Peptide wrwycr Inhibits the Excision of Several Prophages and Traps Holliday Junctions inside Bacteria

Abstract

Peptide inhibitors of phage lambda site-specific recombination were previously isolated by screening synthetic combinatorial peptide libraries. These inhibitors cause the accumulation of complexes between the recombinase and the Holliday junction intermediate of several highly divergent tyrosine recombinases. Peptide WRWYCR and its D-amino acid derivative bind to the center of protein-free junctions and prevent their resolution either by site-specific recombinases or by junction resolvases or helicases. With lesser affinity, the peptides also bind to branched DNA molecules that mimic replication forks. The peptides are bactericidal to both gram-positive and gram-negative bacteria, presumably because they can interfere with DNA repair and with chromosome dimer resolution by the XerC and XerD tyrosine recombinases. In order to test the correspondence between their mechanism in vivo and in vitro, we have tested and shown peptide wrwycr's ability to inhibit the excision of several prophages (lambda, P22, Gifsy-1, Gifsy-2, Fels-1, Fels-2) and to trap Holliday junction intermediates of phage lambda site-specific recombination in vivo. In addition, we found that the peptide inhibits replication of the Salmonella prophage Fels-1 while integrated in the chromosome. These findings further support the proposed mechanistic basis for the antimicrobial activity of the peptide and its use as a tool to dissect strand exchange-dependent DNA repair within cells.Bacteriophage lambda uses a phage-encoded integrase (Int) to catalyze the site-specific recombination reaction that integrates its chromosome into and excises it out of the Escherichia coli chromosome (e.g., see references 2 and 34). We have previously identified and characterized hexapeptides that inhibit site-specific recombination by the phage lambda Int in vitro by binding to the Holliday junction (HJ) intermediates of the reaction and preventing their resolution (4,7,13,22). The most potent of these peptides (WRWYCR, KWWCRW, and related peptides) were subsequently found to be bactericidal, very likely due to their causing the accumulation of DNA breaks and inhibiting chromosome segregation (18; C. Gunderson and A. Segall, unpublished data). In vivo, however, the D-amino acid forms of the peptides (wrwycr and kwwcrw) were more potent than their L-form counterparts, presumably because they resist peptidases (18).The question remains whether these peptides block sitespecific recombination and accumulate HJ inside bacterial cells. Int is the archetype of a large family of site-specific recombinases that use a tyrosine nucleophile for sequential transesterification reactions. The LT2 strain of Salmonella enterica serovar Typhimurium has four naturally occurring prophages (bacteriophages integrated in its chromosome): Gifsy-1, Gifsy-2, Fels-1, and Fels-2 (11, 15, 36). Each of these prophages encodes an Int-like tyrosine recombinase and can be induced to excise and replicate in a manner very similar to that of phage lambda. DNA damage is the predominant signal that leads to ...

Escherichia coli enterobactin synthesis and uptake mutants are hypersensitive to an antimicrobial peptide that limits the availability of iron in addition to blocking Holliday junction resolution

“…Another explanation may be the presence of greater concentrations of intracellular iron after wrwycr treatment. Our best current hypothesis is that the effects of wrwycr on DNA repair (Gunderson et al, 2009) and on iron availability are independent, albeit potentially synergistic.…”

“…We have identified synthetic hexapeptides that bind Holliday junctions as disulfidebridged dimers and inhibit Holliday junction resolution (Boldt et al, 2004;Kepple et al, 2005). One of these peptides, with the sequence wrwycr (we use the D-amino acid form to limit biological degradation), is a potent, broad-spectrum antimicrobial with the ability to inhibit, in vivo, mechanisms of DNA recombination and damage repair that proceed through a Holliday junction intermediate (Gunderson & Segall, 2006;Gunderson et al, 2009;L. Marcusson and other authors, unpublished data).…”

An Antimicrobial Peptide That Targets DNA Repair Intermediates In Vitro Inhibits Salmonella Growth within Murine Macrophages

“…Peptide wrwycr and its close relatives stabilize HJ intermediates of phage lambda site-specific recombination in E. coli and inhibit the tyrosine recombinase-dependent excision of prophages in both E. coli and Salmonella enterica LT2 (23). Treatment of E. coli with the peptide also resulted in the accumulation of filamentous cells and a 10-fold increase in anucleate cells, suggesting that the loss of bacterial viability was due to the combined effects of DNA breaks and chromosome segregation defects (22).…”

Hexapeptides That Inhibit Processing of Branched DNA Structures Induce a Dynamic Ensemble of Holliday Junction Conformations

“…6, A-D) reveals a narrow range of FRET values for conformational fluctuations, which neighbors IsoII in its mean FRET value, without evidence for discrete states. As a further test of the generality of peptideinduced conformational changes, we incubated the GC junction with the peptide WRWYCR, which also inhibits Holliday junction resolution (29,31,33). The effects of WRWYCR on GC junction behavior were analogous to those of KWWCRW (data not shown).…”

“…Indeed, through screens of a combinatorial library, short synthetic hexapeptides with these properties have been identified (23)(24)(25)(26). These peptides have been shown to impede the unwinding of branched DNA substrates by the RecG helicase of Escherichia coli, to interfere with Holliday junction resolution by the RuvABC complex, and to inhibit site-specific recombination by tyrosine recombinases, with accumulation of the Holliday junction intermediate (23,24,(27)(28)(29)(30)(31).…”