Site-specific recombination systems are important in controlling development and gene expression in a diverse array of organisms ranging from bacteria to humans. The long range goal of this project is to understand how bacteriophage X carries-out site-specific recombination. The findings will have a significant impact on other site-specific recombination systems because many of their integrases are related to the lambda Int protein. Biochemical and genetic approaches will be used to characterize the protein-protein and protein-DNA interactions that occur both during the assembly of recombination complexes (intasomes) and the process of strand-cleavage and exchange. The phage-encoded integrase (Int) protein is a central actor in intasome formation and strand exchange. Int mutants will be isolated and characterized in assays that will determine the defects of the individual proteins in the recombination pathway. The host-encoded integration host factor (IHF) also participates in intasome formation by inducing bends in the DNA. We will isolate mutants that have amino acid substitutions of residues that, as suggested by our previous genetic studies, interact with DNA. Such mutants will also provide information that will be useful in interpreting data derived from physical studies on the protein. The phage-encoded excisionase (Xis) and the host- encoded factor for inversion stimulation (FIS) promote excisive recombination. Xis interacts cooperatively with Int and FIS. We will characterize the mechanism(s) of cooperative protein-protein interactions by isolating and characterizing Xis mutants that are defective in interacting with Int or FIS.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Illinois Urbana-Champaign
Schools of Arts and Sciences
United States
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Kolakowski, Adam J; Gardner, Jeffrey F (2016) The N-terminus of IntDOT forms hydrophobic interactions during Holliday Junction resolution. Plasmid 87-88:10-16
Ringwald, Kenneth; Gardner, Jeffrey (2015) The Bacteroides thetaiotaomicron protein Bacteroides host factor A participates in integration of the integrative conjugative element CTnDOT into the chromosome. J Bacteriol 197:1339-49
Hopp, Crystal M; Gardner, Jeffrey F; Salyers, Abigail A (2015) The Xis2d protein of CTnDOT binds to the intergenic region between the mob and tra operons. Plasmid 81:63-71
Wood, Margaret M; Gardner, Jeffrey F (2015) The Integration and Excision of CTnDOT. Microbiol Spectr 3:MDNA3-0020-2014
Keeton, Carolyn M; Park, Jiyeon; Wang, Gui-Rong et al. (2013) The excision proteins of CTnDOT positively regulate the transfer operon. Plasmid 69:172-9
Laprise, Jennifer; Yoneji, Sumiko; Gardner, Jeffrey F (2013) IntDOT interactions with core sites during integrative recombination. J Bacteriol 195:1883-91
Keeton, Carolyn M; Hopp, Crystal M; Yoneji, Sumiko et al. (2013) Interactions of the excision proteins of CTnDOT in the attR intasome. Plasmid 70:190-200
Keeton, Carolyn M; Gardner, Jeffrey F (2012) Roles of Exc protein and DNA homology in the CTnDOT excision reaction. J Bacteriol 194:3368-76
Kim, Seyeun; Gardner, Jeffrey F (2011) Resolution of Holliday junction recombination intermediates by wild-type and mutant IntDOT proteins. J Bacteriol 193:1351-8
Kim, Seyeun; Swalla, Brian M; Gardner, Jeffrey F (2010) Structure-function analysis of IntDOT. J Bacteriol 192:575-86

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