Site-specific DNA binding and bending by the Borrelia burgdorferi Hbb protein.

The Borrelia burgdorferi Hbb protein shows sequence similarity to members of the Escherichia coli HU/integration host factor (IHF) family of DNA accessory factors. We have overexpressed the hbb gene product in E. coli and purified the protein to near homogeneity. Biochemical analyses have revealed that Hbb has unique properties and is neither a strict HU nor IHF analogue. Hbb was found to bind specifically to a site in the putative origin of DNA replication between dnaA and dnaN. DNA footprinting studies have shown that this site is unrelated to the consensus sequence recognized by IHF proteins. Hbb induces a dramatic bend (> 126 degrees ) at this site and was also shown to restrain negative supercoils efficiently upon DNA binding. These features of the protein suggest that Hbb may act as a DNA accessory factor that facilitates the assembly of higher order protein-DNA complexes, such as those involved in DNA replication, transcription, recombination, packaging and perhaps other DNA metabolic processes unique to Borrelia.

Mutations in domain III alpha of the Mu transposase: evidence suggesting an active site component which interacts with the Mu-host junction.

A series of point mutations was constructed in domain IIIalpha of the Mu A protein. The mutant transposases were purified and assayed for their ability to promote various aspects of the in vitro Mu DNA strand transfer reaction. All mutants with discernable phenotypes were inhibited in stable synapsis (Type 0 or Type 1 complex formation). In contrast, these mutant proteins were capable of LER formation (a transient early reaction intermediate in which the Mu left and right ends have been synapsed with the enhancer), at levels comparable to wild-type transposase. These proteins therefore comprise a novel class of transposase mutants, which are specifically inhibited in stable transpososome assembly. The defect in these proteins was also uniformly suppressed by either Mn2+, or the Mu B protein in the presence of ATP and target DNA. Striking phenotypic similarities were recognized between the domain IIIalpha transposase mutant characteristics noted above, and those for substrate mutants carrying a terminal base-pair substitution at the point of cleavage on the donor molecule. This phenotypic congruence suggests that the alterations in either protein or DNA are exerting an effect on the same step of the reaction i.e., engagement of the terminal nucleotide by the active site. We suggest that domain IIIalpha of the transposase comprises the substrate binding pocket of the active site which interacts with the Mu-host junction.

A new set of Mu DNA transposition intermediates: alternate pathways of target capture preceding strand transfer.

Mu DNA transposition occurs within the context of higher order nucleoprotein structures or transpososomes. We describe a new set of transpososomes in which Mu B-bound target DNA interacts non-covalently with previously characterized intermediates prior to the actual strand transfer. This interaction can occur at several points along the reaction pathway: with the LER, the Type 0 or the Type 1 complexes. The formation of these target capture complexes, which rapidly undergo the strand transfer chemistry, is the rate-limiting step in the overall reaction. These complexes provide alternate pathways to strand transfer, thereby maximizing transposition potential. This versatility is in contrast to other characterized transposons, which normally capture target DNA only at a single point in their respective reaction pathways.