An Approach to Random Mutagenesis of DNA Using Mixtures of Triphosphate Derivatives of Nucleoside Analogues

Zaccolo, Manuela; Williams, David M.; Brown, D. M.; Gherardi, Ermanno

doi:10.1006/jmbi.1996.0049

Cited by 287 publications

(222 citation statements)

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“…We found instead that nearly all of the changes were those expected for dPTP: 379 A to G and 135 T to C. Fig. 1 compares the frequencies we obtained with those reported in the original description of these two mutagenic nucleotides (20). It should be noted that those authors used high concentrations of dPTP and 8-oxo-GTP (500 M each versus the 10 and 50 M, respectively, which we used), and even when they combined the two, the effects of the 8-oxo-GTP were quite limited, so that A to G and T to C transversions greatly predominated, similar to our results.…”

Section: Selection Of Pad4 Mutants Thatsupporting

confidence: 53%

“…The PCR product was digested with EcoRI and HindIII and cloned into pBluescript II SK(Ϫ) vector (Agilent Technologies, La Jolla, CA). The resulting plasmid construct was used as DNA template for PCR mutagenesis using the nucleotide analogues 8-oxo-dGTP and dPTP (17,20). The PCR (a total volume of 25 l) contained 2.5 units of Takara Ex TaqDNA polymerase (Clontech); 1 M each of the forward and reverse primers used above; 2 mM MgCl 2 ; 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 400 M each of dATP, dCTP, dGTP, and dTTP; 50 M 8-oxo-dGTP; and 10 M dPTP.…”

Section: Methodsmentioning

confidence: 99%

“…Because dPTP causes A to G and T to C transitions and 8-oxo-GTP causes A to C and T to G transversions (20), a broad spectrum of amino acid substitutions should be generated, although codons containing G and C will be less affected. We found instead that nearly all of the changes were those expected for dPTP: 379 A to G and 135 T to C. Fig.…”

Section: Selection Of Pad4 Mutants Thatmentioning

confidence: 99%

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Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

Chen

Liu

Leysath

et al. 2016

Journal of Biological Chemistry

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The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates the translocation of the two enzymatic moieties of the toxin to the cytosol. Two PA receptors are known, with capillary morphogenesis protein 2 (CMG2) being the more important for pathogenesis and tumor endothelial marker 8 (TEM8) playing a minor role. The C-terminal PA domain 4 (PAD4) has extensive interactions with the receptors and is required for binding. Our previous study identified PAD4 variants having enhanced TEM8 binding specificity. To obtain PA variants that selectively bind to CMG2, here we performed phage display selections using magnetic beads having bound CMG2. We found that PA residue isoleucine 656 plays a critical role in PA binding to TEM8 but has a much lesser effect on PA binding to CMG2. We further characterized the role of residue 656 in distinguishing PA binding to CMG2 versus TEM8 by substituting it with the other 19 amino acids. Of the resulting variants, PA I656Q and PA I656V had significantly reduced activity on TEM8-expressing CHO cells but maintained their activity on CMG2-expressing CHO cells. The preference of these PA mutants for CMG2 over TEM8 was further demonstrated using mouse embryonic fibroblast cells and mice deficient in the CMG2 and/or the TEM8 receptors. The structural basis of the alterations in the receptor binding activities of these mutants is also discussed.Anthrax toxin is composed of three nontoxic proteins: protective antigen (PA), 3 lethal factor (LF), and edema factor (EF), which are released from Bacillus anthracis as monomers and assemble into toxic complexes on the surface of animal cells (1-3). PA is the moiety which binds to cell surface receptors and facilitates entry of the enzymatic EF and LF moieties into the host cell cytosol. Two anthrax toxin receptors have been identified; capillary morphogenesis protein 2 (CMG2, or anthrax toxin receptor 2) is the physiologically relevant toxin receptor mediating most of the in vivo toxicity of the toxin, whereas tumor endothelial marker 8 (TEM8, or anthrax toxin receptor 1) functions only as a minor anthrax toxin receptor (4 -8). Upon binding to the toxin receptors, PA is cleaved at the sequence 164 RKKR 167 by cell surface furin or furin-like proteases (9, 10), yielding the C-terminal 63-kDa fragment (PA63), which assembles to form a ring-shaped oligomer. The PA63 oligomer then binds LF and/or EF, and the toxin complex is internalized by receptor-mediated endocytosis. LF and EF are translocated into the cytosol from early or late endosomes to exert their cytotoxic effects. Therefore, PA plays the crucial role in anthrax toxin pathogenesis, serving as the delivery vehicle for translocation of LF and EF into the cytosol of the cell.The absolute requirement for cell surface proteolytic activation of PA for the toxin's action prompted our group and others to reengineer PA to make it depend on tumor-associated proteases for activation, thereby achieving high specificity for tumors (11)(12)(13)(14)(15). In a recent example, ...

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Section: Selection Of Pad4 Mutants Thatsupporting

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Section: Selection Of Pad4 Mutants Thatmentioning

confidence: 99%

See 1 more Smart Citation

Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

Chen

Liu

Leysath

et al. 2016

Journal of Biological Chemistry

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“…One set of library conditions utilized 10 ng of wtFRB template, each oligonucleotide primer at 0.5 M, 8 mM MgCl 2 , 0.2 mM dATP and dGTP, 1.0 mM dCTP and dTTP, 2.5 units Taq polymerase, and either 0.5 or 0.05 mM MnCl 2 . For the second set of conditions, the non-natural nucleotides 8-oxodGTP and dPTP were utilized to encourage misincorporation (18). For the second FRB library, a mixed template was generated consisting of equimolar ratios of wtFRB, FRB21, and FRB22 as well as a FRB21/FRB22 double mutant.…”

Section: Methodsmentioning

confidence: 99%

The Rapamycin-binding Domain of the Protein Kinase Mammalian Target of Rapamycin Is a Destabilizing Domain

Edwards

Wandless

2007

Journal of Biological Chemistry

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Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506-and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.A common approach for studying complex biological processes is to perturb a protein of interest and observe subsequent changes to the biological system. The classical approach of disrupting a gene and studying the resultant phenotype is problematic for examining essential genes. As a result, several conditional techniques have been developed including the tetracycline/doxycycline and Cre recombinase/lox P systems, where control is exerted at the transcriptional level (1, 2). At the post-transcriptional level, RNA interference (RNAi) 2 is becoming rapidly used as a method of gene silencing (3), but RNAi lacks tunability and does not affect existing protein levels. An ideal perturbation method would directly affect the protein with a cell-permeable probe that rapidly and reversibly targets a specific protein of interest.Small molecules have been very useful as conditional perturbants; however, the lack of specificity of many of these reagents can make it difficult to unambiguously interpret results. One approach to ensure specificity is to chemically modify a small molecule with a large substituent, and this technique has been widely used to interrogate specific protein tyrosine kinases that possess a complementary cavity-forming mutation (4, 5). An alternative system has recently been developed using a cellperm...

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“…Mutagenized intein genes were cloned into the wild-type RB69 DNA polymerase gene, and the resultant transformants were grown on master plates in the absence of phage. The pol-2 Tli intein gene was mutagenized by incorporation of dPTP and 8-oxo-dGTP, which stimulate transitions and transversions during PCR amplification (14). The primers used were TliPol-2F and TliPol-2R, and the PCR protocol was as described previously (1), except that the concentration of each nucleotide analog was 5 M. PCR products were purified, digested with StuI and SacII, and cloned into StuI/SacII-digested pTli Pol-2/I A .…”

mentioning

confidence: 99%

Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

Cann

Amaya

Southworth

et al. 2004

Appl Environ Microbiol

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A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing. The effectiveness of the system was tested by screening for thermosensitive splicing mutants. Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins.Over the last decade, intensive research has yielded important insights into the mechanism by which biological activity is restored to proteins invaded by inteins (4,5,12,13). Unlike introns, which are removed from RNA before translation, inteins are cotranslated with the invaded protein to form a precursor polypeptide (4, 5, 13). The intein is then self-catalytically excised from the precursor, with concomitant ligation of the upstream (N extein) and downstream (C extein) flanking polypeptides (4, 5), to yield two proteins: the mature host protein and the intein. Inteins are often found in active sites and conserved motifs of proteins indispensable to cell metabolism (6,8,9). Inteins are therefore new antimicrobial targets in pathogens with inteins in essential proteins since splicing must occur for normal cell function. To this end, rapid methods are desired to screen for compounds capable of blocking protein splicing. Three in vivo methods for detection of splicing and nonsplicing intein variants were recently published (1,3,11).This report describes a new genetic selection system for the identification of splicing and nonsplicing intein variants inserted into phage RB69 DNA polymerase. The method is based on growth versus lysis of Escherichia coli cells infected with conditionally defective T4 gp43 Ϫ phage, which contains amber mutations in the T4 DNA polymerase gene (gene 43) that render the phage inviable in nonsuppressor strains. As a result, colony formation is observed with T4-susceptible E. coli strains lacking amber suppressors, such as ER2566. Plasmidborne DNA polymerase from the closely related phage RB69 can complement this defect in T4 gp43 Ϫ phage, resulting in cell lysis (10). This system for controlling phage viability was converted into a genetic selection system for protein splicing by in-frame insertion of an intein gene into the active site of the plasmid-encoded RB69 DNA polymerase gene (Fig. 1), rendering the RB69 DNA polymerase inactive in the absence of protein splicing. T4 gp43 Ϫ phage viability would then require protein splicing to produce active RB69 DNA polymerase (Fig. 2).The pol-2 Tli intein gene portion of Thermococcus litoralis (Vent) DNA polymerase (7) was cloned into the homologous active-site, region

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An Approach to Random Mutagenesis of DNA Using Mixtures of Triphosphate Derivatives of Nucleoside Analogues

Cited by 287 publications

References 0 publications

Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

The Rapamycin-binding Domain of the Protein Kinase Mammalian Target of Rapamycin Is a Destabilizing Domain

Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

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