DNA sequence specificity of a naphthylquinoline triple helix-binding ligand.

We have examined the effect of a naphthylquinoline triplex-binding ligand on the formation of intermolecular triplexes on DNA fragments containing the target sites A6G6xC6T6 and G6A6xT6C6. The ligand enhances the binding of T6C2, but not T2C6, to A6G6xC6T6 suggesting that it has a greater effect on TxAT than C+xGC triplets. The complex with T6C2 is only stable below pH 6.0, confirming the requirement for protonation of the third strand cytosines. Antiparallel triplexes with GT-containing oligonucleotides are also stabilised by the ligand. The complex between G5T5 and A6G6xC6T6 is stabilised by lower ligand concentrations than that between T5G5 and G6A6xC6T6. The ligand does not promote the interaction with GT-containing oligonucleotides which have been designed to bind in a parallel orientation. Although the formation of antiparallel triplexes is pH independent, we find that the ligand has a greater stabilising effect at lower pH, suggesting that the active species is protonated. The ligand does not promote the binding of antiparallel GA-containing oligonucleotides at pH 7.5 but induces the interaction between A5G5 and G6A6xT6C6 at pH 5.5. Ethidium bromide does not promote the formation of any of these triplexes and destabilises the interaction of acridine-linked pyrimidine-containing third strands with these target sites.     

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking.

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), through an "indirect-readout" mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3' to the primary-kink site, G:C(7), through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C(7). Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A(6) to specificity for C:G(6), and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181-->Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A(6). The Glu181-->Asp substitution does not eliminate hydrogen-bond formation with G:C(7), and thus does not eliminate direct-readout-based specificity for G:C(7).     

DNA strand scission activity of metalloporphyrins

The iron porphyrin derivatives, iron (III) meso-tetra(4-N-methylpyridyl)-porphine (Fe(III)T4MPyP), aceto-iron (III) meso-tetra(3-N-methylpyridyl)porporphine (AcO-Fe(III)T3MPyP), and iron (III) meso-tetra(p-sulfonatophenyl)-porphine (Fe(III)TSPP), have been shown to induce strand scissions in DNA. Incubation of these porphyrins with PM2 DNA results in the conversion of circular supercoiled DNA to the nicked circular duplex form. The presence of dithiothreitol increases the extent of the nicking reaction. Fe(III)TSPP, which, unlike Fe(III)T4MPyP and AcO-Fe(III)T3MPyP, does not bind to DNA, is the least effective of the three porphyrins in inducing strand scissions in PM2. Both Fe(III)T4MPyP and AcO-Fe(III)T3MPyP induce strand scissions in cellular DNA of pre-labeled HeLa S₃ cells while Fe(III)TSPP has a very limited effect.     

Strand scission of superhelical and linear duplex DNAs by the antitumor protein macromomycin. Relationship of in vitro DNA damage to cell growth inhibition.

Macromomycin, a protein antitumor drug, was found to cause strand scissions in vitro in superhelical PM2 and SV40 DNA as well as linear duplex lambda DNA. DNA damage appeared to be single rather than double-strand scissions, and there is an indication that DNA breaks occur at some preferential base sites. The DNA breaks were predominantly true single-strand scissions as opposed to alkali-labile bonds. The cutting reaction was inhibited by low temperature (0 degrees C) and reached a maximum at 45 degrees C. The reaction was not affected by 2-mercaptoethanol, although EDTA did cause a slight decrease in the reaction rate. MgCl2 was found to be an effective inhibitor of the strand scission activity of the drug. The rate of DNA cutting was linear over a wide range of DNA substrate levels. There appeared to be a correlation between the drug's ability to damage DNA and to inhibit cell growth in that similar losses of these two activities occurred as the drug was thermally denatured.     

Histochemical detection of DNA strand scissions in mammalian cells by in situ nick translation

A method to visualize in situ of single strand scissions of DNA in fixed mammalian cells has been developed. Using the nuclear nick translation with biotin-labeled dUTP followed by binding to avidin-biotin-peroxydase complex, the nuclei of HeLa cells which had been treated with a DNA-damaging antibiotic bleomycin were specifically stained, implicating that the histochemical detection of single strand scissions (nicks) of DNA in fixed cells was completed without destroying the morphology, and without using autoradiography.     

DNA Intercalation and Photoinduced Cleavage by 4-Nitro(N-Hexylamine)1,8-Naphthalimide

Abstract

A novel intercalator, 4-nitro(N-hexylamine)1,8-naphthalimide, was synthesised and its DNA binding and photoinduced DNA cleavage properties were studied. The DNA unwinding results show that it binds through intercalation. Absorption and fluorescence spectroscopy reveal the preference for A/T base pairs as compared to G/C base pairs for the binding. The intercalator produces photoinduced single strand scissions in double helical DNA.     

Formation of endonuclease III-sensitive sites as a consequence of oxygen radical attack on DNA

Exposure of the plasmid pBR 322 to the aerobic xanthine oxidase reaction introduced single strand scissions and endonuclease III-sensitive sites. The latter may be residues of thymine glycol. Both forms of DNA damage were completely prevented by superoxide dismutase or catalase, whereas bovine serum albumin was much less effective. Mannitol and benzoate, added as scavengers of HO., and desferrioxamine or diethylene triamine pentaacetate, added to sequester Fe(III), also protected. These results indicate a metal-catalyzed interaction of O2- with H2O2, which produces HO. which, in turn, causes DNA strand scission and oxidation of thymine residues to thymine glycol. Plasmid isolated from aerobically-incubated cells contained more strand scissions and endonuclease III-sensitive sites than did plasmid from anaerobically-incubated cells, and a low molecular weight scavenger of O2- prevented the damage seen with the aerobic cells. Genetic defects in AP endonucleases rendered E. coli more susceptible to the dioxygen-dependent lethality of plumbagin, which mediates O2- production. Similarly, plasmid DNA, within the endonuclease-deficient cells, exhibited more strand scissions and endonuclease III-sensitive sites upon aerobic exposure to plumbagin than did endonuclease-sufficient cells, and a low molecular weight scavenger of O2- was protective. These results are consistent with the conclusions that strand scissions and formation of endonuclease III-sensitive sites are among the consequences of exposure of DNA to O2- plus H2O2, both in vitro and in vivo.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

Influence of DNA structure on DNA polymerase beta active site function: extension of mutagenic DNA intermediates

In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K(m), dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C approximately A/T > T/A approximately C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K(d)((dGTP)) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.     

DNA cleavage specificity of a group of cationic metalloporphyrins

The ability of a group of water-soluble metalloporphyrins to cleave DNA has been investigated. Incubation of Mn3+, Fe3+, or Co3+ complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2T4MPyP) with DNA in the presence of ascorbate, superoxide ion, or iodosobenzene results in DNA breakage. Comparisons between the rates of porphyrin autodestruction with the rates of strand scission of covalently closed circular PM2 DNA indicate that the porphyrins remain intact during the cleavage process. Analysis of the porphyrin-mediated strand scissions on a 139-base-pair restriction fragment of pBR322 DNA using gel electrophoresis/autoradiography/microdensitometry reveals that the minimum porphyrin cleavage site is (A X T)3. The cleavage pattern within a given site was found to be asymmetric, indicating that porphyrin binding and the strand scission process are highly directional in nature. In addition to an analysis of the mechanism of porphyrin-mediated strand breakage in terms of the DNA cleavage mechanism of methidium-propyl-iron-EDTA and Fe-bleomycin, the potential of the cationic metalloporphyrins as footprinting probes and as new "reporter ligands" for DNA is presented and discussed.     

Processive action of T4 endonuclease V on ultraviolet-irradiated DNA.

The action of the dimer-specific endonuclease V of bacteriophage T4 was studied on UV-irradiated, covalently-closed circular DNa. Form I ColE1 DNA preparations containing average dimer frequencies ranging from 2.5 to 35 pyrimidine dimers per molecule were treated with T4 endonuclease V and analysed by agarose gel electrophoresis. At all dimer frequencies examined, the production of form III DNA was linear with time and the double-strand scissions were made randomly on the ColE1 DNA genome. Since the observed fraction of form III DNA increased with increasing dimer frequency but the initial rate of loss of form I decreased with increasing dimer frequency, it was postulated that multiple single-strand scissions could be produced in a subset of the DNA population while some DNA molecules contained no scissions. When DNA containing an average of 25 dimers per circle was incubated with limiting enzyme concentrations, scissions appeared at most if not all dimmer sites in some molecules before additional strand scissions were produced in other DNA molecules. The results support a processive model for the interaction of T4 endonuclease V with UV-irradiated DNA.     

DNA microstructural requirements for neocarzinostatin chromophore-induced direct strand cleavage.

The microstructural requirements for optimal interaction of neocarzinostatin chromophore (NCS-C) with DNA have been investigated using a series of hexadeoxyribonucleotides with modified bases such as O6-methyl G (MeG), I, 5-methyl C (MeC), U, or 5-Bromo U (BrU) at specific sites in its preferred trinucleotide 5'GNaNb3':5'Na,Nb,C3' (Na = A, C, or T). Results show that MeG:C and G:MeC in place of G:C improve direct strand cleavage at the target Nb (Nb = T greater than A much greater than C greater than G), whereas MeC:G and C:MeG in place of Na:Nb, hinder cleavage. The optimal base target at Nb appears to be determined by its ability to form T:A type base pairing instead of C:G type. The observed differences in DNA strand cleavage patterns can be rationalized by induced changes in target site structure and are compatible with a model for NCS-C:DNA interaction in which the naphthoate moiety intercalates between 5'GNa3', and the activated tetrahydro-s-indacene, lying in the minor groove, abstracts a hydrogen atom from C-5' of Nb.     

DNA contacts stimulate catalysis by a poxvirus topoisomerase

Eukaryotic type 1B topoisomerases act by forming covalent enzyme-DNA intermediates that transiently nick DNA and thereby release DNA supercoils. Here we present a study of the topoisomerase encoded by the pathogenic poxvirus molluscum contagiosum. Our studies of DNA sites favored for catalysis reveal a larger recognition site than the 5'-(T/C)CCTT-3' sequence previously identified for poxvirus topoisomerases. Separate assays of initial DNA binding and covalent complex formation revealed that different DNA sequences were important for each reaction step. The location of the protein-DNA contacts was mapped by analyzing mutant sites and inosine-substituted DNAs. Some of the bases flanking the 5'-(T/C)CCTT-3' sequence were selectively important for covalent complex formation but not initial DNA binding. Interactions important for catalysis were probed with 5'-bridging phosphorothiolates at the site of strand cleavage, which permitted covalent complex formation but prevented subsequent religation. Kinetic studies revealed that the flanking sequences that promoted recovery of covalent complexes increased initial cleavage instead of inhibiting resealing of the nicked intermediate. These data 1) indicate that previously unidentified DNA contacts can accelerate a step between initial binding and covalent complex formation and 2) help specify models for conformational changes promoting catalysis.     

Effect of neocarzinostatin-induced strand scission on the template activity of DNA for DNA polymerase I.

Neocarzinostatin (NCS), an antitumor protein antibiotic that causes strand scissions of DNA both in vitro and in vivo, is shown to lower the template activity of DNA for DNA polymerase Iin vitro. There is a correlation between the extent of strand scission and the degree of inhibition, maximal inhibition of the polymerase reaction being obtained under conditions promoting maximal strand scission. These effects can be related to the concentrations of NCS and of 2-mercaptoethanol and are maximized by pretreatment of the DNA with drug. Results from polymerase assays in which the amount of drug-treated DNA template was varied at a constant level of the enzyme suggest that the sites associated with NCS-induced breaks are nonfunctional in DNA synthesis but bind DNA polymerase I. The binding of the enzyme to the inactive sites is further confirmed using [203 Hg] polymerase. It is shown that the lowering of the template activity of DNA by NCS under conditions of strand scission is due to the generation of a large number of inactive sites that block, competitively, the binding of DNA polymerase to the active sites on the template. Furthermore, the inhibition of DNA synthesis, which depends on the extent of strand breakage and on the relative amounts of template and enzyme, can be reversed by increasing the levels of template or polymerase. The finding that DNA synthesis directed by poly [d(A-T)] is much more sensitive to NCS than that primed by poly [d(G-C)] suggests that the drug preferentially interacts at regions containing adenine and/or thymine residues.     

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: DNA binding specificity based on energetics of DNA kinking.

The catabolite activator protein (CAP) makes no direct contact with the consensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specificity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at position 7 form a T:A/G:C step, which is known to be associated with DNA flexibility. It has been proposed that specificity for T:A at position 6 is a consequence of formation of the DNA kink between positions 6 and 7, and of effects of the T:A(6)/G:C(7) step on the geometry of DNA kinking, or the energetics of DNA kinking. In this work, we determine crystallographic structures of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes containing T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this system does not involve differences in the geometry of DNA kinking but, rather, solely differences in the energetics of DNA kinking. We further infer that the main determinant of DNA conformation in this system is protein-DNA interaction, and not DNA sequence.     

Investigation of site-specific DNA binding with nicking endonuclease Nt.BspD6I at single molecule level by atomic force microscopy

Nicking endonuclease Nt.BspD6I is a heterodimeric restriction endonuclease, one subunit of which exhibits specific nicking activity. It gets bound to double-stranded DNA and makes a break (nick) in one chain at a distance of 4 nucleotides from the binding site. In this work, for visualization of the specific binding and protein landing site an atomic force microscopy was used. In five minutes after incubation of DNA solution with nicking endonuclease, the DNA molecules with associated proteins which located at the expected binding site and "shared" DNA strand into two segments (approximately, 1/3 and 2/3 of length) were observed in the images. In addition, near the binding site DNA molecule had a height corresponding to a single-stranded DNA molecule, which was in good agreement with single-stranded cleavage by nickase in the course of complex formation.     

SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha.

Purified SV40 large T antigen and purified DNA polymerase alpha-primase form a complex detectable by ELISA and by a modified immunoblotting technique. The interaction is specific for the large catalytic subunit of polymerase alpha. The amino terminal 83 amino acids of T antigen are both necessary and sufficient for binding to the polymerase. However, antibody epitopes located in the carboxy terminal ATPase domain of T antigen are masked in the polymerase-T antigen complex, and complex formation is inhibited by an antibody directed against the carboxy terminus of T antigen, suggesting that this region of T antigen, though not required for binding, is in close proximity to the bound polymerase. The affinity of human DNA polymerase alpha for T antigen is approximately 10-fold greater than that of polymerase alpha from calf thymus, consistent with the interpretation that polymerase alpha is at least in part responsible for the primate-specific replication of SV40 DNA in vivo and in vitro. The results suggest that specific protein-protein interaction between DNA polymerase alpha and T antigen plays an important role in viral DNA replication.     

Peptide nucleic acid-anthraquinone conjugates: strand invasion and photoinduced cleavage of duplex DNA.

A bis-peptide nucleic acid (PNA)-anthraquinone imide (AQI) conjugate has been synthesized and shown to form strand invasion complexes with a duplex DNA target. The two arms of the bis-PNA each consist of five consecutive thymine residues and are linked by a flexible, hydrophilic spacer. Probing with potassium permanganate reveals that the bis-PNA complexes to duplex DNA at A5.T5sites with local displacement of the T5DNA strand. The 5 bp sequence targeted by the PNA is the shortest strand invasion complex reported to date. Irradiation of the strand invasion complex results in asymmetric cleavage of the displaced strand, with more efficient cleavage at the 3'-end of the loop. This result indicates that the bis-PNA binds to the DNA such that the C-terminal T5sequence forms the strand invasion complex, leaving the N-terminal T5sequence to bind by triplex formation, thereby placing the AQI closer to the 3'-end of the displaced strand, consistent with the observed photocleavage pattern. The ability of the PNA to directly report its binding site by photoinduced cleavage could have significant utility in mapping the secondary and tertiary structure of nucleic acids.     

DNA recognition and binding by the Euplotes telomere protein.

The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.