Tn552 transposase purification and in vitro activities.
The Staphylococcus aureus transposon Tn552 encodes a protein (p480) containing the 'D,D(35)E' motif common to retroviral integrases and the transposases of a number of bacterial elements, including phage Mu, the integron-containing element Tn5090, Tn7 and IS3. p480 and a histidine-tagged derivative were overexpressed in Escherichia coli and purified by methods involving denaturation and renaturation. DNase I footprinting and gel binding assays demonstrated that p480 binds to two adjacent, directly repeated 23 bp motifs at each end of Tn552. Although donor strand cleavage by p480 was not detected, in vitro conditions were defined for strand transfer activity with transposon end fragments having pre-cleaved 3' termini. Strand transfer was Mn(2+)-dependent and appeared to join a single left or right end fragment to target DNA. The importance of the terminal dinucleotide CA-3' was demonstrated by mutation. The in vitro activities of p480 are consistent with its proposed function as the Tn552 transposase.
Rowland
S J
SJ
Genetics Laboratory, University of Glasgow, UK.
Sherratt
D J
DJ
Stark
W M
WM
Boocock
M R
MR
D001483:Base Sequence; D002352:Carrier Proteins; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015321:Gene Rearrangement; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity; D019895:Transposases
D002352:Carrier Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; D019895:Transposases