Functional analysis of the catalytic triad of the hAT-family transposase TcBuster

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 hae...

Full description

Saved in:
Bibliographic Details
Published in:Plasmid 2021-03, Vol.114, p.102554-102554, Article 102554
Main Authors: Woodard, Lauren E., Williams, Felisha M., Jarrett, Isria C., Wilson, Matthew H.
Format: Article
Language:English
Subjects:
(3−10): Transposon
Animals
Catalytic triad
DNA Transposable Elements
hAT
Humans
Localization
Mutation
Plasmids
Rodlet
Transposase
Transposases - genetics
Transposases - metabolism
Citations: Items that this one cites
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 
ISSN:0147-619X
1095-9890
1095-9890
DOI:10.1016/j.plasmid.2021.102554