Binding Parameters of Integration Host Factor to Four-Way Holliday Junction DNA

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Previous work in the Mukerji lab, characterized the binding properties of structurally similar but sequence non-specific DNA-binding protein HU, to duplex and Holliday junction DNA (4WJ). This research demonstrated that HU binds the central region of the 4WJ with nanomolar affinity and prefers the stacked form of the 4WJ with two HU dimers binding one junction. Given the similarities in structure and function of HU and IHF, we elected to study the binding affinity of IHF to the 4WJ. We have compared the IHF binding affinity for 4WJ DNA relative to that of duplex DNA with and without a consensus sequence. We determined that IHF has a 1:1 binding stoichiometry to the 4WJ. All binding assays established that IHF binds to the 4WJ lacking a consensus sequence with 2 nM KD. Moreover, anisotropy binding measurements demonstrated that IHF binds to the 4WJ with similar affinity as to H1 duplex DNA.

    Item Description
    Name(s)
    Thesis advisor: Mukerji, Ishita
    Date
    April 15, 2010
    Extent
    99 pages
    Language
    eng
    Genre
    Physical Form
    electronic
    Rights and Use
    In Copyright – Non-Commercial Use Permitted
    Digital Collection
    PID
    ir:1190