Volume 13, Issue 2, Pages 279-290 (January 2004) Healing the Wounds Inflicted by Sleeping Beauty Transposition by Double-Strand Break Repair in Mammalian Somatic Cells  Zsuzsanna Izsvák, Eva E Stüwe, Dora Fiedler, Andrea Katzer, Penny A Jeggo, Zoltán Ivics  Molecular Cell  Volume 13, Issue 2, Pages 279-290 (January 2004) DOI: 10.1016/S1097-2765(03)00524-0

Figure 1 Sleeping Beauty Transposition in Rodent Cell Lines that Are Defective Either in NHEJ Repair Factors or in Other Repair Pathways The numbers in parentheses below the bars represent numbers of antibiotic-resistant cell colonies obtained in the absence of transposase per numbers obtained in the presence of transposase per 104 transfected cells. Complementation was done by cotransfection of expression constructs. Transpositional efficiencies in the wild-type cell lines (black columns) are taken as reference points and given a value of 100%. Molecular Cell 2004 13, 279-290DOI: (10.1016/S1097-2765(03)00524-0)

Figure 2 Sleeping Beauty Transposition Has the Capacity to Induce Moderate Cell Death in NHEJ-Deficient Cells Cells were transfected with a transposon donor plasmid together with a transposase-expressing plasmid or a control plasmid expressing β-galactosidase. Each cell line was analyzed for transposition, as in Figure 1, and for transposition-induced cell death with flow cytometry. In the apoptotic assay, cells were stained with both fluorescein-coupled annexin and propidium-iodide. Early apoptotic (EA), late apoptotic (LA), and necrotic (N) cells were sorted based on fluorescence. Transposition in wild-type cells is set to 100%, and cell death in those cells is not measurable. Percentages of transposition events and dying/dead cells in the mutant cells are shown relative to transposition in their wild-type counterparts. For example, transposition in XR-1 cells is about 14% of that measured in the wild-type cell line CHO-K1, and a 12% fraction of transposition events is lost due to cell death. Molecular Cell 2004 13, 279-290DOI: (10.1016/S1097-2765(03)00524-0)

Figure 3 Analysis of Sleeping Beauty Transposon Excision Sites in Wild-Type and Mutant Cells (A) Transposon footprint formation and assay for detection. SB transposition proceeds with staggered cuts at the ends of the transposon, which generate 3′ overhangs at the excision sites. After repair, short transposon footprints are left behind, which can be recovered by PCR using nested primers (arrows) flanking the transposon in the donor plasmid. The agarose gel shows PCR products obtained from the cell lines indicated after transfection of transposon donor and transposase helper constructs. The appearance of an approximately 320 bp band is indicative of transposon excision and subsequent repair of the excision site. The approximately 800 bp band is an artifactual product not related to transposon excision. (B) Sequence analysis of transposon footprints. SB transposition generates 3 nucleotide-long 3′ overhangs at the excision sites, which are converted to transposon footprints by DNA repair. The PCR bands shown in (A) were cloned into plasmid vectors and sequenced. The overall structures of the different types of product recovered are shown. The boxed sequences are microhomologies. Molecular Cell 2004 13, 279-290DOI: (10.1016/S1097-2765(03)00524-0)

Figure 4 Factors of Homologous Recombinational Repair Affect Sleeping Beauty Transposition (A) SB transposition in rodent cell lines defective in HR repair factors. The graphs are organized and labeled as in Figure 1. (B) Transposon footprint formation. The agarose gel shows PCR products obtained from Xrcc3-deficient Irs1SF cells transfected with an empty RNAi vector, Irs1SF cells transfected with an RNAi construct against Ku80 (RNAi-Ku), and Irs1SF cells transfected with the RNAi-Ku construct plus an Xrcc3 expression plasmid. PCR assay, sequence analysis, and labels are as in Figure 3. The arrow marks a dominant PCR product, whose structure is shown on the right. (C) Transposition. The graphs show relative efficiencies of transposition in cells treated as in (B). Molecular Cell 2004 13, 279-290DOI: (10.1016/S1097-2765(03)00524-0)

Figure 5 The Effect of DNA-PKcs on Sleeping Beauty Transposition (A) Effects of adenovirus E4 ORF3 (11k) and ORF6 (34k) gene products on SB transposition. Shown are relative transpositional efficiencies in the absence and presence of adenovirus 11k and 34k gene products. Plasmids of the indicated amounts were cotransfected with transposon donor and transposase-expressing plasmids into AA8 and V3 cells. Efficiency of transposition in the presence of β-galactosidase is taken as reference and given a value of 100%. (B) The effect of overexpression of DNA-PK on SB transposition in mouse 3T3 cells. Shown are numbers of G-418-resistant colonies obtained in 3T3 cells in which human DNA-PK proteins were overexpressed. Overexpression was achieved by either transiently or stably expressing DNA-PK proteins. (C) Kinase-defective mutants of DNA-PKcs rescue efficient SB transposition. Complementation in V3 cells was done by cotransfection of expression constructs. Transpositional efficiencies in the wild-type AA8 cell line is taken as a reference and given a value of 100%. (D) The kinase-dead mutant of DNA-PKcs does not support DSB repair. V3 cells were transfected with plasmids expressing wild-type and K3752M DNA-PKcs constructs and treated with different concentrations of the antibiotic bleomycin. Equal numbers of cells were plated and allowed to form colonies, which were counted. (E) The DNA-PKcs inhibitor LY294002 does not inhibit SB transposition. The graphs show relative transpositional efficiencies in CHO-K1 cells in the absence (100%) and presence of different concentrations of LY294002. (F) LY294002 sensitizes wild-type cells to DSBs. CHO-K1 cells were treated with different concentrations of LY294002 and bleomycin. Equal numbers of cells were plated and allowed to form colonies, which were counted. Molecular Cell 2004 13, 279-290DOI: (10.1016/S1097-2765(03)00524-0)

Figure 6 Sleeping Beauty Transposase Interacts with Ku (A) Immunoprecipitation of nuclear protein extracts of HeLa cells. Extracts of HeLa cells and their derivatives stably expressing SB transposase were incubated with a human Ku70 antibody or nonspecific antibodies against α1-antitrypsin, actin, and p15; blotted; and hybridized with an SB antibody. (B) Western hybridizations of nuclear protein extracts prepared from wild-type (CHO-K1) and Ku-deficient (Xrs6) cells stably expressing the SB transposase. Nuclear extracts were blotted and hybridized with a SB antibody (lanes 1 and 2) or with an actin antibody (lanes 3 and 4). (C) Immunoprecipitation of nuclear protein extracts prepared from wild-type and Ku-deficient cells stably expressing the SB transposase. Extracts were incubated with a Ku70 antibody (lanes 1 and 3) or preimmune serum (lane 2), blotted, and hybridized with an SB antibody. Molecular Cell 2004 13, 279-290DOI: (10.1016/S1097-2765(03)00524-0)

Figure 7 Possible Cellular Responses to DSBs Generated by Transposition Transposase-mediated excision of transposons from donor plasmids generates two molecules: the empty donor plasmid and the excised transposon. The transposon ends are bound by transposase molecules, which mediate genomic insertion. The ends of the empty donor plasmids (as well as those of occasional, unintegrated transposons) might be recognized as DNA damage by sensors, such as the ATM/ATR kinases. NHEJ is primarily involved in repair of the excision sites but might also be responsible for the formation of transposon circles (dashed line). HR can also contribute to excision site repair. The transposase (gray oval) interacts with Ku (small dotted circle). After transposon excision, a stable postcleavage complex may form, in which DNA-PKcs (large, star-like object) is proposed to play a scaffold role. Additional proteins might be recruited to the scaffold, thereby influencing the outcome of the transposition reaction. ATM can signal to the cell cycle checkpoint apparatus and can directly be involved in repair. If the single-stranded gaps flanking the transposition intermediates are left unrepaired, they can be converted to DSBs during DNA replication. Unsuccessful repair of any of the DNA damage associated with transposition might result in cell death. Molecular Cell 2004 13, 279-290DOI: (10.1016/S1097-2765(03)00524-0)