Numt-Mediated Double-Strand Break Repair Mitigates Deletions during Primate Genome Evolution

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Abstract
Non-homologous end joining (NHEJ) is the major mechanism of double-strand break repair (DSBR) in mammalian cells. NHEJ has traditionally been inferred from experimental systems involving induced double strand breaks (DSBs). Whether or not the spectrum of repair events observed in experimental NHEJ reflects the repair of natural breaks by NHEJ during chromosomal evolution is an unresolved issue. In primate phylogeny, nuclear DNA sequences of mitochondrial origin, numts, are inserted into naturally occurring chromosomal breaks via NHEJ. Thus, numt integration sites harbor evidence for the mechanisms that act on the genome over evolutionary timescales. We have identified 35 and 55 lineage-specific numts in the human and chimpanzee genomes, respectively, using the rhesus monkey genome as an outgroup. One hundred and fifty two numt-chromosome fusion points were classified based on their repair patterns. Repair involving microhomology and repair leading to nucleotide additions were detected. These repair patterns are within the experimentally determined spectrum of classical NHEJ, suggesting that information from experimental systems is representative of broader genetic loci and end configurations. However, in incompatible DSBR events, small deletions always occur, whereas in 54% of numt integration events examined, no deletions were detected. Numts show a statistically significant reduction in deletion frequency, even in comparison to DSBR involving filler DNA. Therefore, numts show a unique mechanism of integration via NHEJ. Since the deletion frequency during numt insertion is low, native overhangs of chromosome breaks are preserved, allowing us to determine that 24% of the analyzed breaks are cohesive with overhangs of up to 11 bases. These data represent, to the best of our knowledge, the most comprehensive description of the structure of naturally occurring DSBs. We suggest a model in which the sealing of DSBs by numts, and probably by other filler DNA, prevents nuclear processing of DSBs that could result in deleterious repair. Changes to DNA sequence are the major source of variation in evolution. Those changes often arise from damage to DNA that is repaired in a way that fails to restore the original sequence. One type of DNA damage is a chromosomal double-strand break. Such breaks are mostly studied experimentally in model systems, because naturally occurring chromosomal breaks are hard to follow. Here, we used an evolutionary approach to study the repair of naturally occurring chromosomal breaks. Throughout evolutionary history, fragments of the mitochondrial genome, known as numts (nuclear sequences of mitochondrial origin), have been inserted into the nuclear genome. Numts are passively captured into random chromosomal breaks, leaving sequence traces in genomes. Humans and chimpanzees share a recent common ancestor and their genomes share high sequence similarity; therefore, their species-specific numts can be used to follow both some of the break structure and repair mechanisms. Comparing naturally occurring break and repair patterns with experimental repair patterns identified similarities but also highlighted a clear difference. Experimental breaks usually involve deletions, while deletions were significantly less frequent in the numt based repair system. We propose that extra-chromosomal DNA sequences, like numts, play a role in maintaining genome integrity by protecting naturally occurring chromosomal breaks from further deleterious processing.