Functional characterization of arginine 30, lysine 40, and arginine 62 in Tn5 transposase. J Biol Chem 2001 Jun 22;276(25):23135-43
Date
04/03/2001Pubmed ID
11283001DOI
10.1074/jbc.M010748200Scopus ID
2-s2.0-0035933813 (requires institutional sign-in at Scopus site) 16 CitationsAbstract
Three N-terminal basic residues of Tn5 transposase, which are associated with proteolytic cleavages by Escherichia coli proteinases, were mutated to glutamine residues with the goal of producing more stable transposase molecules. Mutation of either arginine 30 or arginine 62 to glutamine produced transposase molecules that were more stable toward E. coli proteinases than the parent hyperactive Tn5 transposase, however, they were inactive in vivo. In vitro analysis revealed these mutants were inactive, because both Arg(30) and Arg(62) are required for formation of the paired ends complexes when the transposon is attached to the donor backbone. These results suggest Arg(30) and Arg(62) play critical roles in DNA binding and/or synaptic complex formation. Mutation of lysine 40 to glutamine did not increase the overall stability of the transposase to E. coli proteinases. This mutant transposase was only about 1% as active as the parent hyperactive transposase in vivo; however, it retained nearly full activity in vitro. These results suggest that lysine 40 is important for a step in the transposition mechanism that is bypassed in the in vitro assay system, such as the removal of the transposase molecule from DNA following strand transfer.
Author List
Twining SS, Goryshin IY, Bhasin A, Reznikoff WSAuthor
Sally S. Twining PhD Assistant Dean, Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
ArginineBase Sequence
DNA
DNA Primers
Hydrolysis
Lysine
Models, Molecular
Transposases