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Title:A study of Escherichia coli integration host factor interaction with DNA
Author(s):Hales, Laura Marie
Doctoral Committee Chair(s):Gardner, Jeffrey F.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Molecular
Biology, Microbiology
Abstract:The sequence elements required for IHF binding to the H$\sp\prime$ and H1 sites in attP of $\lambda$ were examined. The H$\sp\prime$ site naturally contains a dA+dT element 5$\sp\prime$ to the core consensus element WATCAANNNNTTR, while the H1 site does not. It was found that both elements are required for IHF to bind to the H$\sp\prime$ site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site. Placement of a dA+dT element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT element enhances IHF binding.
Mutants of IHF were examined for their ability to bind to various IHF binding sites in vitro and in vivo, and to promote recombination of $\lambda$ in vitro. The relative affinity of mutant IHF proteins was increased by the presence of the dA+dT region, confirming the enhancer-like properties of the dA+dT element. It was also found that these mutant proteins not only retain their DNA-bending ability but make any protein-protein contacts necessary to form a recombination-proficient intasome.
Gel mobility-shift assays using circularly permuted DNA fragments containing IHF sites showed that IHF forms a more compact protein-DNA complex with a site containing a dA+dT element when compared to a site lacking this sequence element. Additionally, artificial placement of a dA+dT element upstream of the H1 site in the genome of $\lambda$ weakened recombination in vitro. Therefore, the precise nucleoprotein structure required for $\lambda$ recombination is disrupted in this modified substrate. These results support the hypothesis that the DNA at an IHF site is bent in a different manner when the dA+dT element is present. It was demonstrated that IHF can form a specific protein-DNA crosslink with binding sites containing or lacking this dA+dT element. These results confirm the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF.
Issue Date:1995
Rights Information:Copyright 1995 Hales, Laura Marie
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9624356
OCLC Identifier:(UMI)AAI9624356

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