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 (2,3), 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 cryoelectron microscopy approaches. Our focus this past year has been to understand at a molecular level how the transposase interacts with its DNA ends as we believe this is the key to understand the unusual octameric assembly of Hermes. We have collected single particle cryo-EM data on full-length Hermes bound to a pair of its transposon ends, and have been able to visualize for the first time how the N-terminal BED domain interacts with subterminal motifs. To aid these efforts, we have also solved an X-ray structure of the BED domain bound specifically to its recognition motif. Surprisingly, we found that two BED domains are bound to one motif and the organization of the complex suggests which protomers of the octamer contribute these BED domains. We are in the process of preparing this work for publication. Another DNA transposition system of interest to us is piggyBac, an active moth transposon. This transposition system arguably has the widest range of current applications by virtue of its ability to function in many cell types and its seamless excision mechanism that - unlike other eukaryotic cut-and-paste transposons - leaves no genomic footprints. 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 systems 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. Our investigation of Helitron mobility has established the uniqueness of the Helitron transposition mechanism and suggests its potential in future, novel genomic applications. We have recently determined the cryo-electron microscopy structure of Helraiser bound to its cleaved 5' Left Terminal Sequence (10), providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals that the transposase features several domains with novel folds yet it also displays unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an a-helix to control strand cleavage and ligation reactions. Both the Hermes and piggyBac transposase generate sealed hairpins during transposition. Hermes forms them on flanking DNA whereas piggyBac (PB) forms them on its transposon ends. An ongoing effort in the lab is to exploit this property to protect DNA ends in various contexts, such as the development of DNA-based vaccines. This may be of use in the optimization of vaccine strategies against human coronaviruses (such as SARS-cov2) and other infections. 2. Hickman et al. (2005) Molecular architecture of a eukaryotic DNA transposase. Nature Struct. Mol. Biol. 12, 715-721. 3. Hickman et al. (2014) Structural basis of transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica. Cell 158, 353-367. 4. Hickman et al. (2018) Structural insights into the mechanism of double strand break formation by Hermes, a hAT family eukaryotic DNA transposase. Nucleic Acids Res. 46, 10286-10301. 5. Morellet et al. (2018) Sequence-specific DNA binding activityof the cross-brace zinc finger motif of the piggyBac transposase. Nucleic Acids Res. 46, 2660-2677. 6. Henssen et al. (2017) PGBD5 promotes site-specific oncogenic mutations in human tumors. Nature Genet. 49, 1005-1014. 7. Chen, Luo, Veach, Hickman, Wilson, and Dyda (2020) Structural basis of seamless excision and specific targeting by piggyBac transposase. Nature Commun. 11, 3446. 8. Grabundzija et al. (2016) A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes. Nature Commun. 7, 10716. 9. Grabundzija, Hickman, and Dyda (2018) Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition. Nature Commun. 9, 1278. 10. Kosek et al., 2021 Mol. Cell (in press)