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Title:Protein/DNA interactions during site-specific recombination
Author(s):Wood, Margaret
Director of Research:Gardner, Jeffrey F.
Doctoral Committee Chair(s):Gardner, Jeffrey F.
Doctoral Committee Member(s):Imlay, James A.; Metcalf, William W.; Shisler, Joanna L.
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):conjugative transposon
site-specific recombination
mobile genetic elements
Abstract:Bacteroides species are one of the most prevalent groups of bacteria present in the human colon. Many strains carry large, integrated elements including conjugative transposons (CTns) and mobilizable transposons (MTns). One such conjugative transposon is CTnDOT, which is 65 kb in size and encodes resistances to tetracycline and erythromycin. In recent years, CTnDOT has been implicated in the spread of antibiotic resistance among gut microbiota. The integration and excision of CTnDOT is mediated by IntDOT, which has been identified as a member of the tyrosine recombinase family. Previous DNase I footprinting experiments demonstrated that IntDOT interacts with five arm-type sites, but exactly how the arm-type sites participate in the integration and excision of CTnDOT was not known. I constructed site-directed mutations in each of the arm-type sites and tested them in in vitro integration and excision assays. In vitro integration assays performed with arm-type site mutants demonstrated that attDOT sequences containing mutations in the L1 or the R1 and R2 or R1 and R2ʹ arm-type sites were defective in integration. Substrates containing mutations in the R1 and L1 arm-type sites were also defective in in vitro excision, however multiple arm-type site mutations did not have a drastic effect on excision. In addition, a sixth arm-type site (R1ʹ) was identified and determined to be required for integration and important for efficient excision. These results suggest that intramolecular interactions are important for CTnDOT integration while the action of accessory factors is more important for excision. My second project has focused on the excision of another Bacteroides mobile genetic element, mobilizable transposon NBU1. Unlike conjugative transposons, mobilizable transposons require genes encoded by a co-resident conjugative element for excision and transfer into a recipient cell. The integration of NBU1 requires IntN1, which has been identified as a tyrosine recombinase, as well as Bacteroides host factor BHFa. Excision of NBU1 is a more complicated process, involving five element-encoded proteins (IntN1, Orf2, Orf2x, Orf3 and PrmN1) as well as a Bacteroides host factor and a cis-acting DNA sequence. Little is known about what role the proteins play in excision, although IntN1 and Orf2x have been shown to be the only proteins absolutely required for detectable excision. Orf2x has a putative helix-turn-helix motif and interacts specifically with the excisive attachment site attL. I purified IntN1 and partially purified Orf2x, then performed DNase I footprinting experiments with fluorescently-labeled DNA containing the NBU1 attachment sites attL and attR. The results demonstrate that IntN1 interacts with two core-type sites flanking the region of cleavage and strand exchange on attL and attR as well as six arm-type sites. Two of the arm-type sites are located immediately downstream of the attL core, which is a unique feature of the NBU1 system. In vitro integration assays demonstrated that the DR1a, DR1b, DR3a and DR3b arm-type sites are required for in vitro integration. In addition, we have identified one Orf2x binding site (O1) on attL as well as a dA+dT rich upstream element that is required for Orf2x interactions with O1. Experiments are currently underway to elucidate whether IntN1 and Orf2x interact cooperatively when binding attL during excision.
Issue Date:2013-08-22
URI:http://hdl.handle.net/2142/45418
Rights Information:Copyright 2013 Margaret Wood
Date Available in IDEALS:2013-08-22
Date Deposited:2013-08


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