Mechanisms of insertions at a DNA double-strand break

• Published in: Molecular cell 2023-07, Vol.83 (14), p.2434-2448.e7
• Main Authors: Min, Jaewon, Zhao, Junfei, Zagelbaum, Jennifer, Lee, Jina, Takahashi, Sho, Cummings, Portia, Schooley, Allana, Dekker, Job, Gottesman, Max E., Rabadan, Raul, Gautier, Jean
• Format: Article
• Language: English
• Subjects: DNA - genetics; DNA Breaks, Double-Stranded; DNA double-strand breaks; DNA End-Joining Repair; DNA Repair; Genomic Instability; Humans; insertions; nuclear actin; recombination; transcription, break-induced replication; Tripartite Motif Proteins - genetics; Ubiquitin-Protein Ligases - metabolism
• ISSN: 1097-2765; 1097-4164; 1097-4164
• DOI: 10.1016/j.molcel.2023.06.016

Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay called insertion and deletion sequencing (Indel-seq) to monitor rearrangements in human cells at the TRIM37 acceptor locus that reports indels stemming from experimentally induced and spontaneous genome instability. Templated insertions, which derive from sequences genome wide, require contact between donor and acceptor loci, require homologous recombination, and are stimulated by DNA end-processing. Insertions are facilitated by transcription and involve a DNA/RNA hybrid intermediate. Indel-seq reveals that insertions are generated via multiple pathways. The broken acceptor site anneals with a resected DNA break or invades the displaced strand of a transcription bubble or R-loop, followed by DNA synthesis, displacement, and then ligation by non-homologous end joining. Our studies identify transcription-coupled insertions as a critical source of spontaneous genome instability that is distinct from cut-and-paste events. [Display omitted] •Insertions at a DSB harbor asymmetrical junctions—one blunt and one with microhomology•Insertions require contact and recombination between donor and acceptor loci•Transcription-coupled break-induced replication drives insertions•Transcription facilitates insertions in cancer genomes Min et al. describe how insertions at a double-strand break are generated by transcription-coupled break-induced replication. This aberrant recombination process introduces a transcription bias for insertions, which is also observed in cancer genomes.