Most of a cell?s DNA is packaged into chromosomes located within the nucleus. An additional small circular genome encoding only a few genes is located in the mitochondria. In some circumstances, cells release extrachromosomal pieces of DNA from mitochondrial DNA (mtDNA), from the nuclear genome or in the form of retrotransposon cDNA (rt-cDNA). There are two consequences of DNA fragment release. First, many organisms activate an innate immune response that may result in chronic inflammation and cell death. This inflammation may lead to severe diseases in human including some cases of heart failure, Parkinson?s disease or aging- related chronic inflammation. Second, released DNA can be inserted at DNA double strand breaks (DSBs) causing genome instability. Insertions of mtDNA, rt-cDNA and chromosome fragments destabilize the genome and are common in cancer, whereas rare gene insertions at the V(D)J locus constitute a pathway of antibody diversification. Despite a large body of evidence showing severe consequences of the release of DNA fragments, it remains unknown what cellular events lead to its generation. The goal of this proposal is to understand the mechanisms of free DNA formation and its insertion at DSBs. We will: (i) develop a high throughput method to capture and sequence free DNA, (ii) understand the mechanism of insertion of free DNA at DSBs by nonhomologous end joining and by alternative end joining, and (iii) decipher the mechanisms of linear DNA fragment release from mitochondria and from the nuclear genome.

R01GM080600

2007-05-01

2025-02-28