191. Identification of a Novel Functional Domain in the Sleeping Beauty Transposase: Towards Alleviating the Restriction of SB Overproduction Inhibition

Abstract

DNA transposons such as Sleeping Beauty (SB) have been shown to be effective vectors for gene therapy. However, one limitation of the SB system is its regulation by a phenomenon known as overproduction inhibition (OPI), in which elevated concentrations of transposase enzyme inhibit the transposition reaction. In an attempt to characterize the mechanism behind this process, we studied the structure/function relationship of the wild-type SB10 protein|[mdash]|focusing on the region linking the N-terminal DNA binding domain and the C-terminal catalytic domain. This area of SB10 is not only highly conserved (55-60%) among the Tc1/Mariner family of transposases, but has also been previously implicated in adversely affecting SB's DNA binding ability. We first tested whether regions downstream of the transposase N-terminus contribute to SB10 DNA binding activity by analyzing various SB deletion mutants in a yeast one-hybrid assay for transposase-transposon interactions. Results show that the ability of the protein to bind transposon DNA increases exponentially in a manner that is proportional with the size of the C-terminal deletion. Mutational analyses of this inter-domain linker region indicate that it is important for transposase function since nearly all mutants analyzed were either completely inactive or 2-3 fold hyperactive in a quantitative transposition assay. To ascertain whether this newly-identified functional domain contributes to OPI, SB10 and five hyperactive mutants were assayed for transposition activity at increasing transposase:transposon ratios. All mutants demonstrated altered OPI titration curves as compared to SB10, with two distinct mutant classes emerging. Class 1 mutants yielded a curve which was shifted towards higher transposase:transposon ratios, but had the same maximum activity as the SB10 curve. In contrast, Class 2 mutants had transposition levels only reaching |[sim]|50% that of SB10. However, these curves were also shifted toward higher amounts of transposase. Based on the similar shift in curves for both types of mutants, we concluded that mutations in the linker domain resulted in an increased OPI threshold. To elucidate the molecular mechanism behind this change in SB regulation, we tested the binding abilities of these linker domain mutants using our yeast one-hybrid assay. We found that all the inactive transposase mutants were able to bind transposon DNA at least as well as SB10 and some of hyperactive mutants exhibited up to a 4-fold change in DNA binding ability. Though some of the mutants did show a dramatic change in transposon binding, we observed no obvious overall correlation between binding and transpositional regulation. However, based on an altered OPI curve of an N-terminal transposase mutant with known increased affinity for the transposon, we suspect that some relationship between binding and regulation exists. Although we do not as yet completely understand the mechanism behind OPI, our results indicate that it is possible to remove some of the stringency of SB self-inhibition. Through continued study of this regulatory process, we hope to eventually overcome this obstacle and improve the efficiency and utility of SB as a vehicle for gene transfer.