Secondary structure forming sequences drive SD-MMEJ repair of DNA double-strand breaks. Issue 22 (7th November 2017)

• Record Type: Journal Article
• Authors: Khodaverdian, Varandt Y.; Hanscom, Terrence; Yu, Amy Marie; Yu, Taylor L.; Mak, Victoria; Brown, Alexander J.; Roberts, Steven A.; McVey, Mitch
• Is Part Of: Nucleic acids research. Volume 45:Issue 22(2017)
• Page Start: 12848
• Page End: 12861
• Publication Date: 2017-11-07
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkx1056
• Languages: English
• ISSNs: 0305-1048

Abstract

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- Sce I endonuclease recognition site into I- Sce I 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- Sce I 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.