumina amplicon sequencing to compare repair junctions. As predicted by the model, we found that small changes in sequences near the I-SceI site had major impacts on the spectrum of repair junctions. Bioinformatic analyses suggest that these repair differences arise from transiently forming loops and hairpins within 30 nucleotides of the break. We also obtained evidence for 'trans SD-MMEJ,' involving at least two consecutive rounds of microhomology annealing and synthesis across the break site. These results highlight the importance of sequence context for alt-EJ repair and have important implications for genome editing and genome evolution.

Figure 1.

The SD-MMEJ model for alternative end joining repair. (A) Loop-out mechanism with DNA unwinding prior to loop formation. (B) Snap-back mechanism with DNA resection prior to hairpin formation. Both mechanisms utilize annealing of break-proximal primer repeats (P2) to break-distal primer repeats (P1), which primes nascent synthesis that can lead to insertions (black) and the creation of new microhomologous sequences (MH1, green). For loop-out SD-MMEJ, P1 and P2 are direct repeats, while for snap-back SD-MMEJ they are inverted repeats. Repair concludes with unwinding of secondary structures, annealing of nascent microhomologies with MH2 sequences on the other side of the break, fill-in synthesis, and ligation. For the repair events shown here, the inserted sequence becomes part of new, longer direct or inverted repeats. Not shown are the trimming of non-homologous flap intermediates when P2 and MH2 are not directly adjacent to the break site, or deletion junctions with no net insertion that are formed when P1 and MH1 are directly adjacent to each other.

Figure 2.

A high-throughput SD-MMEJ assay. (A) In vivo extract system. Purified plasmid constructs are injected into dechorionated embryos expressing I-SceI. Following incubation to allow for cutting and repair, plasmids are recovered and sequenced directly by Sanger sequencing (not shown) or prepared for high throughput amplicon sequencing. (B) Diagrams showing the predicted effects of right-side flanking sequence changes on SD-MMEJ repair. Yellow highlighting indicates the I-SceI recognition site. In Iw7, blue text corresponds to a GGCC direct and inverted repeat that can participate in snap-back SD-MMEJ (shown) or loop-out SD-MMEJ (not shown). M1 has a change in the first GGCC repeat (red) that is postulated to prevent formation of the hairpin (shown) or loop (not shown) used during the creation of a prominent SD-MMEJ product. M2 has two additional alterations (orange) that are postulated to promote the formation of a 9-nt inverted repeat that can be used to prime snap-back synthesis during SD-MMEJ. M3 has two additional changes (green) that are predicted to act as the start of a microhomology primer. Following synthesis of 4 nt, the nascent DNA could anneal to a CCTGT microhomology on the left side of the break.

Figure 3.

Effects of single nucleotide changes in I-SceI flanking sequences on SD-MMEJ outcomes. (A) Types of inaccurate repair junctions. ABJ = apparent blunt join; MHJ = microhomology junction; InDel = insertion/deletion junction. (B) Overall SD-MMEJ consistency. Each bar represents the percentage of reads for each junction determined to be SD-MMEJ consistent. (C) SD-MMEJ consistency by repair junction type.

Figure 4.

Changes in I-SceI flanking sequence alter the deletion boundaries. Deletion boundaries for inaccurate repair events for (A) Iw7, (B) M1, (C) M2 and (D) M3. Deletion boundaries are defined as the first non-ambiguous base accurately aligned to the left and right of the I-SceI break. SD-MMEJ consistent junctions are represented by darker colors. + indicates all bases to the right or left of the indicated sequence. The arrow in (B) shows the effect of the C→A mutation on the deletion boundary at a prominently used primer repeat.

Figure 5.

The prevalence of repeat motifs utilized in SD-MMEJ consistent deletion junctions depends on the I-SceI flanking sequence. Repeat motifs contain the P1 primer repeat, the adjacent MH1 microhomology repeat, and any intervening sequence. Colors correspond to the frequency that each base is found in a repeat motif, with warmer colors indicating greater frequencies. Red vertical lines indicate the TTAT/AATA overhangs produced by I-SceI cutting.

Figure 6.

Changes in I-SceI flanking sequence influence the secondary structures utilized during SD-MMEJ repair. Individual repeat motifs in SD-MMEJ consistent deletion junctions, with their relative prevalence indicated by color. Percent SD-MMEJ consistent deletion reads was calculated by dividing the total number of reads for each repeat motif by the total number of SD-MMEJ consistent deletion reads. Repeat motifs corresponding to loop-out mechanisms are indicated by straight lines, while snap-back mechanisms are indicated by hatched lines. Red vertical lines indicate the TTAT/AATA overhangs produced by I-SceI. (A) The red box highlights snap-back synthesis products utilizing a GGCC direct repeat, while the blue box highlights loop-out synthesis using the GCGG/CCGC inverted repeat. (B) The red box highlights a CTAG repeat motif created by both snap-back and loop-out repair mechanisms. (C) The red box highlights repeat motifs resulting from snap-back synthesis utilizing part/all of a 6 nt TCCACT/AGTGGA inverted repeat or an 8 nt CTAGTGGA direct repeat. The blue box indicates repeat motifs resulting from snap-back synthesis using part/all of a 9 nt CTAGTGGAC/GTCCACTAG inverted repeat. (D) The arrow indicates a repeat motif with a CAGG microhomology that anneals with CCTG on the left side of the break.

Figure 7.

The prevalence of repeat motifs utilized in SD-MMEJ consistent single-step insertions depends on the I-SceI flanking sequence. Repeat motifs contain the P1 primer repeat, the adjacent MH1 microhomology repeat, and any DNA between these repeats (the insertion). Colors correspond to the frequency that each base in found in a repeat motif, with warmer colors indicating greater frequencies. Red vertical lines indicate the TTAT/AATA overhangs produced by I-SceI cutting. Trans SD-MMEJ occurs with microhomology annealing across the break site, followed by synthesis, dissociation of the nascent DNA, and reannealing at new microhomologies prior to completion of repair.

Figure 8.

Specific primer repeats are preferentially used during SD-MMEJ for single-step insertions. All possible primer pairs (P1 and P2) are shown for the Iw7 construct, along with the type of SD-MMEJ event for each primer set. Primer pairs do not consider overall synthesis length and microhomology formation, therefore, different junctions may have the same primer pair. Colors correspond to the frequency that each primer pair is utilized, with warmer colors indicating greater frequencies. Loop-out = solid outline and connecting line; snap-back = dashed outline and connecting line; trans = dotted outline. Red vertical lines indicate the TTAT/AATA overhangs produced by I-SceI cutting. Red boxes indicate the loop-out priming sites ablated in the M4 plasmid construct.

Figure 9.

Models for SD-MMEJ consistent indel junctions. Shown are primer repeats (blue), microhomology repeats (green), insertions (boxes), and final repeat motif (underlined). (A) Iw7 loop-out event. Unwinding/resection of DNA on the right side of the break allows primer repeats to anneal, followed by limited synthesis, dissociation, and annealing of microhomology repeats. Endonuclease/flap cleavage events are not shown for simplicity. (B) M1 trans SD-MMEJ event. The two-ended arrow indicates transition of the TTA primer repeat to a microhomology repeat. This junction can also be explained by a snap-back mechanism on the left side of the break. (C) M2 snap back event. Asterisks indicate engineered sequence changes utilized during repair. Note that 3′→5′ resection or endonuclease activity following the annealing of primer repeats is required prior to initial synthesis.