Dna2 nuclease deficiency results in large and complex DNA insertions at chromosomal breaks

Insertions of mobile elements(1-4), mitochondrial DNA(5) and fragments of nuclear chromosomes(6) at DNA double strand breaks (DSBs) threaten genome integrity and are common in cancer(7-9). Insertions of chromosome fragments at V(D)J loci can stimulate antibody diversification(10). The origin of insertions of chromosomal fragments and the mechanisms that prevent such insertions remain unknown. Here we found the first mutant, lacking evolutionarily conserved Dna2 nuclease, that shows frequent insertions of ~0.1-1.5 kb long sequences into DSBs with many events carrying multiple DNA fragments joined together. Sequencing of ~500 DNA inserts revealed that they originate from Ty retrotransposons (~8%), rDNA (~15%) and from throughout the genome with preference for fragile regions such as origins of replication, R-loops, centromeres, telomeres or replication fork barriers. Inserted fragments are not lost from their original loci and therefore represent duplications. These duplications depend on nonhomologous end-joining (NHEJ) and Pol4. We propose a model in which alternative processing of DNA structures arising in Dna2-deficient cells can result in the release of DNA fragments and their capture at DSBs.Similar DNA insertions at DSBs are expected in any cells with linear extrachromosomal DNA fragments.