Holliday junction-binding peptides inhibit distinct junction-processing enzymes

Abstract

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...

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

“…The peptides have been characterized extensively and inhibit recombination by binding to the HJ intermediate and by preventing further catalysis (4,20,21), probably by altering the three-dimensional structure of the junction, moving the path of the phosphodiester backbone away from Int's active site (16). Because it binds HJ and, with lesser affinity and stability, other branched DNA intermediates (21), the peptide wrwycr inhibits a number of HJ processing enzymes in a structure-selective manner (20,21). This peptide has also been shown to have antimicrobial activity (18) and inhibits Salmonella growing inside macrophages (L. Su, D. Hall, and A. Segall, unpublished data).…”

New Peptide Inhibitors of Type IB Topoisomerases: Similarities and Differences Vis-a-vis Inhibitors of Tyrosine Recombinases

“…DTT severely curtails all of their activities, strongly suggesting that monomer peptides assemble into at least dimers and in some cases into higher multimers via disulfide bonds. Binding of the WRWYCR to Holliday junctions and inhibition of Int recombination is severely reduced by DTT 20,35 . Both cysteines within peptide WYCRCK are very important to its activity.…”

Determination of Antibiotic Hypersensitivity among 4,000 Single-Gene-Knockout Mutants of Escherichia coli

“…While these lines of investigation show great promise, additional approaches are constantly being sought to yield a new generation of useful antimicrobial compounds. For instance, focusing on species-specific antibiotics rather than broad-spectrum antibiotics can result in important new agents (38), as could targeting bacterial transcription factors (5) or different processes, such as Holliday junction processing (21,31) and quorum sensing (24), or even targeting host factors that support pathogen growth (33). Another approach, examined here, involves potentiating existing antibiotics by identifying targets for increasing susceptibility to specific antimicrobials.…”

“…However, the spread of antibiotic-resistant microorganisms has reached an alarming point (1,11,35), prompting renewed efforts to find new antibiotics by detecting new targets through genomics, altering existing antibiotics, screening chemical (e.g., see reference 9) or peptide (21,31) libraries for specific inhibitors (e.g., see reference 9), or finding new sources of antibiotics via metagenomics (e.g., see reference 53). While these lines of investigation show great promise, additional approaches are constantly being sought to yield a new generation of useful antimicrobial compounds.…”