Publication Date
1-7-2025
Journal
Nucleic Acids Research
DOI
10.1093/nar/gkae1159
PMID
39673516
PMCID
PMC11724301
PubMedCentral® Posted Date
12-3-2024
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
DNA Breaks, Double-Stranded, Replication Protein A, Saccharomyces cerevisiae Proteins, Saccharomyces cerevisiae, DNA End-Joining Repair, Mutagenesis, Insertional, Exodeoxyribonucleases, Mutation, RecQ Helicases, Flap Endonucleases
Abstract
Formation of templated insertions at DNA double-strand breaks (DSBs) is very common in cancer cells. The mechanisms and enzymes regulating these events are largely unknown. Here, we investigated templated insertions in yeast at DSBs using amplicon sequencing across a repaired locus. We document very short (most ∼5-34 bp), templated inverted duplications at DSBs. They are generated through a foldback mechanism that utilizes microhomologies adjacent to the DSB. Enzymatic requirements suggest a hybrid mechanism wherein one end requires Polδ-mediated synthesis while the other end is captured by nonhomologous end joining (NHEJ) or by alternative end joining (Alt-EJ). This process is exacerbated in mutants with low levels or mutated RPA (rtt105Δ; rfa1-t33) or extensive resection deficiency (sgs1Δ exo1Δ). Templated insertions from various distant genomic locations also increase in RPA mutants as well as in rad27Δ and originate from fragile regions of the genome. Among complex insertions, common events are insertions of two sequences, originating from the same locus and with inverted orientation. We propose that these inversions are also formed by microhomology-mediated template switching. Together, we propose that a shortage of RPA, typical in cancer cells, may be a factor that stimulates the formation of templated insertions.
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