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Publication
Functional analysis of the catalytic triad of the hAT-family transposase
TcBuster.
Authors Woodard LE, Williams FM, Jarrett IC, Wilson MH
Submitted By Submitted Externally on 12/3/2021
Status Published
Journal Plasmid
Year 2021
Date Published 3/1/2021
Volume : Pages 114 : 102554
PubMed Reference 33476638
Abstract TcBuster is a hAT-family DNA transposon from the red flour beetle, Tribolium
castaneum. The TcBuster transposase is of interest for genome engineering as it
is highly active in insect and mammalian cells. To test the predicted catalytic
triad of TcBuster, each residue of the catalytic triad of a
haemagglutinin-tagged TcBuster transposase was individually mutated to a
structurally conserved amino acid. Using a drug-resistant colony assay for
transposon integration, we found that the D223N, D289N, and E589Q mutants of
TcBuster transposase were inactive in human cells. We used a modified chromatin
immunoprecipitation assay to determine that each mutant maintained binding to
TcBuster transposon inverted repeat elements. Although the catalytic mutants
retained their transposon binding properties, mutants displayed altered
expression and localization in human cells. None of the catalytic mutants formed
characteristic TcBuster transposase rodlet structures, and the D223N and D289N
mutants were not able to be detected by immunofluorescence microscopy.
Immunoblot analysis demonstrated that the E589Q mutant is less abundant than
wild-type TcBuster transposase. Cells transfected with either TcBuster or
TcBuster-E589Q transposase were imaged by structured illumination microscopy to
quantify differences in the length of the transposase rodlets. The average
length of the TcBuster transposase rodlets (N = 39) was 3.284 µm while the E589Q
rodlets (N = 33) averaged 1.157 µm (p < 0.0001; t-test). The catalytic triad
mutations decreased overall protein levels and disrupted transposase rodlet
formation while nuclear localization and DNA binding to the inverted repeat
elements were maintained. Our results may have broader implications for the
overproduction inhibition phenomenon observed for DNA transposons.




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