Characterization of Exc, a novel protein required for the excision of Bacteroides conjugative transposon.
Conjugative transposons are integrated elements that excise from the chromosome, then transfer by conjugation to a recipient in which they integrate once again. Recently, a gene, designated exc, was shown to be essential for excision of the Bacteroides conjugative transposon (CTnDOT) from the chromosome. The deduced amino acid sequence of Exc had low amino acid sequence similarity to DNA topoisomerase III, an enzyme that relaxes DNA supercoils. This similarity raised the question of whether Exc protein was a topoisomerase and, if so, whether topoisomerase activity might contribute to the excision process. Here, we demonstrate that Exc does have topoisomerase activity in vitro. Exc relaxed supercoiled DNA, had a conserved tyrosine as its active site and required magnesium ions for its relaxation activity. However, although mutation of the catalytic tyrosine of Exc to phenylalanine abolished the ability of the enzyme to relax DNA supercoils in vitro, the mutation did not abolish the ability of the protein to mediate excision in vivo. This surprising result suggests that CTnDOT excision does not rely on the topoisomerase activity of Exc in vivo.
Sutanto
Yuri
Y
Department of Microbiology, University of Illinois, Urbana, Illinois 61801, USA.
Shoemaker
Nadja B
NB
Gardner
Jeffrey F
JF
Salyers
Abigail A
AA
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001439:Bacteroides / Q000235:genetics* / Q000378:metabolism; D003227:Conjugation, Genetic; D026942:DNA Topoisomerases / Q000378:metabolism; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010957:Plasmids; D014443:Tyrosine / Q000378:metabolism
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004270:DNA, Circular; D014443:Tyrosine; D026942:DNA Topoisomerases