Double-strand break repair (DSBR) by homologous recombination repairs double strand breaks produced by ionizing radiation and this DNA repair process is conserved from bacteriophage to humans. In all organisms, homologous DSBR requires the DNA strand exchange activity of an ortholog of the E. coli RecA protein. The RecA family is the most highly sequence-conserved family of DNA repair proteins, which exhibits strong conservation of global structure and function in promoting homologous recombination and DSBR transactions. Nevertheless, RecAs for which biochemical data are available exhibit surprising diversity in DNA binding properties, catalysis, and allosteric mechanisms, while others appear to be specialized recombination mediators. Does this diversity/specialization reflect different evolutionary paths towards RecA function? Also, to what extent does RecA biochemical diversity/specialization reflect the environmental demands placed on the DNA recombination and repair systems of a host organism? The work proposed in this Project intends to address these questions by studying the functional and structural diversity of RecA enzymes.
In SPECIFIC AIM 1 we will compare biochemical and structural properties of divergent members of the RecA family. Target enzymes identified by computational methods will be cloned, expressed and purified. Using high throughput methods, DNA-binding and catalytic properties of each RecA ortholog will be determined. Variations in biochemical properties will be correlated with Phylogenetic and/or predicted structural variations within the RecA enzyme family. RecA orthologs representing distinct Phylogenetic and/or biochemical classes will be crystallized in the presence/absence of bound nucleotide, polynucleotide, and mediator protein ligands, and their high resolution X-ray structures will be determined.
In SPECIFIC AIM 2 we will study the allosteric mechanisms of divergent RecA enzymes. The roles of key amino acid residues in the induction of the high-affinity ssDNA-binding state will be determined for selected RecA orthologs. The ability of cognate recombination mediator proteins (RMPs) to induce high-affinity RecA-ssDNA binding will also be examined in targeted systems.
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