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.

Identification of large-scale genomic rearrangements during wheat evolution and the underlying mechanisms

“…the InDel borders showed only micro-homology (<10 bp), which is not sufficient to serve as a template for homologous recombination (Bennetzen et al 2005 Khodaverdian et al 2017).…”

“…Filler DNA can be produced when the 3' ends formed at the break site invade a template, such that synthesis is primed based on a short region of homology. Following one or more rounds of template-dependent synthesis, the newly synthesized DNA can join the second end of the DSB, resulting in template insertion (Gorbunova and Levy 1997;Yu and McVey 2010;Vu et al 2014;Khodaverdian et al 2017). The template for filler DNA synthesis seems more often to be found in cis, namely on the same molecule, rather than in trans, i.e, on another molecule (Wessler et al 1990;Gorbunova and Levy 1997;Khodaverdian et al 2017).…”

“…Following one or more rounds of template-dependent synthesis, the newly synthesized DNA can join the second end of the DSB, resulting in template insertion (Gorbunova and Levy 1997;Yu and McVey 2010;Vu et al 2014;Khodaverdian et al 2017). The template for filler DNA synthesis seems more often to be found in cis, namely on the same molecule, rather than in trans, i.e, on another molecule (Wessler et al 1990;Gorbunova and Levy 1997;Khodaverdian et al 2017). It was proposed that limited DNA synthesis can lead to the presence of microhomology between the DSB ends, which can then be used for DSB repair via synthesisdependent microhomology-mediated end-joining (SD-MMEJ) (Gorbunova and Levy 1997;Yu and McVey 2010;Vu et al 2014;Khodaverdian et al 2017).…”

“…In eukaryotic cells, DSB repair occurs through two main processes, homologous recombination and NHEJ. In plants, DSB repair occurs more frequently via NHEJ than via homologous recombination(Gorbunova and Levy 1997).NHEJ pathways for DSB repair can be divided as canonical non-homologous end-joining (C-NHEJ) and microhomology-mediated end-joining (MMEJ) processes(Khodaverdian et al 2017;Ranjha et al 2018). The C-NHEJ and MMEJ pathways are template independent-mechanisms and thus can generate a wide range of chromosomal rearrangements, including large deletions and template insertions(Gorbunova and Levy 1997;Ceccaldi et al 2016;Ranjha et al 2018).…”

Parp3 promotes long-range end-joining in murine cells

“…Highthroughput analysis of amplicon deep sequencing can facilitate the rapid interrogation of repair phenotypes (21)(22)(23). We used this approach to address an aspect of the long-standing question of PARP enzyme redundancy in the context of DNA repair.…”