Tn7 transposition: Target DNA recognition is mediated by multiple Tn7-encoded proteins in a purified in vitro system

A cell‐free system of Tn3 transposition and transposition immunity

“…Bacteriophage Mu and transposon Tn7 also exhibit transposition immunity, which requires ATP hydrolysis (Adzuma & Mizuuchi 1988;Bainton et al 1993). Both elements encode transposase, which binds specifically to their own terminal ends, but has no ATPase activity.…”

“…Both elements encode transposase, which binds specifically to their own terminal ends, but has no ATPase activity. MuB protein (another protein encoded by Mu), and the TnsC protein (another protein encoded by Tn7), have nonspecific DNA-binding activity and have ATPase activity which is involved in target DNA selection and transposition immunity (Adzuma & Mizuuchi 1988;Bainton et al 1993). As for Mu, MuA protein, bound to a target in the end region of Mu DNA, enhances the ATPase activity of the MuB protein, which is also bound to the target, thus causing dissociation of the MuB protein from the target.…”

Recognition of triple-helical DNA structures by transposon Tn7

“…To generate transposition targets containing triplex DNA, several intermolecular triplex targets were constructed by annealing short (13-15 bp) TFOs to plasmids ranging in size from 2.5 to 5.2 kb. To increase stability of these pyrimidine-motif (i.e., Y⅐R⅐Y) triplexes under in vitro transposition reaction conditions (14), TFOs contained nucleoside analogs 5-methylcytosine (19,20) and 8-oxoadenine (21) to negate the low pH requirement of Hoogsteen hydrogen bond formation (22,23), and they also had a psoralen intercalator (24) to allow covalent attachment to the plasmid by photoactivated crosslinking. Once annealed and crosslinked, nearly 80% of the duplex plasmids contained a TFO attached by mono adduct.…”

“…Analysis of this low level of recombination by TnsABC reveals that Tn7 inserts into many different target sites (14). Transposition by TnsABC A225V , a TnsC gain-of-function allele that provides for recombination in the absence of either TnsD or TnsE, occurs at much higher frequency, approaching the level of TnsABC ϩ D recombination (18).…”

“…Reconstitution of a cell-free TnsABC ϩ D transposition system (14) has allowed a molecular definition of the Tn7 transposition pathway and of the specific roles of each of these Tn7-encoded proteins. Tn7 moves through a cut-and-paste mechanism ( Fig.…”