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Title:Structure-function analysis of IntDOT, the CTnDOT integrase
Author(s):Kim, Seyeun
Director of Research:Gardner, Jeffrey F.
Doctoral Committee Chair(s):Gardner, Jeffrey F.
Doctoral Committee Member(s):Slauch, James M.; Blanke, Steven R.; Vanderpool, Carin K.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Tyrosine recombinase family
Bacteroides conjugative transposon (CTnDOT)
CTnDOT integrase (IntDOT)
Abstract:IntDOT is an enzyme required for the recombination reactions that promote movement of CTnDOT that is an integrative conjugative element (ICE) found in Bacteroides spp. IntDOT has been classified in the tyrosine recombinase family. However, unlike most other tyrosine recombinases that require sequence identity in the region where strand exchanges occur, IntDOT recombines overlap sequences that contain mismatched base pairs. Therefore, IntDOT uses different mechanisms from those of other tyrosine recombinases. IntDOT has three DNA binding domains, the N terminus arm (N) domain, the core binding (CB) domain, the catalytic (CAT) domain. The CAT and CB domain bind to the core type sites and the N domain binds to the arm-type sites that are distantly located from the core-type sites. The CAT domain is the most conserved domain among members of the tyrosine recombinase family and contains six catalytic signature residues, R1KHIRIIHIIY, IntDOT, however, has serine residue in the place of the first catalytic arginine, R1. I performed a mutational analysis of IntDOT protein and isolated mutants with substitutions in all three domains of IntDOT proteins, I constructed an E.coli indicator strain that employs the lacI gene as a reporter. The in vivo integration assay using the E.coli indicator strain enables screening for IntDOT mutants from a large population containing random mutations. I isolated twenty five IntDOT mutants that are defective in integration. Four substitutions contained substitutions int eh N domain, two contained substitutions in the CB domain, and the nineteen contained substitutions in the CAT domain. Biochemical assays such as DNA binding, cleavage, and ligation were used to characterize IntDOT functions to determine which steps in the recombination pathway were defective. The results show that the IntDOT N domain is not required for cleavage and ligation but is important for binding to the arm-type sites. In the CAT domain, residue A352 is likely to be important for positioning the catalytic tyrosine, Y381, in the active site. Residue R285 could provide the missing function of the R1 residue in catalysis. Durign recombination, four IntDOT monomers perform two sets of sequential strand exchanges. The first set of strand exchanges forms the Holliday Junction (HJ) intermediate and the second set of strand exchanges resolves the HJ to complete the recombination reaction. During formation and resolution of the HJ, the four IntDOT monomers communicate with each other and coordinate the resolution fo the HJ intermediate. To study protein-protein interactions formed between IntDOT monomers, I developed an HJ in vitro resolution assay using synthetic HJs. I constructed two different types of HJs by designing oligos lacking the arm-type sites but containing the core-type sites. One HJ contains identical overlap sequences as si found in most tyrosine recombinase systems. The other HJ contains mismatched overlap sequences as found in natural attDOT and attB sites. I analyzed resolution of two tyes of HJs by wild type IntDOT and mutant IntDOT proteins. Wild type IntDOT resolves the HJ with identical overlap sequences into both substrates and recombinants but resolves the HJ with mismatched overlap sequences only into substrates. The results indicate that IntDOT and a host factor must form a nucleoprotein complex on attDOT to promote the processing of HJ intermediates through the region of heterology. Charaterization of HJ resolution by IntDOT mutant proteins showed that three residues, V95, R295, and S368 are important residues for protein-protein interactions in HJ resolution. V95 is a residue in the coupler region between the N terminal and CB domain. The R295 and S368 residues in the CAT domain are on the surface of protein that can likely make protein-protein interactions. I hypothesized that the IntDOT coupler and the N domain interact using charged residues as shown in the interaction between the lambda Int coupler and N domain. To test my hypothesis, I performed a site directed mutational analysis of charged residues in the IntDOT coupler region and N domain. The results indicate that IntDOT coupler is important for HJ resolution, but charged residues in the IntDOT N domain are not involved in protein-protein interactions. It is possible that hydrophobic residues including V95 residue in the IntDOT coupler makes protein-protien interactions.
Issue Date:2011-01-14
Rights Information:Copyright 2010 Seyeun Kim
Date Available in IDEALS:2011-01-14
Date Deposited:2010-12

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