Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells

Abstract

Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.

Illegitimate DNA integration in mammalian cells

“…97,103 However, the clearest demonstration of the link between DNA repair mechanisms and illegitimate integration comes from studies that find that inducing a chromosomal DSB at a defined genomic locus (by expression of a site-specific endonuclease) promotes illegitimate integration at that site. 29,108 These experiments clearly show that most integration events are probably mediated by the cellular DNA repair machinery.…”

“…NHEJ often involves short sequence homology between the joined ends, and additions or deletions of approximately 25 nucleotides or less at the junctions. 34,36,37 Similar processes have been shown to occur during chromosomal double-strand break (DSB) repair, 27,29,38 which again suggests that extrachromosomal and chromosomal repair are mediated by similar cellular machinery.…”

“…HR has been demonstrated to play an important role in concatamer formation, 41,42 and some studies have shown that cells that participate in an extrachromosomal HR event are more likely to integrate DNA. [29][30][31] In support of a link between both types of DNA repair and integration, cells that incorporated DNA by illegitimate integration were also 80-fold more likely to integrate additional DNA by gene targeting, a process dependent upon homologous recombination. 98 Induction of genomic DNA damage by various means, such as treatment of cells with various Topo I and II inhibitors, 85 restriction enzymes, crosslinking agents (eg psoralen), or UV damage, often leads to an increased frequency of illegitimate DNA integration, supporting the above model.…”

“…[21][22][23][24][25][26] The end products of extrachromosomal homologous recombination are similar to those obtained during chromosomal homologous recombination, suggesting that at least some mechanisms are shared by the two processes. [27][28][29][30][31] Transfected DNA can also be mutated at high frequency (on the order of 1%) by homology-independent means including point mutations, deletions, and more complex rearrangements such as insertion of genomic DNA. [32][33][34] (see also our unpublished work).…”

The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair

“…Illegitimate integration sites are not random but probably depend on open chromatin associated with transcription or repair, allowing access of foreign DNA and integration factors to the genomic DNA (10)(11)(12). Illegitimate integration resembles nonhomologous end-joining (NHEJ) repair, because it is enhanced by DNA damage, especially double-strand breaks (DSBs); it is homology-independent and involves end joining (12)(13)(14). In addition, there are several reports of reduced integration of retroviral DNA and Arabidopsis T elements in NHEJ-defective cells (15)(16)(17)(18).…”