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.

Architectural protein IHF modulates Tn10 transposition in vitro. IHF stimulates transposon excision. Also, separately, IHF forces transposon end/target DNA interactions into a constrained pathway, "channeling," that yields only unknotted intratransposon inversion circles. Negative supercoiling influences both effects, differently. We infer that IHF is an architectural catalyst: it promotes initial transpososome assembly and is then ejected from the transpososome. IHF then rebinds, altering transpososome conformation to promote channeling. We also infer that the developing transpososome is a molecular spring: DNA provides basic elasticity; a conformational change in transposase provides force; and IHF and/or supercoiling provide conformational inputs. In vivo, IHF is a sensory transducer of chromosomal supercoiling status: with supercoiling absent, IHF is "supercoiling relief factor"; with supercoiling present, stimulation and channeling comprise a homeostatic pair such that modest changes in chromosome condition strongly influence transpositional outcome.

We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 ts mutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast to scMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (⌬mex67) is synthetically lethal with the rae1-167 mutation and accumulates poly(A) ؉ RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of the rae1-167 ⌬mex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149-505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149-505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149-505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

Tn10 transposition, like all transposition reactions examined thus far, involves assembly of a stable protein-DNA transpososome, containing a pair of transposon ends, within which all chemical events occur. We report here that stable Tn10 pre-cleavage transpososomes occur in two conformations: a folded form which contains the DNA-bending factor IHF and an unfolded form which lacks IHF. Functional analysis shows that both forms undergo double strand cleavage at the transposon ends but that only the unfolded form is competent for target capture (and thus for strand transfer to target DNA). Additional studies reveal that formation of any type of stable transpososome, folded or unfolded, requires not only IHF but also non-specific transposase-DNA contacts immediately internal to the IHF-binding site, implying the occurrence of a topo- logically closed loop at the transposon end. Overall, transpososome assembly must proceed via a folded intermediate which, however, must be unfolded in order for intermolecular transposition to occur. These and other results support key features of a recently proposed model for transpososome assembly and morphogenesis.

The cer-Xer dimer resolution system of plasmid ColE1 is highly selective, acting only at sites on the same molecule and in direct repeat. Recombination requires the XerCD recombinase and accessory proteins ArgR and PepA. The Escherichia coli chromosome dimer resolution site dif and the type II hybrid site use the same recombinase but are independent of ArgR and PepA and show no site selectivity. This has led to the proposal that ArgR and PepA are responsible for the imposition of constraint. We describe here the characterization of a novel class of "conditionally constrained' multimer resolution sites whose properties support this hypothesis. In the presence of ArgR and PepA, plasmids containing conditionally constrained sites are monomeric, but in their absence, extensive multimerisation is seen. A mutant ArgR derivative (ArgR110), which is defective in cer-mediated dimer resolution, remains able to prevent plasmid multimerisation by a conditionally constrained site. This implies that the accessory factors block recombination in trans rather than ensuring rapid multimer resolution. When the distance between the ArgR and XerCD binding sites in a conditionally constrained site was altered by a non-integral number of helical turns, the site became unconstrained. Constraint was restored by the insertion of a full helical turn.