Anti-cancer binary system activated by bacteriophage HK022 integrase.
ABSTRACT: The binary system presented in this work is based on the bacteriophage HK022 integrase recombinase that activates the expression of a silenced Diphtheria toxin gene, both controlled by the cancer specific hTERT promoter. Using a lung cancer mice model, assays of different apoptotic and anti-apoptotic factors have demonstrated that the Integrase based binary system is highly specific towards cancer cells and more efficient compared to the conventional mono system whose toxin is directly expressed under hTERT. In a mice survival test, this binary system demonstrated longer persistence compared to the untreated and the mono treated ones. The reason underlying the advantage of this binary system over the mono system seems to be an overexpression of various hTERT suppressing factors induced by the mono system.
Project description:Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system.
Project description:Bacteriophage ? N protein binds boxB RNA hairpins in the nut (N utilization) sites of immediate early ? transcripts and interacts with host factors to suppress transcriptional termination at downstream terminators. In opposition to ? N, the Nun protein of HK022 binds the boxBs of coinfecting ? transcripts, interacts with a similar or identical set of host factors, and terminates transcription to suppress ? replication. Comparison of N-boxB and Nun-boxB nuclear magnetic resonance (NMR) structural models suggests similar interactions, though limited mutagenesis of Nun is available. Here, libraries of Nun's arginine-rich motif (ARM) were screened for the ability to exclude ? coinfection, and mutants were assayed for Nun termination with a boxB plasmid reporter system. Several Nun ARM residues appear to be immutable: Asp26, Arg28, Arg29, Arg32, Trp33, and Arg36. Asp26 and Trp33 appear to be unable to contact boxB and are not found at equivalent positions in ? N ARM. To understand if the requirement of Asp26, Trp33, and Arg36 indicated differences between HK022 Nun termination and ? N antitermination complexes, the same Nun libraries were fused to the activation domain of ? N and screened for clones able to complement N-deficient ?. Mutants were assayed for N antitermination. Surprisingly, Asp26 and Trp33 were still essential when Nun ARM was fused to N. Docking suggests that Nun ARM contacts a hydrophobic surface of the NusG carboxy-terminal domain containing residues necessary for Nun function. These findings indicate that Nun ARM relies on distinct contacts in its ternary complex and illustrate how protein-RNA recognition can evolve new regulatory functions.? N protein interacts with host factors to allow ? nut-containing transcripts to elongate past termination signals. A competing bacteriophage, HK022, expresses Nun protein, which causes termination of ? nut transcripts. ? N and HK022 Nun use similar arginine-rich motifs (ARMs) to bind the same boxB RNAs in nut transcripts. Screening libraries of Nun ARM mutants, both in HK022 Nun and in a ? N fusion, revealed amino acids essential to Nun that could bind one or more host factors. Docking suggests that NusG, which is present in both Nun termination and N antitermination, is a plausible partner. These findings could help understand how transcription elongation is regulated and illustrate how subtle differences allow ARMs to evolve new regulatory functions.
Project description:HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active 'attB' sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native 'attB' sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native 'attB' sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.
Project description:We report the sequence of a region of the PR operon of lambdoid phage HK022 and an analysis of the proteins it encodes. This region has DNA sequence elements and open reading frames that resemble those found in phages lambda, P22, and phi 80. The open reading frames encode homologs of the lambda CII transcription activator, the P22 DNA replication proteins, and a fourth protein of unknown function.
Project description:Bacteriophage O276 is a laboratory-generated hybrid that carries the immunity region of bacteriophage HK022 and all remaining genes from phage ?. Its construction was instrumental in the discovery of RNA-mediated antitermination, an intriguing alternative to the protein-mediated mechanism of transcription antitermination found in most lambdoid phages.
Project description:The Nun protein of coliphage HK022 excludes superinfecting lambda phage. Nun recognizes and binds to the N utilization (nut) sites on phage lambda nascent RNA and induces transcription termination. Overexpression of Nun from a high-copy plasmid is toxic for Escherichia coli, despite the fact that nut sites are not encoded in the E. coli genome. Cells expressing Nun cannot exit stationary phase. Toxicity is related to transcription termination, since host and nun mutations that block termination also suppress cell killing. Nun inhibits expression of wild-type lacZ, but not lacZ expressed from the Crp/cAMP-independent lacUV5 promoter. Microarray and proteomic analyses show that Nun down-regulates crp and tnaA. Crp overexpression and high indole concentrations partially reverse Nun-mediated toxicity and restore lacZ expression.
Project description:The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage ? N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the ? nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP ?' subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5' edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.
Project description:Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (?-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin.
Project description:Coliphage HK022 Nun blocks superinfection by coliphage ? by stalling RNA polymerase (RNAP) translocation specifically on ? DNA. To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of Escherichia coli RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP ? and ?' subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.
Project description:Temperate coliphage HK022 requires integration host factor (IHF) for lytic growth. The determinant responsible for this requirement was identified as a new gene (roi) located between genes P and Q. This gene encodes a DNA-binding protein (Roi) containing a helix-turn-helix motif. We have shown that Roi binds a site within its own gene that is closely linked to an IHF binding site. By gel retardation experiments, we have found that IHF binding stabilizes the interaction of Roi with its gene. We have isolated three independent phage mutants that are able to grow on an IHF- host. They carry different mutations scattered in the roi gene and specifying single amino-acid changes. The interactions of all three Roi mutant proteins with the Roi binding site differed from that of the wild type. Roi displays strong similarities, in its C-terminal half, to two putative DNA-binding proteins of bacteriophage P1: Ant1 and KilA. The mode of action of the Roi protein and the possibility that IHF is modulating the expression and/or the action of Roi are discussed.