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 so-called "hAT" superfamily, which has active members in plants and insects. We have been studying a representative member of this superfamily, Hermes (2-4), a transposon that is active not only in the house fly from which it was isolated but also in other insects such as Aedes aegypti, the mosquito species that transmits yellow fever. A close relative of Hermes, the Herves transposon, is active in the malaria vector Anopheles gambiae. An active insect transposon is particularly interesting because it offers the potential to produce transgenic insects for controlling medically significant pests. Hermes transposition employs a mechanism in which excision is accompanied by hairpin formation on the DNA flanking the transposon, as also seen for the RAG1/2 recombinase of the adaptive immune system. We are continuing our investigation into the mechanism of Hermes DNA transposition using both crystallographic and cryo-electron microscopy (cryo-EM) approaches. To do this, we have determined three structures: the X-ray structure of the N-terminal BED domain bound specifically to its recognition motif, the cryo-EM structure of the transpososome bound to two transposon Left Ends, and the cryo-EM structure of the transpososome bound to two transposon Right Ends. The collective snapshots reveal that the full-length Hermes octamer extensively interacts with its transposon Left End through multiple BED domains contributed by three Hermes protomers whereas the Right End is not bound by any BED domains. This suggests a transposition mechanism in which the Left End is first captured by the transposase and then the Right End, nearby in the genome (and therefore three-dimensional space), can be located and bound despite its substantially weaker affinity. This work has recently been published (5). Another DNA transposition system of interest to us is piggyBac, a cut-and-paste DNA transposon. This transposition system, originally found 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. 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 (6). 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 transposaseDNA 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. This work has recently been published (7), and we continue to explore the use of this modified piggyBac system for genomic applications. We are also extending this line of investigation to other members of the PiggyBac-Like Elements (PBLE) superfamily. Another DNA transposition system of interest to us is piggyBac, acut-and-paste DNA transposon. This transposition system, originally found 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. It can insert exogenous DNA as large as 100 kilobases in the genome, which simplifies a number of gene and cell therapy approaches. Many aspects of its transposition mechanism are not yet well-understood. Using single particle cryo-EM, we have recently determined its structure in two states illuminating how piggyBac works: one when the piggyBac transposase (PB) is bound to its hairpinned transposon ends and the other after it has inserted these ends into target DNA (7). 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. One unanticipated discovery is that there is a structural "echo" between how PB binds the transposition intermediate hairpins and how it binds its specific TTAA target site. The tetranucleotide is recognized in the single-stranded form by an intricate network of interactions that are dominated by an omega loop of the catalytic domain of the transposase. We are continuing our efforts to understand the early steps in the transposition pathway using structural approaches. 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 (8), from the genome of the little brown bat (Myotis lucifugus). We have shown that for transposition, the donor site must be double-stranded and that single-stranded donors do not suffice (9). Nevertheless, replication and integration assays reveal that only one of the transposon donor strands is used. We have also recently determined the cryo-EM structure of Helraiser bound to its cleaved 5' Left Terminal Sequence (10), 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 continue to explore. We have also recently determined the cryo-EM structure of Helraiser bound to its cleaved 5' Left Terminal Sequence (10), 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 continue to explore. 2. Hickman et al. (2005) Nature Struct. Mol. Biol. 12, 715-721. 3. Hickman et al. (2014) Cell 158, 353-367. 4. Hickman et al. (2018) Nucleic Acids Res. 46, 10286-10301. 5. Lannes et al. (2023) Nature Commun. 14, 4470. 6. Chen et al. (2020) Nature Commun. 11, 3446. 7. Luo*, Hickman* et al. (2023) Nucleic Acids Res. 50, 13128-13142. 8. Grabundzija et al. (2016) Nature Commun. 7, 10716. 9. Grabundzija, Hickman, and Dyda (2018) Nature Commun. 9, 1278. 10. Kosek et al. Mol. Cell 81, 4271-4286.