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...

Nuclear factor I (NFI) or its isolated DNA‐binding domain (NFI‐BD) enhances initiation of adenovirus DNA replication up to 50‐fold at low concentrations of the precursor terminal protein‐DNA polymerase (pTP‐pol) complex. Both in solution and when bound to DNA, NFI‐BD can form a complex with pTP‐pol. To investigate the mechanism of enhancement by NFI, we determined the stability of a functional preinitiation complex formed in vitro between pTP‐pol and the origin. Challenge experiments with a distinguishable template containing an identical origin revealed that in the absence of NFI, this preinitiation complex was very sensitive to competition for pTP‐pol. Addition of NFI‐BD increased the half‐life of the complex at least 10‐fold and led to the formation of a template‐committed preinitiation complex. In agreement with this, binding of pTP‐pol to origin DNA in band‐shift assays was enhanced by NFI. By DNase I footprinting we show that the specificity of binding as well as induction of structural changes in origin DNA by pTP‐pol are increased by NFI. These results indicate that NFI, by binding and positioning pTP‐pol, stabilizes the complex between pTP‐pol and the core origin, and thus enhances initiation of DNA replication.

Initiation of adenovirus DNA replication is strongly enhanced by two transcription factors, nuclear factor I (NFI) and nuclear factor III (NFIII/oct-1). These proteins bind to two closely spaced recognition sequences in the origin. We produced NFI and NFIII/oct-1, as well as their biologically active, replication-competent DNA-binding domains (NFI-BD and the POU domain), in a vaccinia virus expression system and purified these polypeptides to apparent homogeneity. By DNase I footprinting and gel retardation, we show that the two proteins, as well as their purified DNA-binding domains, bind independently and without cooperative effects to their recognition sequences. By using a reconstituted system consisting of the purified viral proteins (precursor terminal protein-DNA polymerase complex (pTP-pol) and DNA-binding protein, we show that NFIII/oct-1 or the POU domain stimulates DNA replication in the absence of NFI or NFI-BD and vice versa. When added together, the enhancing effect of the two transcription factors was independent and nonsynergistic. Interestingly, stimulation by NFI or NFI-BD was strongly dependent on the concentration of the pTP-pol complex. At low pTP-pol concentrations, NFI or NFI-BD stimulated up to 50-fold, while at high concentrations, the stimulation was less than twofold, indicating that the need for NFI can be overcome by high pTP-pol concentrations. In contrast, stimulation by NFIII/oct-1 or the POU domain was much less dependent on the pTP-pol concentration. These data support a model in which NFI enhances initiation through an interaction with pTP-pol. Glutaraldehyde cross-linking experiments indicate contacts between pTP-pol and NFI but not NFIII/oct-1. The site of interaction is located in the NFI-BD domain.

Guanosine triphosphate (GTP)-binding proteins are involved in controlling a wide range of fundamental cellular processes. In vitro studies have indicated a role for GTP during Drosophila P element transposition. Here we show that P element transposase contains a non-canonical GTP-binding domain that is critical for its ability to mediate transposition in Drosophila cells. Moreover, a single amino acid substitution could switch the nucleotide binding-specificity of transposase from GTP to xanthosine triphosphate (XTP). Importantly, this mutant protein could no longer function effectively in transposition in vivo but required addition of exogenous xanthine or xanthosine for reactivation. These results suggest that transposition may be controlled by physiological GTP levels and demonstrate that a single mutation can switch the nucleotide specificity for a complex cellular process in vivo.

Initiation of adenovirus DNA replication in vitro minimally requires the viral TP-DNA template and the precursor terminal protein-DNA polymerase heterodimer (pTP-pol). Optimal initiation occurs in the presence of the cellular transcription factors NFI and Oct-1 and the viral DNA binding protein (DBP). We have studied the influence of these three stimulatory proteins on the kinetics of formation of the pTP-dCMP initiation complex. NFI increases the Vmax of the reaction but does not affect the apparent Km for dC-TP. This indicates that NFI acts by enlarging the amount of active initiation complex in agreement with its stabilizing effect on binding of pTP-pol to the template. Similar kinetic effects were observed for Oct-1. Since Oct-1 does not stabilize binding of pTP-pol to the origin this suggests that Oct-1 increases the rate of pTP-dCMP formation. DBP stimulates the initiation reaction in two ways. First, it moderately increases the Vmax at suboptimal NFI concentrations, which is related to its enhancing effect on binding of NFI to the origin. Second, a much larger stimulation was caused by DBP itself based on a reduction of the Km for dCTP, which was independent of the concentration of pTP-pol or NFI. The Km for dCTP during initiation is lower than during elongation.