Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Zinder Laboratory


This thesis analyzes the interaction of two DNA-binding proteins with the plus strand replication origin of bacteriophage f1. The origin has a bipartite structure consisting of a required core origin region and an adjacent A +T- rich enhancer sequence that potentiates replication approximately 100-fold. The core origin binds the initiator protein, and the enhancer contains three binding sites for the E. coli integration host factor (IHF). Both activator proteins bend the DNA sequence to which they bind, implying that together they wrap the origin DNA into a higher order structure that is active in initiation. The replication initiator protein of bacteriophage f1 (gene II protein) is a multifunctional protein that participates in DNA replication at a number of levels. The gene II protein binds to the core origin in a novel two-step fashion. The first binding step involves interaction of two gene II protein molecules with an inverted repeat (β- γ) at the center of the core origin to form a binding intermediate, complex I. The second binding step involves addition of two protein molecules to complex I, resulting in formation of the functional complex, complex II. Of these two protein molecules, one binds to and contacts repeat δ, the other gene II protein molecule protects the nicking site in a sequence-independent fashion. The sequence protected in complex II corresponds to the core origin sequence as determined previously by in vivo analyses. The enhancer independent mutation mp1 in gene II protein (met40 ile) increases the cooperativity with which the protein binds to the core origin to form complex II. A model is presented for the binding reaction involving both protein-DNA and protein-protein interactions. A major finding of this thesis is that IHF activates f1 DNA replication by binding to three sites within the replication enhancer. The growth defect of f1 in IHF mutants was shown to be at the level of DNA replication. This growth defect of f1 in IHF mutants is suppressed by an initiator mutation (mp1) that also suppresses the lack of the replication enhancer, indicating that the enhancer is the genetic site of action of IHF.


A thesis submitted to the faculty of the Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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