Analysis of Nucleoprotein Complexes Formed by E. coli RecA Protein Using Affinity Cleavage

Author: Baliga, Ramesh

Year: 1996

Degree: Dissertation (Ph.D.)

Advisor: Dougherty, Dennis A.

Committee Members: Richards, John H.; Dougherty, Dennis A.; Dervan, Peter B.; Parker, Carl Stevens

Option: Chemistry; Biology

DOI: 10.7907/vgxz-vf87

Abstract

Homologous recombination, a process ubiquitous to most living cells, involves the exchange of strands between two molecules of double-stranded DNA at sites of sequence homology, resulting in the formation of hybrid products. One of the best studied model systems for in vitro studies of homologous recombination is the three-strand reaction catalyzed by the Escherichia coli RecA protein. In the presence of appropriate cofactors, RecA protein polymerizes on single-stranded DNA to form nucleoprotein filaments which can then bind to homologous sequences on duplex DNA. This thesis describes the use of affinity cleavage with oligonucleotides carrying an EDTA moiety appended at a single position, to study the binding of RecA• oligonucleotide filaments on duplex DNA. Chapter One provides a general overview of protein-DNA recognition, chemical approaches to molecular recognition of B-form DNA (including oligonucleotide-directed triple helix formation) and what is currently known about the role of RecA protein in homologous recombination and DNA repair. Chapter Two describes the technique of affinity cleavage and its optimization for RecA• oligonucleotide filaments. Chapter Three describes the use of affinity cleavage to observe cooperative binding of nucleoprotein filaments to adjacent sites on duplex DNA and an investigation into the basis for the observed cooperativity. In Chaper Four affinity cleavage with oligonucleotides carrying a single thymidine-EDTA residue at an internal site is used to determine the groove location of the incoming strand, in the threestranded complex formed by recognition of a homologous site on duplex DNA. Finally, in Chapter Five the results from various chemically modified substrates of the three-strand reaction are presented in support of a mechanism of homologous recognition that does not involve formation of a pre-exchange triple helical intermediate.

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