Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit.
AuthorsMuth GW, Hennelly SP, Hill WE
JournalBiochemistry
PubMed ID10747796
'Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem. The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous. This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of ... More
Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent.
AuthorsEbright RH, Ebright YW, Pendergrast PS, Gunasekera A
JournalProc Natl Acad Sci U S A
PubMed ID2158096
'Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA ... More
Structure of the Escherichia coli Fis-DNA complex probed by protein conjugated with 1,10-phenanthroline copper(I) complex.
AuthorsPan CQ, Feng JA, Finkel SE, Landgraf R, Sigman D, Johnson RC
JournalProc Natl Acad Sci U S A
PubMed ID8127871
'The Escherichia coli Fis (factor for inversion stimulation) protein functions in many diverse biological systems including recombination, transcription, and DNA replication. Although Fis is a site-specific DNA-binding protein, it lacks a well-defined consensus recognition sequence. The electrophoretic mobility of Fis-DNA complexes, along with considerations of the Fis crystal structure, indicates ... More
Cleavage of 16S rRNA within the ribosome by mRNA modified in the A-site codon with phenanthroline-Cu(II).
AuthorsBucklin DJ, van Waes MA, Bullard JM, Hill WE
JournalBiochemistry
PubMed ID9201941
'Cleavage of 16S rRNA was obtained through mRNA modified at position +5 with the chemical cleavage agent 1,10-o-phenanthroline. In the presence of Cu2+, and after addition of reducing agent to the modified mRNA-70S complex, cleavage of proximal nucleotides within the 16S rRNA occurred. Primer extension analysis of 16S rRNA fragments ... More
Sequence-specific and hydrolytic scission of DNA and RNA by lanthanide complex-oligoDNA hybrids.
AuthorsKomiyama M
JournalJ Biochem (Tokyo)
PubMed ID8576074
'Totally synthetic and sequence-specific nucleases and ribonucleases, which hydrolyze DNAs and RNAs selectively at target sites, have been prepared. Lanthanide ions, which efficiently hydrolyze the phosphodiester linkages in nucleic acids, are attached to the 5''-end of synthetic DNA oligomers (sequence-recognizing sites) by use of an iminodiacetate ligand. Under physiological conditions, ... More
Transforming the Escherichia coli Trp repressor into a site-specific nuclease.
AuthorsSutton CL, Mazumder A, Chen CH, Sigman DS
JournalBiochemistry
PubMed ID8476849
'The Escherichia coli Trp repressor has been converted into an operator-specific nuclease by alkylating cysteine-49, inserted by site-directed mutagenesis, with 5-(iodoacetamido)-1,10-phenanthroline. In the presence of copper ion and thiol, high yields (> 50%) of double-stranded breaks of DNA are observed after a 20-min reaction. The high cleavage efficiency of this ... More
Sequence-specific recognition and cleavage of duplex DNA via triple-helix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate.
AuthorsFrançois JC, Saison-Behmoaras T, Barbier C, Chassignol M, Thuong NT, Hélène C
JournalProc Natl Acad Sci U S A
PubMed ID2557624
'Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine.homopyrimidine sequences by forming local triple helices. Phenanthroline was covalently attached to the 5'' end of an 11-mer homopyrimidine oligonucleotide of sequence d(TTTCCTCCTCT). Simian virus 40 DNA, which contains a single target site for this oligonucleotide, was used ... More
Oxygen-dependent cleavage of DNA by the 1,10-phenanthroline . cuprous complex. Inhibition of Escherichia coli DNA polymerase I.
AuthorsSigman DS, Graham DR, D'Aurora V, Stern AM
JournalJ Biol Chem
PubMed ID387784
DNA-binding proteins as site-specific nucleases.
AuthorsPan CQ, Landgraf R, Sigman DS
JournalMol Microbiol
PubMed ID8065254
DNA-binding proteins can be converted into site-specific nucleases by linking them to the chemical nuclease 1,10-phenanthroline-copper. This can be readily accomplished by converting a minor groove-proximal amino acid to a cysteine residue using site-directed mutagenesis and then chemically modifying the sulphydryl group with 5-iodoacetamido-1,10-phenanthroline-copper. These chimeric scission reagents can be ... More
Chemical conversion of a DNA-binding protein into a site-specific nuclease.
AuthorsChen CH, Sigman DS
JournalScience
PubMed ID2820056
The tryptophan gene (trp) repressor of Escherichia coli has been converted into a site-specific nuclease by covalently attaching it to the 1,10-phenanthroline-copper complex. In its cuprous form, the coordination complex with hydrogen peroxide as a coreactant cleaves DNA by oxidatively attacking the deoxyribose moiety. The chemistry for the attachment of ... More
Anisotropy-based sensing with reference fluorophores.
AuthorsLakowicz JR, Gryczynski I, Gryczynski Z, Dattelbaum JD
JournalAnal Biochem
PubMed ID10036147
We describe a new approach to fluorescence sensing based on measurements of steady-state anisotropies in the presence of reference fluorophores with known anisotropies. The basic concept is that the anisotropy of a mixture reflects a weighted average of the anisotropies of the emitting species. By use of reference fluorophores the ... More
Regions of 16S ribosomal RNA proximal to transfer RNA bound at the P-site of Escherichia coli ribosomes.
AuthorsBullard JM, van Waes MA, Bucklin DJ, Rice MJ, Hill WE
JournalBiochemistry
PubMed ID9477963
Unmodified uridines have been randomly replaced by 4-thiouridines in transfer RNAPhe (tRNAPhe) transcribed in a T7 RNA polymerase system. These 4-thiouridines serve as conjugation sites for attachment of the cleavage reagent 5-iodoacetamido-1,10-o-phenanthroline (IoP). In a reducing environment, when complexed with Cu2+, 1,10-o-phenanthroline causes cleavage of nearby nucleic acids. We show ... More
Photochemically and chemically activatable antisense oligonucleotides: comparison of their reactivities towards DNA and RNA targets.
AuthorsGodard G, François JC, Duroux I, Asseline U, Chassignol M, Nguyen T, Hélène C, Saison-Behmoaras T
JournalNucleic Acids Res
PubMed ID7527139
Dodecadeoxyribonucleotides derivatized with 1,10-phenanthroline or psoralen were targeted to the point mutation (G<-->U) in codon 12 of the Ha-ras mRNA. DNA and RNA fragments, 27 nucleotides in length, and containing the complementary sequence of the 12mers, were used to compare the reactivity of the activatable dodecamers (cleavage of the target ... More
Nuclease activity of 1,10-phenanthroline-copper. New conjugates with low molecular weight targeting ligands.
AuthorsChen CH, Mazumder A, Constant JF, Sigman DS
JournalBioconjug Chem
PubMed ID7679292
The chemical nuclease activity of 1,10-phenanthroline-copper depends on DNA sequence because the coordination complex has affinity for DNA. In order to target this efficient nucleolytic activity, it is essential to override its inherent specificity. The minimal size of ligands capable of redirecting the specificity has been investigated. A conjugate (HOP) ... More
Optimizing the targeted chemical nuclease activity of 1,10-phenanthroline-copper by ligand modification.
AuthorsGallagher J, Chen CH, Pan CQ, Perrin DM, Cho YM, Sigman DS
JournalBioconjug Chem
PubMed ID8853454
Our interest in improving the efficiency of targeted scission reagents has prompted us to study the influence of ring substituents on the nuclease activity of 1,10-phenanthroline-copper conjugated to oligonucleotides and DNA-binding proteins. Since methyl substitution at all but the 2 and 9 positions enhances the copper-dependent chemical nuclease activity of ... More