RPA and Rad27 limit templated and inverted insertions at DNA breaks

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). This process is exacerbated in mutants with low levels or mutated RPA (rtt105Δ; rfa1-t33) or extensive resection mutant (sgs1Δ exo1Δ). Templated insertions from various distant genomic locations also increase in these mutants as well as in rad27Δ and originate from fragile regions of the genome. Among complex events, common events are insertions of two sequences, originating from the same locus and with inverted orientation. Evidence suggests that inversions are also formed by microhomology-mediated template switching. Taken together, our results suggest that limited RPA, typical in many cancers, may contribute to the high level of microhomology-mediated templated insertions.