Asymmetrical cleavages ofSleeping Beautytransposons generate multiple excised transposon fragments during transposition

Abstract

1Although the Sleeping Beauty (SB) transposon is the most validated DNA transposon used as a 2 gene delivery vehicle in vertebrates, many details of the excision and integration steps in the transposition 3 process are unclear. We have probed in detail the products of the excision step and apparent selective 4 integration of a subset of those products during transposition. The standard model of SB transposase-5 mediated transposition includes symmetrical cleavages at both ends of the transposon for excision and 6 re-integration in another DNA sequence. In our analysis of excised transposon fragments (ETFs), we 7 found evidence for the requirement of certain flanking sequences for efficient cleavage and a significant 8 rate of asymmetrical cleavage during the excision process that generates multiple ETFs. Our results 9 suggest that the cleavage step by SB transposase is not as precise as indicated in most models. Repair 10 of the donor ends can produce eight footprint sequences (TACTGTA, TACAGTA, TACATA, TACGTA, 11 TATGTA, TACTA, TAGTA and TATA). Our data also suggest that mismatch repair (MMR) is not an 12 essential requirement for footprint formation. Among the twenty liberated ETFs, only eight appear to 13 effectively re-integrate into TA sites distributed across the genome, supporting earlier findings of unequal 14 rates of excision and reintegration during SB transposition. These findings may be important in 15 considerations of efficiency of SB transposon remobilization, selection of TA integration sites and 16 detection of SB excision and integration loci, all of which may be important in human gene therapy. 17 18 20 31 poorly understood because i) in vitro SB transposition assays remain unsuccessful; ii) the crystal structure 32 of full-length SB transposase has not been resolved, although the NMR structure of the SB11 DNA-33 binding domain (33) and crystal structure of the SB100X catalytic domain (11) have been reported; and iii) 34 compared to other Tc1/mariner transposons, the inverted terminal repeat (ITR) sequences that comprise 35 the termini of SB transposons are considerably longer and each has two transposase-binding, direct 36 4 repeat (DR) sequences (Figure 1A) (7). Hence, the ITRs of SB transposons are called IR/DRs for their 1 unusual structure. This last feature introduces several questions about the coordination of transposase 2 enzymes in both the cleavage and integration reactions; e.g., whether dimers or tetramers form to affect 3 either or both steps. 4In the standard model, SB transposon excision results from staggered cuts at both ends of the 5 transposon that leaves a 3-bp single stranded overhanging sequence in both the donor DNA and the 6 excised transposon. When the wounded DNA is repaired, either of two 7-bp canonical footprints, 7 TACAGTA and TACTGTA, that differ in the fourth position (underlined) are left due to ambiguous repair of 8 the mismatch at this position ( Figure 1B) (20). The model suggests that SB transposase precisely cleaves 9 the donor DNA and the targeted integra...