Tn10 transposase mutants with altered transpososome unfolding properties are defective in hairpin formation.
Transposition reactions take place in the context of higher-order protein-DNA complexes called transpososomes. In the Tn10 transpososome, IHF binding to an "outside end" creates a bend in the DNA that allows the transposase protein to contact the end at two different sites, the terminal and subterminal binding sites. Presumably this helps to stabilize the transposase-end interaction. However, the DNA loop that is formed must be unfolded at a later stage in order for the transposon to integrate into other DNA molecules. It has been proposed that transpososome unfolding also plays a role in transposon excision. To investigate this possibility further, we have isolated and characterized transposase mutants with altered transpososome unfolding properties. Two such mutants were identified, R182A and R184A. Both mutants fail to carry out hairpin formation, an intermediate step in transposon excision, specifically with outside end-containing substrates. These results support the idea that transpososome unfolding and excision are linked. Also, based on the importance of residues R182 and R184 in transpososome unfolding, we propose a new model for the Tn10 transpososome, wherein both DNA ends of the transpososome make subterminal contacts with transposase.
Humayun
Saima
S
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Wardle
Simon J
SJ
Shilton
Brian H
BH
Pribil
Patrick A
PA
Liburd
Janine
J
Haniford
David B
DB
D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D019895:Transposases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D046911:Macromolecular Substances; C473501:Tn10 transposase; D019895:Transposases