Orf protein modulates phage and bacterial pathways of genetic recombination

Abstract

The emergence of novel pathogenic organisms due to the acquisition of virulencedeterminants from bacteriophages has generated significant interest in the pathways responsible for genomic rearrangements. Phage λ encodes its own recombination system, the Red system, comprising Exo, β and γ proteins. In addition, A encodes another recombinase, Orf, which participates in the initial stages of genetic exchange and supplies a function equivalent to that of the Escherichia coli RecFOR proteins. This thesis focuses on determining the function of Orf in phage and bacterial recombination pathways by analysing its impact on recombinases encoded by λ and E. coli. Experiments revealed that Orf interacts with bacterial and phage recombination proteins in the initial exchange step of recombination, modulating the activities of both Exo and RecA. Orf, along with β, attenuates the 5'-3' exonuclease activity of Exo, a feature that depends largely on the ability of Orf to bind DNA. Orf also facilitates loading of RecA onto ssDNA pre-coated with SSB but only if a ssDNA:dsDNA intersection is incorporated in the substrate. A motif similar to that found at the BRCA2-Rad51 interface may be responsible for Orf mimicking a RecA monomer to initiate nucleoprotein filament formation. Significantly, this would direct recombination down the bacterial RecA pathway of break restoration rather than the phage Red pathway with potentially important consequences for the outcome of the ~xchange reaction.