Four cationic polymers used to deliver DNA into cultured dendrimer generally appeared as clusters in electron cells: polylysine, intact polyamidoamine dendrimer, fracmicrographs; their diameters in solution were larger than tured polyamidoamine dendrimer and polyethylenimine, 1000 nm, which suggests that their toroidal complexes are examined for their ability to interact with DNA. Comaggregate in solution. The cationic polymers bind to DNA plexes between the polymers and DNA were examined in a stoichiometry that is nearly 1:1 in primary amines to using electron microscopy. Similar toroidal structures with DNA phosphates. The apparent binding of all cationic polydiameters of 55 ± 12 nm were formed from all of the catmers to DNA decreases linearly with increasing ionic ionic polymers with DNA. The DNA complexes of the fracstrength, up to 0.8 M NaCl. Thus, at the concentrations tured dendrimer and polyethylenimine were observed as studied, these polymers interact electrostatically with DNA single, distinct units; their apparent diameters in solution forming a unit structure with toroidal morphology; the as measured by dynamic light scattering ranged from 90 extent of aggregation of the unit structures in solution to 130 nm. The DNA complexes of polylysine and intact depends upon the characteristics of the individual polymer.

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...

P transposable elements in Drosophila are members of a larger class of mobile elements that move using a cutand-paste mechanism. P-element transposase uses guanosine triphosphate (GTP) as a cofactor for transposition. Here, we use atomic force microscopy (AFM) to visualize protein-DNA complexes formed during the initial stages of P-element transposition. These studies reveal that GTP acts to promote assembly of the first detectable noncovalent precleavage synaptic complex. This initial complex then randomly and independently cleaves each P-element end. These data show that GTP acts to promote protein-DNA assembly, and may explain why Pelement excision often leads to unidirectional deletions.