P element transposases in Drosophila melanogaster and other Eukaryotes

Transposable elements are mobile genetic sequences that are found in the genomes of nearly all organisms. DNA transposons constitute a major class of transposable elements and can move throughout the host genome by a cut-and-paste mechanism catalyzed by an encoded transposase protein. Although transposase activity can be detrimental to the host, numerous examples of host benefit have been documented. Over evolutionary time transposon-related sequences and proteins have been adapted to serve a wide range of cellular functions, a process termed transposon domestication./nThe Drosophila P element is one well-studied example of a eukaryotic DNA transposable element. Although the encoded P element transposase protein has been biochemically characterized, it exhibits several features that distinguish it from the other characterized DNA transposases. Namely, P element transposase requires a guanosine triphosphate (GTP) cofactor and generates unusually long 17 nucleotide staggered DNA breaks at the transposon ends during transposition. To gain insight into the molecular basis of these distinguishing features we determined the cryo-EM structure of the Drosophila P element transposase strand transfer complex (STC) to 3.6 Å - a nucleoprotein complex in which the transposase protein is bound to P element donor DNAs covalently joined to a target DNA. Our structure reveals that the STC is dimeric, the P element donor DNAs adopt a highly unusual DNA geometry and further reveals a function for GTP in positioning the P element ends into the transposase active site for catalysis. This structure provides the first view of the P element superfamily of eukaryotic DNA transposases, offers new insights in P element transposition and implies a transposition pathway that is mechanistically distinct from other cut-and-paste DNA transposases./nFurthermore, bioinformatic and biochemical analysis have identified C2CH DNA binding domain termed the THAP domain. This novel and evolutionarily conserved domain is found across a wide range of animal genomes, including vertebrates, invertebrates, Drosophila P element transposase, in primates and in 12 human genes. Of the 12 THAP domain containing genes in humans, THAP9 is homologous to the entirety of Drosophila P element transposase, still has DNA transposase activity, but lacks the hallmarks of an active DNA transposable element. maintained. The evidence implies that THAP9 has likely been domesticated/adapted by the cell in early chordates from an ancient THAP9-like P element transposon, such as those found in Ciona. However, a cellular function for THAP9 has not been identified. In an attempt to elucidate a cellular function for THAP9, we carried out genome-editing in human embryonic stem cells (hESCs) to either knockout or epitope tag the endogenous THAP9 gene. Disruption of THAP9 did not produce overt phenotypic changes in hESCs and did not affect differentiation into fibroblast-like cells, indicating that THAP9 is likely not required for the hESC maintenance. However, endogenously epitope tagged THAP9 is translated, can be immunoprecipitated and localizes to the nucleus in hESCs. To determine potential THAP9 human genome cleavage and binding sites, we raised an antibody to purified, recombinant human THAP9 protein, performed direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing, or BLESS, to detect potential DNA cleavage site, a method used successfully to find Cas9 off-target genomic cleavage sites and ChIP-Nexus experiment, a chromatin immunoprecipitation method similar to ChIP-Exo. The ongoing analysis and comparison of both the BLESS and ChIP-Nexus sequencing data should identify genomic binding sites, potential genomic DNA cleavage sites, motifs associated with human THAP9 DNA binding and cleavage and should uncover a cellular function for the human THAP9 gene./nWhile these projects are essentially independent of one another, they all relate to P element DNA transposases. Together, they hopefully contribute to a deeper understanding of the mechanisms of P element transposition and the expanding roles that transposase-related proteins play in the context of cellular function in human cells.

Advisor: Rio, Donald C