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 ( rtt105 Δ; rfa1 -t33) or extensive resection mutant ( sgs1 Δ exo1 Δ). Templated insertions from various distant genomic locations also increase in these mutants as well as in rad27 Δ 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. Taken together, we propose that a shortage of RPA typical in cancer cells is one possible factor stimulating the formation of templated insertions.

Yeast EndoG prevents genome instability by degrading cytoplasmic DNA.

In metazoans release of mitochondrial DNA or retrotransposon cDNA to cytoplasm can cause sterile inflammation and disease 1. Cytoplasmic nucleases degrade these DNA species to limit inflammation 2,3. It remains unknown whether degradation these DNA also prevents nuclear genome instability. To address this question, we decided to identify the nuclease regulating transfer of these cytoplasmic DNA species to the nucleus. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. Nuclear mtDNA (NUMTs) and retrotransposon cDNA insertions increase dramatically in nondividing stationary phase cells. Yeast EndoG (Nuc1) nuclease limits insertions of cDNA and transfer of very long mtDNA (>10 kb) that forms unstable circles or rarely insert in the genome, but it promotes formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of cytoplasmic DNA to nucleus in aging or during meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs can originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating cytoplasmic DNA play a role in preserving genome stability.

Yeast EndoG prevents genome instability by degrading cytoplasmic DNA.

In metazoans release of mitochondrial DNA or retrotransposon cDNA to cytoplasm can cause sterile inflammation and disease. Cytoplasmic nucleases degrade these DNA species to limit inflammation. It remains unknown whether degradation these DNA also prevents nuclear genome instability. To address this question, we decided to identify the nuclease regulating transfer of these cytoplasmic DNA species to the nucleus. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. Nu clear mt DNA (NUMTs) and retrotransposon cDNA insertions increase dramatically in nondividing stationary phase cells. Yeast EndoG (Nuc1) nuclease limits insertions of cDNA and transfer of very long mtDNA (>10 kb) that forms unstable circles or rarely insert in the genome, but it promotes formation of short NUMTs (∼45-200 bp). Nuc1 also regulates transfer of cytoplasmic DNA to nucleus in aging or during meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs can originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating cytoplasmic DNA play a role in preserving genome stability.