Analysis of P Element Transposase Protein-DNA Interactions during the Early Stages of Transposition

Abstract

P elements are a family of transposable elements found inDrosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.Mobile genetic elements are ubiquitous among both prokaryotic and eukaryotic organisms (1). Genome sequencing projects have shown that transposable elements make up a substantial fraction of eukaryotic genomes, including 49% of the human genome (2, 3). These mobile elements can lead to mutations and genome rearrangements and appear to play a role in genome evolution (4, 5). The mechanisms by which transposons are mobilized can be grouped based upon whether there is a DNA or an RNA intermediate (1,4,5). P elements use a cut-and-paste mechanism with a DNA intermediate, related to those used by the Tn5, Tn10, and Tn7 prokaryotic transposons and the eukaryotic Tc1/mariner family (6 -8). Studies of P element transposition in vitro have demonstrated cofactor requirements for both GTP and magnesium ions (Mg 2ϩ ) (6, 9 -11). Although divalent metal ions are universally required cofactors for transposase proteins (1, 12, 13), the P element reaction is unique among this family of polynucleotidyltransferases in the use of GTP as a cofactor (9). Recent studies have shown that GTP promotes pre-cleavage synaptic complex assembly between the P element termini and the transposase protein (14). Previous in vitro studies had also shown that nonhydrolyzable GTP analogs support both the P element DNA cleavage and joining reactions (9). Furthermore, GTP does not effect the binding of P element transposase to the high affinity sites near the transposon termini, as assayed by DNase I footprinting (9, 15). Whereas some transposon systems have been studied with respect to the assembly state of their transposase, the oligomeric state of the active P element transposase protein and how it initially interacts with P element DNA are unknown. In cases where it has been studied, other transpos...

DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

“…It binds to DNA double-strand breaks to form a synapsis and recruits other proteins such as the ligase complex (17)(18)(19)(20). As a central event of transposition, PEC formation is also characterized by a synapsis between both ends of the transposon (21,22), and we thus addressed the question of whether PBase interacting with DNA-PK contributes to PEC formation.…”

Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis

“…The cleaved P-element ends remain tightly bound by the transposase enzyme (10,16). The flanking donor site DNA is released after P-element cleavage (16) and needs to be efficiently repaired by endogenous DNA repair mechanisms. Engineered somatic mobilization of as few as 15-20 small nonautonomous P elements by transposase leads to a temperature-dependent pupal lethality due to extensive DNA damage and apoptosis (17).…”

“…The P-element transposase catalyzes DNA cleavage within the 31-bp TIRs to create 17-nt 3′ single-strand extensions at both the donor site and the transposon ends (14,15). The cleaved P-element ends remain tightly bound by the transposase enzyme (10,16). The flanking donor site DNA is released after P-element cleavage (16) and needs to be efficiently repaired by endogenous DNA repair mechanisms.…”

Mechanisms of DNA Transposition

“…The active form of Hermes is a ring-shaped octamer in which eight N-terminal site-specific DNA binding domains are available to interact with these interior sites while presenting the two transposon TIRs to the catalytic sites of one of the dimers of the octameric assembly (91; Figure 6E). The P element transposase, which also has asymmetric ends (Majumdar & Rio, this volume), is reportedly tetrameric both prior to DNA binding and upon end synapsis (84). Sleeping Beauty, a resurrected vertebrate transposase of the Tc1/mariner family (117), is also proposed to form a tetrameric transpososome (118).…”