Characterization of the integrase of NBU1, a Bacteroides mobilizable transposon.
NBU1 is a Bacteroides mobilizable transposon (MTn) that is integrated within the host chromosome and requires CTnDOT functions for its excision and transfer into a new host. The NBU1 integrase IntN1 has been classified as a tyrosine recombinase based on the presence of conserved residues. We created alanine mutants of the residues R291, K314, H393, R396, H419 and the conserved substitution Y429F and tested them for integration efficiency. The results suggest that these residues in IntN1 are important for integration, and Y429 could be the catalytic nucleophile. We employed suicide substrates and partially purified IntN1 to determine the positions of IntN1 cleavage within the 14 bp common core region that is identical in both NBU1 att sites. We show that IntN1 makes 7 bp staggered cuts on the top and bottom strands. From previous mutational analysis of the att sites, we show that two specific mutations near the site of bottom strand cleavage within this 7 bp region increased integration, and mutations of the two bases near top strand cleavage site had no effect on integration. These results indicate that IntN1 lacks the strict requirement for homology between the recombining sites seen with other tyrosine recombinases. We also show that phosphorothioate substitutions at the cleavage site and 1 bp downstream inhibited cleavage by IntN1. This differs from other studied tyrosine recombinases where inhibition occurs by substitutions at the cleavage site only.
Rajeev
Lara
L
Department of Microbiology, University of Illinois, Urbana, IL 61801, USA. rajeev@uiuc.edu
Salyers
Abigail A
AA
Gardner
Jeffrey F
JF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001439:Bacteroides / Q000201:enzymology* / Q000235:genetics*; D004251:DNA Transposable Elements; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D019426:Integrases