David S. Sigman

Sequence-specific modification of guanosine in DNA by a C60-linked deoxyoligonucleotide: Evidence for a non-singlet oxygen mechanism

Abstract A C60-linked deoxyoligonucleotide (C60-DON-1) was prepared from bromoacetate 3. This C60-oligonucleotide conjugate was hybridized to a complementary single-stranded DNA. This system reacted with light and oxygen to damage only guanosines in the single-stranded region which are closest to C60. The damage did not involve 1O2 as the active species but rather resulted from a single electron-transfer mechanism between guanosine and 3C60, as shown by comparison experiments with eosin-attached DON-1 and by the use of singlet oxygen quenchers.

Chemical nuclease activity of 5-phenyl-1,10-phenanthroline-copper ion detects intermediates in transcription initiation by E. Coli RNA polymerase

The nuclease activity of the copper complex of 5-phenyl-1,10-phenanthroline (5-phi-OP-Cu) detects conformational changes in the lac UV-5 promoter caused by E. Coli RNA polymerase. The template strand in melted regions of initiation complexes upstream of the site of nucleotide triphosphate incorporation is very reactive. In open and elongation complexes, downstream scission sites (e.g. +4 and +5 for the open complex) on both strands are observed. The patterns of both downstream cutting sites suggest an atypical double helix at the leading edge of the transcription bubble.

1,10-Phenanthroline-cuprous ion complex, a potent inhibitor of DNA and RNA polymerases

Abstract The inhibition by 1,10-phenanthroline of E. coli DNA polymerase I has recently been attributed to the formation in the assay mixtures of a unique and effective inhibitor, the 2:1 1,10-phenanthroline-cuprous ion complex (1). We have now found that this coordination complex is also an effective inhibitor of E. coli DNA dependent RNA polymerase, Micrococcus luteus DNA dependent DNA polymerase, and T-4 DNA dependent DNA polymerase. This conclusion is based either on the requirement of a thiol for 1,10-phenanthroline inhibition or on the reversal of 1,10-phenanthroline inhibition by the non-inhibitory cuprous ion specific chelating agent 2,9-dimethyl-1,10-phenanthroline. 2,2′,2″-Terpyridine is also very effective at relieving 1,10-phenanthroline inhibition. The reversal of 1,10-phenanthroline inhibition should be attempted before it is claimed that 1,10-phenanthroline inhibits any polymerases by coordinating a zinc ion at the active site.

Inhibition of E. coli DNA polymerase I by 1,10-phenanthroline

Abstract A 1,10-phenanthroline-cuprous ion complex is a potent reversible inhibitor of E. coli DNA polymerase I yielding 50% inhibition in the micromolar concentration range. The 2:1 1,10-phenanthroline-cuprous ion complex is most probably the inhibitory species. Complexes of cupric ion and 1,10-phenanthroline have no apparent kinetic effect. The previously reported inhibition of the enzyme by 1,10-phenanthroline (1,2) is most likely due to the formation of this complex from thiols normally added to the assay mixtures and trace amounts of cupric ion invariably present notwithstanding reasonable precaution. The reversible and instantaneous 1,10-phenanthroline inhibition observed for other polymerases may be due to this unique inhibitory species and not coordination of a catalytically important zinc ion at the active site by the chelating agent.

DNA‐binding proteins as site‐specific nucleases

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 used as rare cutters to analyse chromosomal DNA, to test predictions based on high‐resolution nuclear magnetic resonance and X‐ray crystal structures, and to locate binding sites of proteins within genomes.

Identification of a modifier site on acetylcholinesterase with affinity for d-tubocurarine.

Abstract Decamethonium and d-tubocurarine displace N-methylacridinium ion, a potent fluorescent inhibitor of acetylcholinesterase, from the surface of the enzyme. Decamethonium is competitive with N-methylacridinium which indicates that the binding sites for these ligands overlap. However, the displacement of N-methylacridinium ion by d-tubocurarine requires the existence of a binding site for d-tubocurarine in addition to the active site. Since the affinities for d-tubocurarine at both sites are comparable, two well defined ligand binding sites must exist for each catalytic site that is titratable by 7-dimethylcarbamyl-N-methylquinolinium iodide.

Scission of DNA at a preselected sequence using a single-strand-specific chemical nuclease.

BACKGROUND We were interested in developing a protocol for cleaving large DNAs specifically. Previous attempts to develop such methods have failed to work because of high levels of nonspecific background scission. RESULTS R-loop formation was chosen for sequence-specific targeting, a method of hybridization whereby an RNA displaces a DNA strand of identical sequence in 70% formamide using Watson-Crick base-pairing, leading to a three-stranded structure. R-loops are stabilized in aqueous solution by modifying the bases with chemical reagents. The R-loop was cleaved using a novel nuclease prepared from the Thr48-->Cys mutant of the single-strand-specific M-13 gene V protein (GVP), which was alkylated with 5-(iodoacetamido-beta-alanyl)1,10-phenanthroline. The cleavage products of the pGEM plasmid were cloned in to the pCR 2.1-TOPO vector. Adenovirus 2 DNA (35.8 kb; tenfold larger than the pGEM plasmid) was also cleaved quantitatively at a preselected sequence. CONCLUSIONS A new method for cleaving duplex DNA at any preselected sequence was developed. The cleavage method relies on the chemical conversion of M-13 GVP into a nuclease, reflecting GVPs specificity for single-stranded DNA. The GVP chimera is the first example of a semisynthetic secondary structure specific nuclease. The chemical nuclease activity of 1,10-phenanthroline-copper is uniquely suited to this technique because it oxidizes the deoxyribose moiety without generating diffusible intermediates, providing clonable DNA fragments. The protocol could be useful in generating large DNA fragments for mapping the contiguity of probes or defining the exon-intron structure of transcription units.

Mutagenicity of the nuclease activity of 1,10-phenanthroline-copper ion

The nuclease activity of 1,10-phenanthroline-copper functions intracellularly. This was shown by its mutagenicity in the Ames Test using the tester strain TA 102 and the in vivo nicking of plasmids derived from this strain. In vivo DNA strand scission requires all the components essential for the in vitro activity: 1,10-phenanthroline, cupric ion, thiol and hydrogen peroxide. Although 60Co gamma radiation potentiates the nuclease activity of 1,10-phenanthroline-copper ion in vitro via a superoxide dependent pathway, it does not promote significant mutagenesis in vivo at exposure levels below cytotoxicity.

Inhibition of Escherichia coli DNA polymerase I by rose bengal

Abstract The fluorescein dye, rose bengal, inhibits Escherichia coli DNA polymerase I reversibly in the dark and irreversibly in the light. The reversible inhibition, which occurs in the micromolar concentration range, is competitive with respect to the poly(dA-T) template/ primer and noncompetitive with respect to the complementary deoxynucleoside triphosphates. The Hill coefficient for the inhibition by rose bengal is 3.0. Equilibrium dialysis experiments using 131 I-labeled rose bengal have demonstrated direct binding of the inhibitor to the enzyme. No dye binds to poly(dA-T) at concentrations where the inhibition is observed. There are 22 ± 3 rose bengal binding sites per polymerase which can be subdivided into a class of high affinity sites and one of low affinity sites. The high affinity sites (3 μ m ) bind rose bengal with a Hill coefficient of 1.7 and are responsible for the observed inhibition. The low affinity sites (7μ m ) are more numerous (about 16) and bind rose bengal in a noncooperative manner. The displacement of rose bengal from the enzyme by poly(dA-T) at equilibrium confirms the competition between poly(dA-T) and rose bengal inferred from the kinetic data for the polymerization reaction. The inhibition of the 3′,5′ exonuclease activity and the template-directed dATP ⇌ P-P exchange reaction by rose bengal is fully consistent with the interaction of rose bengal at the polynucleotide binding site. The enzyme induces an extrinsic Cotton effect in the visible absorption of rose bengal. The abolition of this Cotton effect by poly(dA-T) further supports the proposed site of binding of the dye.

Limiting rates of ligand association to alcohol dehydrogenase

Abstract Detailed stopped-flow kinetic studies of the association of 2,2-bipyridine, 1,10-phenanthroline, and 5-chloro-1,10-phenanthroline to the zinc ion at the active site of alcohol dehydrogenase have demonstrated that a process with a limiting rate constant of about 200 s−1 restricts the binding of the bidentate chelating agents to the free enzyme. The formation of the enzyme-ligand complexes has been followed by means of the characteristic absorption spectra of the resulting complexes or by the displacement of the fluorescent dye, auramine O. Monodentate ligands, upon binding to the free enzyme or enzyme-NAD+ and enzyme-NADH complexes, do not exhibit a comparable limiting rate. In analogy with simple inorganic systems, these observations have been interpreted in terms of the rate limiting dissociation of an inner sphere water molecule following the rapid formation by the bidentate ligand of an outer sphere complex. The displacement of a water molecule from the zinc ion by 1,10-phenanthroline has been observed in crystallographic studies which have also established that the zinc ion in the enzyme-1,10-phenanthroline complex is pentacoordinate. Monodentate ligands, which are substrate analogs, do not exhibit limiting rates because displacement of water is not required for their addition to a coordinate position which is apparently vacant in the free enzyme. If a water molecule remains bound to the zinc ion in the kinetically competent ternary complex, it could play an essential role in the proton transfer reaction accompanying catalysis.