Structure and function of eukaryotic DNA transposases

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Eukaryotic DNA transposons can be classified into a number of distinct superfamilies, and one of the most widely distributed of these is the piggyBac superfamily. The original piggyBac mobile element was in the genome of the cabbage looper moth, arguably has the widest range of current applications by virtue of its ability to function in many cell types and its simple cut-and-paste mechanism. It can insert exogenous DNA as large as 100 kilobases into the genome, which simplifies a number of gene and cell therapy approaches. Many aspects of its transposition mechanism are not yet well-understood. However, we recently determined its structure in two states: one when the piggyBac transposase (PB) synapsed the two transposon ends and the other right after it inserted these ends into target DNA. The protein forms an asymmetric dimer in which one transposon end interacts with both C-terminal domains of the protein but the other end is much less tightly bound. This observed asymmetry suggested to us possible ways to re-engineer the transpososome with the aim at simplification. With the help of AlphaFold2 structural prediction and biophysical approaches, we defined the role of the transposase N-terminus. We found that phosphorylation of casein kinase II sites in the N-terminus inhibits transposition, most likely by preventing transposase-DNA interactions. Deletion of the region containing these sites releases the inhibition and enhances transposition activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. Based on the cryo-EM structures, we engineered a symmetrized version of the transpososome that showed substantially increased activity on symmetric transposon ends. We continue to explore the use of this modified piggyBac system for genomic applications. Building on this success, we have been extending our line of investigation to other members of the PiggyBac-Like Elements (PBLE) superfamily, including piggyBat, which is to date the only known active DNA transposon found in mammals. Through a combination of in vitro DNase I footprinting experiments and cell culture-based transposition assays, we have been able to rationalize the organization of the piggyBat transposon ends. The results allowed us to determine the cryo-EM structure of a piggyBat pre-synaptic complex containing one bound transposon end at 3.6 Å resolution. Modifications of both the LE and the Right End (RE), elimination of predicted inhibitory N-terminal phosphorylation sites of the transposase, and tandem duplication of its C-terminal DNA binding domain following a design based on the cryo-EM structure increased transposition activity by two orders of magnitude relative to the activity of the wild-type transposon. This system that has been evolving in mammals represents another possibility for exploration for genomic applications, and the results were recently published. Our current efforts are focused on strategies to convert PBLEs to site-specific integrases. The third superfamily of eukaryotic DNA transposons that we have been studying are the Helitrons. These elements must once have been very active, as their remnants are widespread throughout the eukaryotic kingdom. Unlike other known eukaryotic DNA transposons, Helitron insertions in the host genome are not bordered by target site duplications, suggesting a replicative transposition mechanism that differs substantially from the cut-and-paste mode of transposition used by all other currently characterized eukaryotic DNA transposons. We have reconstituted an active Helitron transposon, called Helraiser, from the genome of the little brown bat (Myotis lucifugus). We recently determined the cryo-EM structure of Helraiser bound to its cleaved 5' Left Terminal Sequence, providing insight into its multidomain architecture and function. Our investigation of Helitron mobility has established the uniqueness of the Helitron transposition mechanism and suggests its potential in future, novel genomic applications which we are currently exploring.