Pericentromeric heterochromatin is highly enriched for repetitive sequences prone to aberrant recombination. Previous studies showed that homologous recombination (HR) repair is uniquely regulated in this domain to enable ‘safe’ repair while preventing aberrant recombination. In Drosophila cells, DNA double-strand breaks (DSBs) relocalize to the nuclear periphery through nuclear actin-driven directed motions before recruiting the strand invasion protein Rad51 and completing HR repair. End-joining (EJ) repair also occurs with high frequency in heterochromatin of fly tissues, but how alternative EJ (alt-EJ) pathways operate in heterochromatin remains largely uncharacterized. Here, we induce DSBs in single euchromatic and heterochromatic sites using a new system that combines the DR-white reporter and I-SceI expression in spermatogonia of flies. Using this approach, we detect higher frequency of HR repair in heterochromatin, relative to euchromatin. Further, sequencing of mutagenic repair junctions reveals the preferential use of different EJ pathways across distinct euchromatic and heterochromatic sites. Interestingly, synthesis-dependent microhomology-mediated end joining (SD-MMEJ) appears differentially regulated in the two domains, with a preferential use of motifs close to the cut site in heterochromatin relative to euchromatin, resulting in smaller deletions. Together, these studies establish a new approach to study repair outcomes in fly tissues, and support the conclusion that heterochromatin uses more HR and less mutagenic EJ repair relative to euchromatin.
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Characterization of sequence contexts that favor alternative end joining at Cas9-induced double-strand breaks
Abstract Alternative end joining (alt-EJ) mechanisms, such as polymerase theta-mediated end joining, are increasingly recognized as important contributors to inaccurate double-strand break repair. We previously proposed an alt-EJ model whereby short DNA repeats near a double-strand break anneal to form secondary structures that prime limited DNA synthesis. The nascent DNA then pairs with microhomologous sequences on the other break end. This synthesis-dependent microhomology-mediated end joining (SD-MMEJ) explains many of the alt-EJ repair products recovered following I-SceI nuclease cutting in Drosophila. However, sequence-specific factors that influence SD-MMEJ repair remain to be fully characterized. Here, we expand the utility of the SD-MMEJ model through computational analysis of repair products at Cas9-induced double-strand breaks for 1100 different sequence contexts. We find evidence at single nucleotide resolution for sequence characteristics that drive successful SD-MMEJ repair. These include optimal primer repeat length, distance of repeats from the break, flexibility of DNA sequence between primer repeats, and positioning of microhomology templates relative to preferred primer repeats. In addition, we show that DNA polymerase theta is necessary for most SD-MMEJ repair at Cas9 breaks. The analysis described here includes a computational pipeline that can be utilized to characterize preferred mechanisms of alt-EJ repair in any sequence context.
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- PAR ID:
- 10377359
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 13
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- 7465 to 7478
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/nar/gkac575
