Structure-function analysis of IntDOT, the CTnDOT integrase
type: CreativeWork
description: 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 im…
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 im…
author: Kim, Seyeun
language: en
keywords: Tyrosine recombinase family, Bacteroides conjugative transposon (CTnDOT), CTnDOT integrase (IntDOT)
date published: 2011-01-14T22:46:15Z
date modified: 2026-07-22T22:25:11Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: http://hdl.handle.net/2142/18325