The bacteroides NBU1 integrase performs a homology-independent strand exchange to form a holliday junction intermediate.
The Bacteroides mobilizable transposon NBU1 uses an integrase (IntN1) that is a tyrosine recombinase for its integration and excision from the host chromosome. Previously we showed that IntN1 makes 7-bp staggered cuts within the NBU1 att sites, and certain mismatches within the crossover region of the attN1 site (G(-2)C attN1) or the chromosomal target site (C(-3)G attBT1-1) enhanced the in vivo integration efficiency. Here we describe an in vitro integration system for NBU1. We used nicked substrates and a Holliday junction trapping peptide to show that NBU1 integration proceeds via formation of a Holliday junction intermediate that is formed by exchange of bottom strands. Some mismatches next to the first strand exchange site (in reactions with C(-3)G attBT1-1 or G(-2)C attN1 with their wild-type partner site) not only allowed formation of the Holliday junction intermediate but also increased the rate of recombinant formation. The second strand exchange appears to be homology-dependent. IntN1 is the only tyrosine recombinase known to catalyze a reaction that is more efficient in the presence of mismatches and where the first strand exchange is homology-independent. The possible mechanisms by which the mismatches stimulate recombination are discussed.
Rajeev
Lara
L
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA. rajeev@uiuc.edu
Segall
Anca
A
Gardner
Jeffrey
J
D001439:Bacteroides / Q000235:genetics*; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D045566:DNA, Cruciform / Q000235:genetics*; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D019426:Integrases / Q000737:chemistry / Q000235:genetics; D008957:Models, Genetic; D010957:Plasmids; D011995:Recombination, Genetic; D013997:Time Factors
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D045566:DNA, Cruciform; D019426:Integrases; D019286:Endopeptidase K; D011088:DNA Ligases