The molecular bases of tryptophan replacement effects in RecA: A biochemical and biophysical characterization of single- and null- tryptophan mutant Escherichia coli RecA proteins
Berger, Michael Dean, Jr
Singleton, Scott F.
Doctor of Philosophy
Recombinational DNA repair is an essential component of DNA metabolism and the proteins required for recombinational repair pathways are highly conserved. The Escherichia coli RecA protein is studied as a prototypic strand exchange protein with structural and biochemical similarity to the archaeal RadA and eukaryotic Rad51 family of recombination proteins. Although the RecA protein has been extensively investigated, structural information characterizing the molecular interactions required for DNA binding, pairing, and resolution remain conspiciously unresolved. A detailed molecular model for RecA-mediated strand exchange will provide greater insight into the universal mechanisms of strand exchange and identify similarities and differences between prokaryotic RecA and eukaryotic Rad51 recombination proteins. Sensitive probes of real-time changes in RecA conformation and RecA-DNA interactions are required to monitor strand exchange processes on a relevant time scale. Fluorescence spectroscopy of the RecA protein has been used to elucidate some of the obscure intermediates involved in the strand exchange process. The rapid timescale of changes in tryptophan fluorescence and the precise placement of fluorescence probes in the RecA protein provide a unique opportunity to obtain structural information from active complexes in real-time (nanoseconds-milliseconds) consistent with transient protein intermediates. Signal changes from fluorescent probes could be used in conjunction with mechanistic models to uncover structure-function relationships in RecA's diverse activities. As an initial step toward this goal, we have characterized the fluorescence properties of the two tryptophan residues native to Escherichia coli RecA, we have designed a tryptophanless Escherichia coli RecA mutant that functions like the wild type protein, and we have addressed the additivity of mutational effects on the photophysical properties of the RecA protein. Moreover, in this effort, we have uncovered what may be an important and highly conserved site of interaction between the RecA protein and as yet undescribed protein systems important to in vivo recombinational repair.
Molecular biology; Biochemistry; Biophysics