Characterization of the Integrase of the Bacteroides Mobilizable Transposon NBU1

The Bacteroides mobilizable transposon NBU1 uses an integrase (IntN1) for its integration and excision from the host chromosome. IntN1 has six conserved amino acids in the C-terminal end (RKHRHY) that are characteristic of tyrosine recombinases. I used site-directed mutagenesis to construct alanine mutants of these six residues. I tested the mutant proteins using an E. coli conjugation assay and showed that the mutants were defective for in vivo integration. This confirmed that IntN1 is a tyrosine recombinase. NBU1 integrates site-specifically at the 3' end of the leu-tRNA gene (atIBT1-1) within a 14 bp region which is identically present in the NBU1 attN1 site. I used suicide attBT1-1 substrates to determine that IntN1 makes 7 bp staggered cuts on the top (between bases -9 and -8) and on the bottom (between bases -1 and -2) strands within the 14 bp core region. Previous mutational analysis of the 14 bp showed that 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. This suggested that IntN1 does not require strict homology within the crossover region between the two substrates as seen for other tyrosine recombinases. To analyze IntN1 mechanism further, I developed an in vitro integration system for NBU1 using a supercoiled attN1 plasmid and a radiolabeled linear attBT1-1 fragment. I confirmed that the C(-3)G attBT1-1 had a higher rate of reaction than the wild type in the in vitro system. I used nicked attBT1-1 substrates and a Holliday junction trapping peptide to show that NBU1 integration proceeds via formation of a Holiday 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 attBTI-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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Rajeev, Lara

Contributors dc:contributor

• Gardner, Jeffrey F.

• Biology, Microbiology

Language dc:language

eng

Identifier

(MiAaPQ)AAI3301214

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/86703