The human genome contains ~50 genes that were derived from transposable elements or transposons, and many are now integral components of cellular gene expression programs. The human THAP9 gene is related to the Drosophila P-element transposase. Here, we show that human THAP9 can mobilize Drosophila P-elements in both Drosophila and human cells. Chimeric proteins formed between the Drosophila P-element transposase N-terminal THAP DNA binding domain and the C-terminal regions of human THAP9 can also mobilize Drosophila P elements. Our results indicate that human THAP9 is an active DNA transposase that, although “domesticated,” still retains the catalytic activity to mobilize P transposable elements across species.

P transposable elements were discovered in Drosophila as the causative agents of a syndrome of genetic traits called hybrid dysgenesis. Hybrid dysgenesis exhibits a unique pattern of maternal inheritance linked to the germline-specific small RNA piwi-interacting (piRNA) pathway. The use of P transposable elements as vectors for gene transfer and as genetic tools revolutionized the field of Drosophila molecular genetics. P element transposons have served as a useful model to investigate mechanisms of cut-and-paste transposition in eukaryotes. Biochemical studies have revealed new and unexpected insights into how eukaryotic DNA-based transposons are mobilized. For example, the P element transposase makes unusual 17nt-3′ extended double-strand DNA breaks at the transposon termini and uses guanosine triphosphate (GTP) as a cofactor to promote synapsis of the two transposon ends early in the transposition pathway. The N-terminal DNA binding domain of the P element transposase, called a THAP domain, contains a C 2 CH zinc-coordinating motif and is the founding member of a large family of animal-specific site-specific DNA binding proteins. Over the past decade genome sequencing efforts have revealed the presence of P element-like transposable elements or P element transposase-like genes (called THAP9) in many eukaryotic genomes, including vertebrates, such as primates including humans, zebrafish and Xenopus, as well as the human parasite Trichomonas vaginalis, the sea squirt Ciona, sea urchin and hydra. Surprisingly, the human and zebrafish P element transposase-related THAP9 genes promote transposition of the Drosophila P element transposon DNA in human and Drosophila cells, indicating that the THAP9 genes encode active P element "transposase" proteins.

The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the ␣ subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of G␣ subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a G␣ chimera (designated ␣ T *), comprised mainly of the ␣ subunit of retinal transducin (␣ T ) and the Switch III region from the ␣ subunit of G i1 . As expected, both the ␣ T *(R174C) and ␣ T *(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and G␤␥. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and G␤␥ to undergo GDP-[ 35 S]GTP␥S exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotidebound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P i complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal G␣ subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.The visual phototransduction cascade in retinal rod outer segments consists of the heterotrimeric G protein, transducin, its upstream receptor rhodopsin, and its downstream effector, the cGMP-phosphodiesterase (PDE).2 This is a highly sensitive signaling system, capable of detecting a single photon of light, which excites the sevenmembrane spanning receptor, rhodopsin. Light-activated rhodopsin binds and activates the ␣ subunit of transducin (␣ T ) enabling it to bind GTP. Activated GTP-bound ␣ T then stimulates PDE activity by altering the positions of its regulatory ␥ subunits (␥ PDE ), thereby allowing its catalytic subunits (␣ PDE and ␤ PDE ) to hydrolyze cGMP. The reduction in the cellular levels of this second messenger causes cation-specific cGMP-gated ion channels in the outer segments to close, leading to the hyperpolarization of rod outer segment membranes and the generation of the visual response.

The GDP-GTP exchange activity of the retinal G protein, transducin, is markedly accelerated by the photoreceptor rhodopsin in the first step of visual transduction. The x-ray structures for the ␣ subunits of transducin (␣ T ) and other G proteins suggest that the nucleotide-binding (Ras-like) domain and a large helical domain form a "clam shell" that buries the GDP molecule. Thus, receptor-promoted G protein activation may involve "opening the clam shell" to facilitate GDP dissociation. In this study, we have examined whether perturbing the linker regions connecting the Ras-like and helical domains of G␣ subunits gives rise to a more readily exchangeable state. The sole glycine residues in linkers 1 and 2 were individually changed to proline residues within an ␣ T /␣ i1 chimera (designated ␣ T * ). Both ␣* T linker mutants showed significant increases in their basal rates of GDP-GTP exchange when compared either to retinal ␣ T or recombinant ␣ * T . The ␣ * T linker mutants were responsive to aluminum fluoride, which binds to ␣-GDP complexes and induces changes in Switch 2. Although both linker mutants were further activated by light-activated rhodopsin together with the ␤␥ complex, their activation was not influenced by ␤␥ alone, arguing against the idea that the ␤␥ complex helps to pry apart the helical and Ras-like domains of G␣ subunits. Once activated, the ␣* T linker mutants were able to stimulate the cyclic GMP phosphodiesterase. Overall, these findings highlight a new class of activated G␣ mutants that constitutively exchange GDP for GTP and should prove valuable in studying different G protein-signaling systems.The visual phototransduction cascade in retinal rod outer segments consists of the heterotrimeric G protein, transducin, its upstream receptor rhodopsin and its downstream effector, cGMP-phosphodiesterase (PDE).1 This is a highly sensitive system, capable of detecting and being activated by a single photon of light, which excites the seven-membrane spanning receptor, rhodopsin. Upon the absorption of light, rhodopsin is activated through the photoisomerization of the chromophore, retinal, which is attached through a Schiff base linkage to the seventh membrane-spanning helix of the protein. Light-activated rhodopsin binds and activates the ␣ subunit of transducin (␣ T ) by stimulating its GDP-GTP exchange reaction. Activated GTP-bound ␣ T then stimulates PDE activity by altering the positions of its regulatory ␥ subunits (␥ PDE ) and thereby allowing the ␣ and ␤ catalytic subunits (␣ PDE and ␤ PDE ) to hydrolyze and consequently decrease cellular cGMP concentrations. This causes cation-specific cGMP-gated ion channels in the outer segments to close, leading to the hyperpolarization of rod outer segment membranes and the generation of the visual response. There is an amplification of the signal at every step of this cascade such that a single photon of light can lead to the inhibition of 10 6 -10 7 Na ϩ ions from entering the rod cell. Members of the family of large G proteins are made up of three ...