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

Area of Science:

• Genetics
• Molecular Biology
• Genomics

Background:

• Insertions of mobile elements, mitochondrial DNA, and nuclear chromosome fragments at DNA double-strand breaks (DSBs) threaten genome integrity and are prevalent in cancer.
• The origins and prevention mechanisms of chromosomal fragment insertions at DSBs are largely unknown.

Purpose of the Study:

• To investigate the mechanisms underlying the insertion of DNA fragments at DSBs.
• To identify the genomic origins and characteristics of these inserted fragments.
• To elucidate the cellular pathways involved in preventing such insertions.

Main Methods:

• Characterization of a yeast mutant lacking the evolutionarily conserved Dna2 nuclease.
• Sequencing of approximately 500 DNA inserts found at DSBs.
• Analysis of the dependence of these duplications on nonhomologous end-joining (NHEJ) and Pol4.

Main Results:

• The Dna2-deficient yeast mutant exhibits frequent insertions of DNA sequences (0.1–1.5 kb) into DSBs, often involving multiple joined fragments.
• Inserted fragments originate from Ty retrotransposons (8%), ribosomal DNA (rDNA) (15%), and other genomic regions, with a preference for fragile sites.
• These insertions represent duplications, as the original loci remain intact, and depend on NHEJ and Pol4.

Conclusions:

• Alternative processing of DNA structures in Dna2-deficient cells can lead to fragment release and subsequent capture at DSBs.
• This mechanism provides insight into the origin of chromosomal fragment insertions.
• Similar DNA insertions at DSBs are likely to occur in any cell type with linear extrachromosomal DNA fragments.