Analysis of the binding mechanisms and cellular targets of peptide inhibitors that block site-specific recombination in vitro

Bacteriophage lambda uses the recombination protein Integrase (Int) to incorporate and remove its genome from the chromosomal DNA of E. coli . Intermediates of the recombination reaction are difficult to study because of the high efficiency of the reaction and lack of high-energy cofactor requirements. Previously, peptide libraries were screened in order to isolate inhibitors of the recombination reaction. These peptides function by targeting specific recombination intermediates, but also inhibit the growth of bacterial cells. The exact mechanism by which the peptides function both in vitro and in vivo is not known with certainty.; In this work, I am interested in how peptide inhibitors interact with their target structure and if this can be applied to explain their antibiotic activity. The specific questions I set out to answer include: what is the site of interaction between the peptide inhibitors and recombination intermediates? What role does substrate conformation play during recombination and inhibition of recombination? What types of biochemical interactions are important for peptide binding? Are potential in vivo protein targets inhibited by the peptides? If inhibition of these potential targets is observed, what is the mechanism of inhibition?; I show that peptide inhibitors bind to the center of the protein-bound Holliday junction complex, in agreement with crystal structure data. The data indicate that contacts are mediated by interactions that mimic base stacking. In addition, peptides bind Holliday junctions in the square-planar conformation even in the absence of Int. This opens up the possibility that inhibitors of lambda recombination that trap Holliday junctions may also inhibit many other proteins that process DNA junctions. Indeed, the peptides prevent unwinding of branched DNA substrates by the RecG helicase of E. coli and interfere with the resolution of Holliday junction substrates by the RuvABC complex. The results indicate that inhibition of lambda Integrase, the RecG helicase, and the RuvABC complex all occur by a similar mechanism and imply that there are a number of potential targets for these peptides in vivo. Finally, the results emphasize the importance of Holliday junction conformation for peptide activity and as a determinant for the directionality of catalysis by lambda Int.