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Trapping of phage lambda excisive recombination junction intermediates inside E. coli. The left side of the figure shows the structure of plasmid pLR110 and the orientation of the attL and attR sites. The right panel displays the results of a Southern analysis of plasmid DNA isolated from cells in which Int and Xis expression was induced or not, which were treated with the indicated amounts of peptide wrwyrggrywrw. The probe used was a 496-bp fragment encoding attL from pHN872 (32). The position of the markers for recombination substrate and product fragments and HJs is based on a parallel in vitro reaction. prod, products; rec, recombination; MW, molecular weight; +, present; −, absent.
Source publication
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 der...
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Supercoiled DNA is the relevant substrate for a large number of DNA transactions and has additionally been found to be a favorable form for delivering DNA and protein-DNA complexes to cells. We report here a facile method for stoichiometrically incorporating several different modifications at multiple, specific, and widely spaced sites in supercoil...
Citations
... 4B). The copy number increases described above are indicative of escape replication (also called onion-skin replication) of the defective prophages in a subpopulation of cells in the cultures(108,109). The mechanisms for regulation of their replication are not fully understood, and there is evidence that several including CP4-6 and DLP12 are controlled by the Rho factor (35, 37). ...
In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) which are not being simultaneously translated. It has been proposed that in Rho’s absence toxic RNA-DNA hybrids (R-loops) may be generated from nascent untranslated transcripts; and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E. coli of several such individual antisense regions on a plasmid or the chromosome generates R-loops that, in an RNase H-modulated manner, serve to disrupt genome integrity. Rho inhibition was associated with increased prevalence of antisense R-loops also in Xanthomonas oryzae pv. oryzae and Caulobacter crescentus . Our results confirm the essential role of Rho in several bacterial genera for prevention of toxic R-loops from pervasive yet cryptic endogenous antisense transcripts. Engineered antisense R-looped regions may be useful for studies on both site-specific impediments to bacterial chromosomal replication and the mechanisms of their resolution.
... We then ask whether pyocin induction in strains inactivated for XerC recombinase activity occurs independently of RecA, as in DxerC strains. Finally, we ask how a previously characterized antibacterial Holliday junction-binding, tyrosine recombinase-inhibiting hexapeptide (18)(19)(20) impacts P. aeruginosa cell growth and/or pyocin expression. Our findings reveal that, surprisingly, DNA damage-induced pyocin expression in DxerC cells is independent of RecA but remains dependent on PrtN. ...
... Drug treatment that inhibited XerC to stimulate pyocin expression would likely sensitize cells to fluoroquinolones like ciprofloxacin, imbuing recombinase inhibitors with potential therapeutic utility. Known inhibitors include hexapeptides that bind to Holliday junctions to achieve tyrosine recombinase inhibition (18); these inhibitors also inhibit growth of E. coli cells (19). However, these hexapeptide inhibitors have not been tested for their ability to stimulate pyocin production in P. aeruginosa. ...
... To our knowledge, the only known inhibitors of bacterial recombinases are hexapeptides, which have primarily been characterized in E. coli. These inhibitors, the best known of which is WRWYCR (or wrwycr, constructed from D-amino acids), trap Holliday junctions (including intermediates in XerCD-mediated chromosome dimer resolution), can prevent prophage excision, and thereby inhibit bacterial growth (18)(19)(20). We confirmed that WRWYCR, but not a previously described control hexapeptide, WKHYNY (19), effectively inhibited growth of our P. aeruginosa strains (Fig. 6). ...
The opportunistic pathogen Pseudomonas aeruginosa produces pyocins—intraspecific, interbacterial killing complexes. The canonical pathway for pyocin production involves DNA damage and RecA activation.
... We have previously reported that an antimicrobial peptide, wrwycr in its D isomer form, is able to significantly enhance acid-induced killing of all STEC seropathotypes associated with HUS and does so without increasing Stx release 23 . This peptide wrwycr has been shown to inhibit mechanisms of DNA damage repair and recombination that proceed through Holiday Junction (HJ) intermediates 24,25 . Further studies have expanded the list of potential intracellular bacterial targets of the peptide wrwycr, indicating that it causes cells to phenotypically starve for iron 26 . ...
Infection with Shiga toxin-producing Escherichia coli (STEC) results in hemorrhagic colitis and can lead to life-threatening sequelae including hemolytic uremic syndrome (HUS). Conventional treatment is intravenous fluid volume expansion. Antibiotic treatment is contraindicated, due in part to the elevated risk of HUS related to increased Shiga toxin (Stx) release associated with some antibiotics. Given the lack of effective strategies and the increasing number of STEC outbreaks, new treatment approaches are critically needed. In this study, we used an antimicrobial peptide wrwycr, previously shown to enhance STEC killing without increasing Stx production, in combination with antibiotic treatments. Checkerboard and time-kill assays were used to assess peptide wrwycr-antibiotic combinations for synergistic STEC killing. Cytotoxicity and real-time PCR were used to evaluate Stx production and stx expression, respectively, associated with these combinations. The synergistic combinations that showed rapid killing, no growth recovery and minimal Stx production were peptide wrwycr-kanamycin/gentamicin. Transmission electron microscopy revealed striking differences in bacterial cell morphology associated with various treatments. This study provides proof of principle for the design of an antibiotic-peptide wrwycr combination effective in killing STEC without enhancing release of Shiga toxins. It also offers a strategy for the repurposing of antibiotics for treatment of STEC infection.
... This, in turn, results in the accumulation of misfolded proteins and reduction of cellular viability [87]. Other peptides that target intracellular processes include the hexapeptide WRWYCR, which targets DNA repair mechanisms by binding to Holliday junctions [88]; buforin II, which diffuses through the plasma membrane to bind RNA and DNA [89]; and polyphemusin, which enters E. coli cells without disrupting the membrane [90]. ...
... For example, Lisomer peptides are susceptible to proteolysis. In the case of wrwycr, proteolytic degradation is avoided by converting the L-to the D-isomer form of the peptide [24,27,28]. Nevertheless, most peptides tend to be hydrophilic, and have high molecular weight and poor solubility, making it difficult for them to pass through the gastrointestinal tract wall and blood-brain barriers [29][30][31]. ...
... Nevertheless, the small molecules, specifically, 1609-10 (IC 50 The peptide wrwycr stabilizes Holliday junction intermediates inside bacterial cells [28]. It reduces the viability of different cancer cell lines in a dose-dependent manner and its effects were potentiated by chemotherapeutic agents such as doxorubicin, etoposide and hydroxyurea [34]. ...
Prostate cancer (PCa) is the most common cancer among men and is the second leading cause of cancer-related deaths in males in the United States and Europe. PCa is more common in African-American than Caucasian-American men, potentially because African- American men have a higher expression of androgen receptors. Early treatments include deprivation of androgen and removal of prostate tissues. However, these treatments may be rendered ineffective as the disease progresses to become androgen-independent (the castrate refractory stage) and as the tumor metastasizes. Finding additional and more effective treatments for castrate refractory PCa is highly desirable. Our lab has previously identified peptides that stabilize Holliday junctions (HJs) formed during DNA recombination reactions by inhibiting their resolution. HJs are intermediates in homologous recombination repair (HRR) pathway and cancer cells depend more on HRR than normal cells as they have uncontrolled replication and proliferation. Our most potent peptide, wrwycr, is active as a homodimer. In order to increase the potential of developing chemotherapeutic agents targeting DNA repair, we identified non-peptide small molecule surrogates from over 20 different chemical scaffolds; two types of scaffolds yielded small molecules (1609-3, 1609- 10, 1795-3 and 1795-10) that shared some properties with the peptides, including interactions with HJs. Results of cell viability and cytotoxicity assays have shown that all of the 1609 and 1795 compounds caused cell death in PC3 cells, and that one of the molecules reduces total DNA synthesis, but not total RNA or protein synthesis. Small molecule-induced cell death is lethal and independent of p53-induced apoptosis. In addition, small molecules induce double- stranded DNA breaks, as evident from the activation of DNA damage sensors, γH2AX and p53-binding protein 1 (53BP1). As expected, the downstream signaling checkpoint proteins, Chk1 and Chk2 are activated in consequence. However, no cell cycle arrest was observed. However, contrary to our hypothesis that the small molecules may interfere with double strand break repair, there was no synergy between compound 1795-10 and ionizing radiation.
... 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 recom-binases, with accumulation of the Holliday junction intermediate (23,24,(27)(28)(29)(30)(31). ...
... Furthermore, comparison of the TD plots for the peptide-free and peptide-bound states of the GC junction ( Fig. 6A-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 peptide-induced 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). ...
Holliday junctions are critical intermediates in DNA recombination, repair, and restart of blocked replication. Hexapeptides have been identified that bind to junctions and inhibit various junction-processing enzymes, and these peptides confer anti-microbial and anti-tumor properties. Earlier studies suggested that inhi-bition results from stabilization of peptide-bound Holliday junctions in the square-planar conformation. Here, we use single molecule fluorescence resonance energy transfer (smFRET) and two model junctions, which are AT- or GC-rich at the branch points, to show that binding of the peptide KWWCRW induces a dynamic ensemble of junction conformations that differs from both the square-planar and stacked-X conformations. The specific features of the conformational distributions differ for the two peptide-bound junctions, but both junctions display greatly decreased Mg2+ de-pendence and increased conformational fluctu-ations. The smFRET results, complemented by gel mobility shift and small-angle X-ray scat-tering (SAXS) analyses, reveal structural ef-fects of peptides and highlight the sensitivity of smFRET for analyzing complex mixtures of DNA structures. The peptide-induced confor-mational dynamics suggest multiple stacking arrangements of aromatic amino acids with the nucleobases at the junction core. This confor-mational heterogeneity may inhibit DNA pro-cessing by increasing the population of inactive junction conformations, thereby preventing the binding of processing enzymes and/or resulting in their premature dissociation.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... 97 One candidate peptide was able to block prophage excision by trapping such an intermediate, which also additionally resulted in antimicrobial activity, perhaps because of interference with DNA replication and repair. 98 DNA Transport. For a DNA molecule to transfer between bacteria, it must cross lipid membranes, which is energetically disfavored. ...
... Additionally, the examples of the SOS response, prophage excision, and relaxase function suggest targeting of these processes can potentially lead to the accumulation of toxic intermediates and cell death. 37,98,102 Further, as stress responses are linked to bacterial persistence, biofilm formation, and the expression of virulence factors, targeting evolutionary processes may decrease pathogenicity. 11, 105,106 In the case of HGT-mediated antibiotic resistance, while there is an assumption that all clinically relevant gene transfer events have already occurred by the time a patient shows signs of an infection, we do not know the timing of such events and, at a minimum, preventative strategies for patients at high risk of acquiring new resistant pathogens via HGT could be entertained. ...
Bacteria possess a remarkable ability to rapidly adapt and evolve in response to antibiotics. Acquired antibiotic resistance can arise by multiple mechanisms, but commonly involves altering the target site of the drug, enzymatically inactivating the drug, or preventing the drug from accessing its target. These mechanisms involve new genetic changes in the pathogen leading to heritable resistance. This recognition underscores the importance of understanding how such genetic changes can arise. Here, we review recent advances in our understanding of the processes that contribute to the evolution of antibiotic resistance, with a particular focus on hypermutation mediated by the SOS pathway and horizontal gene transfer. We explore the molecular mechanisms involved in acquired resistance and discuss their viability as potential targets. We propose that additional studies into these adaptive mechanisms can not only provide insights into evolution but also offer a strategy to potentiate our current antibiotic arsenal.
... Subsequently, they were shown to be bacteriocidal to both Gram positive and Gram negative bacteria, presumably because they can interfere with DNA repair and chromosome dimer resolution by XerC/D. They were also shown to inhibit the excision of several different prophages in vivo (60). The in vivo successes of the hexapeptide inhibitors motivated the Segall group to search for therapeutically more useful small molecules with similar activities. ...
The λ Integrase Site-specific Recombination Pathway, Page 1 of 2
Abstract
The λ site-specific recombination pathway has enjoyed the sequential attentions of geneticists, biochemists, and structural biologists for more than 50 years. It has proven to be a rewarding model system of sufficient simplicity to yield a gratifying level of understanding within a single (fortuitously timed) professional career, and of sufficient complexity to engage a small cadre of scientists motivated to peal this onion. The initiating highlight of the genetics phase was the insightful proposal by Allan Campbell for the pathway by which the λ chromosome integrates into, and excises from, the Escherichia coli host chromosome ( 1 ). The breakthrough for the biochemical phase was the purification of λ integrase (Int) and the integration host factor (IHF) by Howard Nash ( 2 , 3 ). The first major step in the structural phase was the cocrystal structure of IHF bound to its DNA target site by Phoebe Rice and Howard Nash ( 4 ). Although the crystal structure of naked Fis protein had been determined earlier ( 5 , 6 ), the full impact of Fis on understanding the fundamentals of the Int reaction did not come until much later ( 7 , 8 ).
... In another example, the hexapeptide WRWYCR, produced in vitro with D-amino acids to limit biological degradation, is a potent, broad-spectrum antimicrobial with the ability to inhibit, in vivo, mechanisms of DNA recombination. It also attacks the machinery of repairment of damaged DNA that proceeds through a Holliday junction intermediate [102,103]. This peptide can inhibit the growth of Salmonella enterica serovar Typhimurium inside macrophages [104]. ...
In the last decades, the indiscriminate use of conventional antibiotics has generated high rates of microbial resistance. This situation has increased the need for obtaining new antimicrobial compounds against infectious diseases. Among these, antimicrobial peptides (AMPs) constitute a promising alternative as therapeutic agents against various pathogenic microbes. These therapeutic agents can be isolated from different organisms, being widespread in nature and synthesized by microorganisms, plants and animals (both invertebrates and vertebrates). Additionally, AMPs are usually produced by a non-specific innate immune response. These peptides are involved in the inhibition of cell growth and of the killing of several microorganisms, such as bacteria, fungi, envelope viruses, protozoans and other parasites. They have many interesting properties as potential antibiotics, such as relatively small sizes (below 25-30 kDa), amphipathic structures, cationic nature, and offer low probability for the generation of microbial resistance. In recent years, many novel AMPs, with very promising therapeutic properties, have been discovered. These peptides have been the base for the production of chemical analogs, which have been designed, chemically synthesized and tested in vitro for their antimicrobial activity. This review is focused on antibacterial (against Gram (-) and Gram (+) bacteria) and antifungal peptides, discussing action mode of AMPs, and recent advances in the study of the molecular basis of their anti-microbial activity. Finally, we emphasize in their current pharmacological development, future directions and applications of AMPs as promising antibiotics of therapeutic use for microbial infections.
... Among the most potent such molecules was the damino acid hexapeptide wrwycr (Trp-Arg-Trp-Tyr-Cys-Arg), whose active form is a disulfide-bridged homodimer. In addition to binding HJs in vitro, peptide wrwycr also binds Holliday junctions in vivo [24] and is synergistically toxic with DNAdamaging agents including mitomycin C, UV, H 2 O 2 , and topoisomerase inhibitors in bacteria ( [25]; Naili, Rostron and Segall, ms. submitted). ...
Effective treatments for cancer are still needed, both for cancers that do not respond well to current therapeutics and for cancers that become resistant to available treatments. Herein we investigated the effect of a structure-selective d-amino acid peptide wrwycr that binds replication fork mimics and Holliday Junction (HJs) intermediates of homologous recombination (HR) in vitro, and inhibits their resolution by HJ-processing enzymes. We predicted that treating cells with HJ-binding compounds would lead to accumulation of DNA damage. As cells repair endogenous or exogenous DNA damage, collapsed replication forks and HJ intermediates will accumulate and serve as targets for the HJ-binding peptides. Inhibiting junction resolution will lead to further accumulation of DNA breaks, eventually resulting in amplification of the damage and causing cell death. Both peptide wrwycr and the related wrwyrggrywrw entered cancer cells and reduced cell survival in a dose- and time-dependent manner. Early markers for DNA damage, γH2AX foci and 53BP1 foci, increased with dose and/or time exposure to the peptides. DNA breaks persisted at least 48 h, and both checkpoint proteins Chk1 and Chk2 were activated. The passage of the cells from S to G2/M was blocked even after 72 h. Apoptosis, however, was not induced in either HeLa or PC3 cells. Based on colony-forming assays, about 35% peptide-induced cytotoxicity was irreversible. Finally, sublethal doses of peptide wrwycr (50-100 µM) in conjunction with sublethal doses of several DNA damaging agents (etoposide, doxorubicin, and HU) reduced cell survival at least additively and sometimes synergistically. Taken together, the results suggest that the peptides merit further investigation as proof-of-principle molecules for a new class of anti-cancer therapeutics, in particular in combination with other DNA damaging therapies.
