Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP protein binds to multiple sites on the ends of P-element DNA, unlike the full-length transposase protein. These sites include the high-affinity transposase binding site, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-bp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding.P-element transposons are mobile DNA elements found in many Drosophila melanogaster strains and have been used extensively as molecular genetic tools (9,25,26). The biological effects of P elements were initially observed in crosses between laboratory fly strains (M strains), which lack functional P elements, and those isolated from natural populations (P strains), which carry active P elements. When a P-strain female is crossed to either an M-strain male or a P-strain male, the progeny are healthy, with a wild-type germ line. However, when an M-strain female is crossed to a P-strain male, the progeny exhibit a series of genetic disorders in the germ line, such as chromosomal rearrangements, mutations, and sterility, collectively referred to as hybrid dysgenesis. It was later discovered that the causative agent of hybrid dysgenesis is the P-element transposon (5, 31). These observations indicate that P elements introduced by a P strain male into the egg of an M strain female mobilize in the germ line of the developing embryo and cause hybrid dysgenesis. In contrast, a P strain female maternally deposits P-element-encoded repressor proteins in her eggs that repress the mobilization of any P elements introduced by the male. This state of repression in P strain females has been termed the P cytotype. Although genetically well characterized, the molecular mechanisms of P-cytotype repression are not known.The P-element transposon contains several sequence elements at its termini that are essential in cis for transposition (Fig. 1A). The transposase binding site contains a 10-bp consensus sequence and is internally located at each end of the P element (10). Interaction between this site and the transposase protein is essential for transposition (10,21). A terminal 31-bp inverted repeat located at each end of the transposon is also necessary for mobilization and is recognized by a Drosophila host protein, IRBP (inverted repeat binding protein) (4,21,28). Finally, there is an internally located 11-bp inverted repeat t...