Holliday junctions (HJ) are the central intermediates in both homologous recombination and site-specific recombination performed by tyrosine recombinases such as the bacteriophage Integrase (Int) protein. Previously, our lab identified peptide inhibitors of Int-mediated recombination that prevent the resolution of HJ intermediates. We now show that two of these inhibitors bind HJ DNA in the square-planar conformation even in the absence of Int protein. The peptides prevent unwinding of branched DNA substrates by the RecG helicase of Escherichia coli and interfere with the resolution of HJ substrates by the RuvABC complex. Our results suggest that these peptides target all proteins that process HJ in the square-planar conformation. These inhibitors should be extremely useful for dissecting homologous recombination and recombination-dependent repair in vitro and in vivo.homologous recombination ͉ recombination-dependent repair ͉ RecG helicase ͉ RuvABC resolvasome ͉ tyrosine recombinase H olliday junctions (HJ), or four-way junctions, are central intermediates in homologous recombination, repair of collapsed replication forks, and reactions performed by the tyrosine recombinase family of site-specific enzymes (1-5). The first two processes are important in all organisms and are involved in the maintenance of chromosome integrity and repair of DNA damage. In the case of diploid organisms, faithful chromosome segregation depends on homologous recombination (6). Sitespecific recombination reactions performed by tyrosine recombinases are also very widespread and control gene expression, regulate plasmid and bacterial chromosome copy number, and mediate lysogeny (3). The presence and disappearance of HJ, their level within cells, and the enzymes that both generate and resolve them are of intense interest. More tools would be extremely useful for both in vitro and in vivo dissection of homologous recombination and repair processes in all organisms.Phage integrase (Int) binds to and mediates strand exchange between pairs of att sites. During recombination, one round of DNA cleavage, strand exchange, and ligation of the top strands of each partner DNA molecule generates a HJ intermediate, which is resolved by a second round of the same catalytic steps (3). These reactions are both rapid and highly reversible, making intermediates very difficult to study.We have identified peptides that inhibit recombination by trapping the protein-bound HJ intermediate and preventing its resolution either to substrates or to products (7-9). The most potent inhibitory peptide, WRWYCR, traps virtually all HJ formed during a reaction and has an IC 50 of 5-20 nM (9). A related peptide, KWWCRW, is very similar to WRWYCR in potency (8). These peptides also inhibit the mechanistically related Cre, XerC and D, and Flp proteins (ref. 9; A.M.S., unpublished results; A. Conway and P. A. Rice, personal communication). Because these proteins share little primary sequence identity, we reasoned that these peptides might interact with free HJ.HJ adopt one...

The bacteriophage integrase catalyzes four site-specific recombination pathways with distinct protein and DNA requirements and nucleoprotein intermediates. Some of these intermediates are very transient and difficult to obtain in significant amounts, due to the high efficiency and processivity of integrase, the lack of requirements for external energy factors or metal ions, and the highly reversible nature of each of the intermediates. We have previously used mixture-based combinatorial libraries to identify hexapeptides that trap 40 -60% of recombination substrates at the Holliday junction stage of the reaction. These inhibitors discriminate between the four pathways, blocking one of them (bent-L recombination) more severely than the others and blocking the excision pathway least. We presume that these differences reflect specific conformational differences of the nucleoprotein intermediates in each pathway. We have now identified new inhibitors of the excision pathway. One of these, WRWYCR, is over 50-fold more potent at inhibiting excision than the previously identified peptides. This peptide stably traps Holliday junction complexes in all recombination pathways mediated by integrase as well as Cre. This finding and other data presented indicate that the peptide's target is a common feature shared by the Holliday junction complexes assembled by tyrosine recombinases. We have taken advantage of reversible inhibition by the active peptides to develop a new assay for Holliday junction resolution. This assay is particularly useful for determining junction resolution rates in cases where complexes directly assembled on junction substrates undergo little or no catalysis.Tyrosine recombinases catalyze site-specific recombination between partner sequences via two rounds of single-stranded cleavage, exchange, and ligation (see Fig. 1) (1, 2). Because catalysis on the "top" strands of each substrate is separated temporally from catalysis on the "bottom" strands, a Holliday junction is made during the first round of catalysis and resolved during the second round. Normally, the Holliday junction is extremely transient, and no more than ϳ2% of substrates accumulate at this stage, making this intermediate recalcitrant to study. We have previously identified several hexapeptides that trap substrates at the Holliday junction stage (3). These peptides stabilize the protein-bound form of the Holliday junction (4, 5). In the case of one of these peptides, WKHYNY, and its derivatives, the initial DNA cleavage event is not affected (5), whereas in the case of the peptide KW-WCRW and its derivatives, initial DNA cleavage is blocked at high concentrations of the peptide (4).Phage integrase (Int), 1 like many of the phage-encoded tyrosine recombinases used to establish lysogeny, performs site-specific recombination between different pairs of DNA substrates: between attP and attB during integration of the phage chromosome into the host's chromosome and between attL and attR during excision (Table I) (2). In addition to these two path...

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 ...

Holliday junctions (HJs) are critical intermediates in many recombination-dependent DNA repair pathways. Our lab has previously identified several hexameric peptides that target HJ intermediates formed in DNA recombination reactions. One of the most potent peptides, WRWYCR, is active as a homodimer and has shown bactericidal activity partly because of its ability to interfere with DNA repair proteins that act upon HJs. To increase the possibility of developing a therapeutic targeting DNA repair, we searched for small molecule inhibitors that were functional surrogates of the peptides. Initial screens of heterocyclic small molecule libraries resulted in the identification of several N-methyl aminocyclic thiourea inhibitors. Like the peptides, these inhibitors trapped HJs formed during recombination reactions in vitro, but were less potent than the peptides in biochemical assays and had little antibacterial activity. In this study, we describe the screening of a second set of libraries containing somewhat larger and more symmetrical scaffolds in an effort to mimic the symmetry of a WRWYCR homodimer and its target. From this screen, we identified several pyrrolidine bis-cyclic guanidine inhibitors that also interfere with processing of HJs in vitro and are potent inhibitors of Gram-negative and especially Gram-positive bacterial growth. These molecules are proof-of-principle of a class of compounds with novel activities, which may in the future be developed into a new class of antibiotics that will expand the available choices for therapy against drug-resistant bacteria.