Ligand-binding specificity of the RecA nucleoprotein filament: Homologous DNA, metal ions, and nucleotides
Lee, Andrew Michael
Singleton, Scott F.
Doctor of Philosophy
The Escherichia coli RecA protein is a remarkable protein, playing key roles in initiating the SOS response to DNA damage, restarting stalled replication forks, and participating in recombinational DNA repair by binding single-stranded DNA and pairing it with homologous duplex DNA. We investigated the ligand binding specificity of the RecA nucleoprotein filament, specifically its interaction with homologous DNA strands, divalent metal ions, and nucleotide cofactors. We probed the kinetics of the mechanism of homology recognition by RecA using substituted oligonucleotide substrates to disrupt complete homology. Using this system, we were able to lend support to a base-pairing model proposed in the literature for the process of homology recognition and to provide unique structural and mechanistic insights not previously noted in investigations of the strand exchange mechanism of RecA. We also determined that RecA is inhibited by divalent metals in a novel fashion, as zinc(II), copper(II), and mercury(II) all inactivate RecA in vitro by initiating aggregation of the protein. A mechanistic hypothesis was developed for the action of metal-ligand complexes on RecA, and the potential use for metal-ligand complexes as inhibitors of RecA activities is discussed herein. We demonstrated the first proof of small-molecule inhibition of RecA activities. Using negative design, we developed several putative nucleotide analog inhibitors of RecA, and found that N6-(1-naphthyl)-ADP is a potent and highly specific inhibitor of ReCA function. In this report, we discuss the inhibitory properties of this compound on RecA and the implications for the development of novel antibiotic therapies. Finally, we investigated the role of the nucleotide cofactor binding site of RecA in the determination of substrate specificity and also activation of the RecA NPF. The D100R RecA protein was found to have reduced activity relative to wild-type in SOS induction, DNA damage repair, and ATPase assays, while neutrally charged residues at position 100 generated hyperactive RecA proteins. We postulated that RecA uses the Asp100 residue to discriminate against GTP activation and maintain selectivity for ATP binding, and that RecA uses Coulombic forces to communicate NTP binding cooperatively to both the protein conformational changes and DNA binding required for active NPF formation.