We present evidence that excision of the nonreplicative transposon Tn10 involves three distinct chemical steps, first-strand nicking, hairpin formation, and hairpin resolution. This three-step mechanism makes it possible for a single protein-active site to cleave two DNA strands of opposite polarity, as appears to be the case in this reaction. We infer the existence of alternating bifunctionality within the active site with suitable modulation of substrate components between steps. DNA double-strand breaks are also made by a "hairpin mechanism" in V(D)J recombination, possibly reflecting the same basic constraints faced in the Tn10 system. Similarities in the basic chemical steps in Tn10 transposition and V(D)J recombination suggest that the V(D)J mechanism may have evolved from a bacterial transposition system.

Site-directed mutagenesis and detailed fluorescence studies were used to study the structure and dynamics of recombinant human proapolipoprotein (proapo) A-I in the lipid free state and in reconstituted high-density lipoprotein (rHDL) particles. Five different mutants of proapoA-I, each containing a single tryptophan residue, were produced in bacteria corresponding to each of the naturally occurring Trp residues (position -3 in the pro-segment, 8, 50, 72, and 108) in the N-terminal half of the protein. Structural analyses indicated that the conservative Phe-Trp substitutions did not perturb the conformation of the mutants with respect to the wild-type protein. Steady-state fluorescence studies indicated that all of the Trp residues exist in nonpolar environments that are highly protected from solvent in both the lipid-free and lipid-bound forms. Time-resolved lifetime and anisotropy studies indicated that the shape of the monomeric form of proapoA-I is a prolate ellipsoid with an axial ratio of about 6:1. In addition, the region surrounding Trp 108 appears to be more mobile than the rest of the protein in the lipid-free state. However, in rHDL particles, no significant domain motion was detected for any of the Trp residues. The results presented in this work are consistent with a model for monomeric lipid-free proapoA-I in which the N-terminal half of the molecule is organized into a bundle of helices.

The transposase family of proteins mediate DNA transposition or retroviral DNA integration via multistep phosphoryl transfer reactions. For Tn10 and phage Mu, a single active site of one transposase protomer catalyzes the successive transposition reaction steps. We examined phosphorothioate stereoselectivity at the scissile position for all four reaction steps catalyzed by the Tn10 transposase. The results suggest that the first three steps required for double-strand cutting at the transposon end proceed as a succession of pseudo-reverse reaction steps while the 3' end of the transposon remains bound to the same side of the active site. However, the mode of substrate binding to the active site changes for the cut transposon 3' end to target DNA strand joining. The phosphorothioate stereoselectivity of the corresponding steps of phage Mu transposition and HIV DNA integration matches that of Tn10 reaction, indicating a common mode of substrate-active site interactions for this class of DNA transposition reactions.

No abstract