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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