Nonhomologous-end-joining factors regulate DNA repair fidelity during Sleeping Beauty element transposition in mammalian cells.

Herein, we report that the DNA-dependent protein kinase (DNA-PK) regulates the DNA damage introduced during Sleeping Beauty (SB) element excision and reinsertion in mammalian cells. Using both plasmid- and chromosome-based mobility assays, we analyzed the repair of transposase-induced double-stranded DNA breaks in cells deficient in either the DNA-binding subunit of DNA-PK (Ku) or its catalytic subunit (DNAPKcs). We found that the free 3 overhangs left after SB element excision were efficiently and accurately processed by the major Ku-dependent nonhomologous-end-joining pathway. Rejoining of broken DNA molecules in the absence of Ku resulted in extensive end degradation at the donor site and greatly increased the frequency of recombination with ectopic templates. Therefore, the major DNA-PK-dependent DNA damage response predominates over more-error-prone repair pathways and thereby facilitates high-fidelity DNA repair during transposon mobilization in mammalian cells. Although transposable elements were not found to be efficiently circularized after transposase-mediated excision, DNA-PK deficiency supported more-frequent transposase-mediated element insertion than was found in wild-type controls. We conclude that, based on its ability to regulate excision site junctional diversity and transposon insertion frequency, DNA-PK serves an important protective role during transpositional recombination in mammals. The use of transposable elements as genetic tools has contributed significantly to our understanding of biological systems. The Tc1/mariner elements are likely the most widespread transposons in nature and can transpose in species other than their hosts, making them potential tools for functional genomics in diverse organisms, including vertebrates (48). However, most naturally occurring Tc1/mariner-like transposons are nonfunctional due to the accumulation of inactivating mutations (36). Although no single active element has ever been identified in vertebrates, an active Tc1-like transposon called Sleeping Beauty (SB) was recently reconstructed from pieces of defective fish elements (23). SB functions in a variety of vertebrate species, including human and mouse cells (24), and is the most active member of the Tc1/mariner family (16). Moreover, this element has been applied recently to gene discovery in the mouse germ line (13, 14, 16, 21) and has been shown to promote stable in vivo delivery of therapeutic genes in somatic tissues of adult mice (41, 65, 66). Based on emerging interest in further developing such tools for basic research and therapeutic applications, studies which elucidate the molecular mechanisms involved in transposition and its regulation in mammals remain of significant importance. Each end of SB contains an inverted repeat (IR)-direct repeat (DR) structure consisting of two short DRs within an 230-bp imperfect terminal IR. These DRs (30 bp) serve as core binding sites for the element-encoded transposase (23), and the presence of both sites within an individual IR is required for efficient transposition (24). In addition to the DRs, the left IR of SB contains a half binding site, termed HDR, which acts as a transpositional enhancer-like sequence (25). Specific binding to the DRs is mediated by an N-terminal, paired-domain-like DNA-binding domain of the transposase (23, 25). The C-terminal, catalytic domain of the transposase is responsible for all DNA cleavage and strand transfer reactions and is characterized by the presence of a conserved amino acid triad, the DDE motif. This catalytic triad is found in a large group of recombinases, including many eukaryotic and bacterial transposases, retroviral integrases, and the RAG1 V(D)J recombinase, involved in immunoglobulin gene rearrange