DNA binding and cleavage by enediyne antitumor antibiotics, esperamicin and dynemicin

Esperamicin A1 and dynemicin represent a new class of DNA binding/damaging agents characterized by unique ten-membered ene-diyne core. The former antitumor antibiotic shows high DNA cleavage activity in the presence of dithiothreitol, whereas the latter in the presence of NADPH. Esperamicin A1 favorably breaks T and C bases at oligopyrimidine regions such as 5'-CTC, 5'-TTC, and 5'-TTT sequences. Dynemicin attacks the bases at 3'-side of purine residues such as 5'-AG, 5'-AT, and 5'-GC sequences. These drugs also present good circumstantial evidence for minor groove interaction of B-DNA.     

Photo-induced DNA cleavage reaction characteristics of propargylic sulfones possessing anthraquinone chromophore

DNA cleavage potency of propargylic sulfones possessing anthraquinone chromophore 1 under UV-irradiation was evaluated in comparison with the dark reaction. 1 showed inefficient DNA cleavage activity, while having considerably strong DNA binding ability. This result is accounted for by spatial conditions that the activated alkylating allenic site of intercalated 1 could not effectively approach to DNA bases, most probably guanine moiety, and thereby led to insufficient DNA strand cleavage. In contrast, the DNA cleavage activity of 1 was notably enhanced upon UV-irradiation (lambda(ex)=365 nm) followed by incubation. Under UV-irradiation, further DNA cleavage were occurred primary at 5'-G of GG steps within DNA. A DNA cleavage mechanism for 1, by which photo-induced one-electron oxidation of 5'-G of GG steps may occur along with ordinary alkylation, has been proposed.     

Synthesis of novel aminoglycosides via allylic azide rearrangement for investigating the significance of 2′-amino group

Using allylic azide rearrangement, a convenient method has been developed for the synthesis of 2′,3′-dideoxyaminoglycosides that are, otherwise, difficult to be prepared. The antibacterial activity of these novel aminoglycosides also confirms the indispensable role of 2′-NH₂ group for both neomycin and kanamycin classes of aminoglycosides. A novel structural motif containing the hexylaminocarbonyl groups at O-5 and/or O-6 of 2′,3′-dideoxyneamine could lead to the production of new aminoglycosides against resistant bacteria.     

Synthesis ofγ,δ-unsaturated amino acids via ester enolate Claisen rearrangement of chelated allylic esters

Summary Deprotonation ofN-protected amino acid allylic esters with LDA at –78°C and subsequent addition of a metal salt presumably results in the formation of a chelated metal enolate which undergoes Claisen rearrangement upon warming up to room temperature, giving rise to unsaturated amino acid. Many different metal salts can be used for chelation, but in general the best results are obtained with zinc chloride. Due to the fixed enolate geometry, as a result of chelate formation, and a strong preference for thechair like transition state, the rearrangement proceeds with a high degree of diastereoselectivity. This methodology can be applied to acyclic as well as to cyclic substrates, and even to peptides, and allows for the synthesis of amino acids containing quaternary carbon centers.     

Reductively activated cleavage of DNA mediated by o, o'-diphenylenehalonium compounds

o,o'-Diphenylenehalonium (DPH) cations represent a novel class of DNA-affinic compounds characterized by binding constants within the range of 10(5)-10(6) M(-1). The maximum binding capacity of 2-2.5 base pairs per DPH cation and about 30% hypochromic reduction in the optical absorption of DPH cations upon binding to DNA suggest intercalation as a likely binding mode. In a DNA-bound form, DPH cations induce strand breaks upon reduction by radiation-produced electrons in aqueous solutions. In keeping with this mechanism, the cleavage is strongly inhibited by oxygen and is not affected by OH radical scavengers in the bulk. The yields of DPH-mediated base release significantly exceed the yield of base release caused by hydroxyl radical (in the absence of scavenger) in anoxic solutions. The yields are weakly dependent on DNA loading within the range from 5 to 50 base pairs per intercalator, which indicates the ability of excess electrons in DNA to react with a scavenger separated by tens of base pairs from the electron attachment site. The question regarding the mechanism by which the distant reactants reach each other in DNA remains unanswered, although it most likely involves electron hopping rather than a single-step long-distance tunneling. The latter conclusion is based on our finding that the electron affinity of DPH cations does not affect their properties as electron scavengers in DNA as would be expected if the direct long-distance tunneling is involved.     

Single strand DNA cleavage reaction of duplex DNA by Drosophila topoisomerase II

A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.     

Pyrimidine dimer dependent cleavage of single-stranded DNA by T4 UV endonuclease

T4 UV endonuclease cleaves double- and single-stranded DNA with equal specificity for photo-pyrimidine dimers. Thus, the enzyme can be used for mapping and quantifying pyrimidine dimers in single-stranded DNA as well as in double-stranded DNA. Mapping of pyrimidine dimers shows that rates of UV-dimerization are not only affected by 5', 3' adjacent bases, but also by position within pyrimidine tracts. Di-pyrimidines at 3' ends of tracts are more photoreactive than those at 5' ends.     

Synthesis and DNA damaging ability of enediyne model compounds possessing photo-triggering devices

Enediyne model compounds possessing photo-triggering devices were developed. These enediynes afforded biradicals by UV irradiation and showed DNA cleaving activity. The DNA damage was confirmed to be mainly caused by the biradical, not singlet oxygen.     

Synthesis of pacidamycin analogues via an Ugi-multicomponent reaction

The second-generation synthesis of 3'-hydroxypacidamycin D (2) has been accomplished via an Ugi-four component reaction at a late stage of the synthesis. This approach provided ready access to a range of analogues including diastereomers of the diaminobutylic acid residue and hybrid-type analogues of mureidomycins. Biological evaluations of these analogues indicated that the stereochemistry at the diaminobutylic acid residue has a crucial impact on both the MraY biochemical inhibition and whole-cell antibacterial activity.     

Cloning of an activated human ret gene with a novel 5' sequence fused by DNA rearrangement.

By transfecting a high-Mr DNA from human stomach cancer into NIH3T3 cells, a transforming sequence that showed homology with the human ret gene was identified. The transforming sequence was found to be generated by a DNA rearrangement in the human ret proto-oncogene. This rearrangement was suggested to have occurred during the transfection procedure. The nucleotide sequences of cDNAs of the rearranged ret gene and deduced amino acid (aa) sequences revealed that the rearrangement had resulted in recombination of the 3' segment of the ret proto-oncogene with a segment of an unknown human sequence, and that the recombination had generated a novel gene encoding a fusion protein of 435 aa. The rearrangement was presumed to be responsible for activation of the ret gene.     

Immobilization of DNA by UV irradiation and its utilization as functional materials.

The water-insoluble DNA film was successfully prepared by UV irradiation. The DNA film was stable in water. It could effectively accumulated the DNA-binding intercalating materials, such as ethidium bromide, dibenzo-p-dioxin and benzo[a]pyrene, in their aqueous solutions. On the other hand, DNA was immobilized onto nonwoven cellulose fabrics, also by the UV irradiation. The DNA immobilized cloth was found to bind silver ions. The DNA-cloth containing silver ion showed antibacterial activity. The water-insoluble DNA prepared by UV irradiation has a potential ability to serve as biomaterials for medical, engineering and environmental objects.     

DNA targeting and cleavage by an engineered metalloprotein dimer

Nature has illustrated through numerous examples that protein dimerization has structural and functional advantages. We previously reported the design and characterization of an engineered “metallohomeodomain” protein (C2) based on a chimera of the EF-hand Ca-binding motif and the helix–turn–helix motif of homeodomains (Lim and Franklin in Protein Sci. 15:2159–2165, 2004). This small metalloprotein binds the hard metal ions Ca(II) and Ln(III) and interacts with DNA with modest sequence preference and affinity, yet exhibits only residual DNA cleavage activity. Here we have achieved substantial improvement in function by constructing a covalent dimer of this C2 module (F2) to create a larger multidomain protein. As assayed via fluorescence spectroscopy, this F2 protein binds Ca(II) more avidly (25-fold) than C2 on a per-domain basis; in gel shift selection experiments, metallated F2 exhibits a specificity toward 5′-TAATTA-3′ sequences. Finally, Ca₂F2 cleaves plasmid DNA and generates a linear product in a Ca(II)-dependent way, unlike the CaC2 monomer. To the best of our knowledge this activation of Ca(II) in the context of an EF-hand binding motif is unique and represents a significant step forward in the design of artificial metallonucleases by utilizing biologically significant metal ions.     

Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide.

Several classes of antitumor drugs are known to stabilize topoisomerase complexes in which the enzyme is covalently bound to a terminus of a DNA strand break. The DNA cleavage sites generally are different for each class of drugs. We have determined the DNA sequence locations of a large number of drug-stimulated cleavage sites of topoisomerase II, and find that the results provide a clue to the possible structure of the complexes and the origin of the drug-specific differences. Cleavage enhancements by VM-26 and amsacrine (m-AMSA), which are representative of different classes of topoisomerase II inhibitors, have strong dependence on bases directly at the sites of cleavage. The preferred bases were C at the 3' terminus for VM-26 and A at the 5' terminus for m-AMSA. Also, a region of dyad symmetry of 12 to 16 base pairs was detected about the enzyme cleavage positions. These results are consistent with those obtained with doxorubicin, although in the case of doxorubicin, cleavage requires the presence of an A at the 3' terminus of at least one the pair of breaks that constitute a double-strand cleavage (Capranico et al., Nucleic Acids Res., 1990, 18: 6611). These findings suggest that topoisomerase II inhibitors may stack with one or the other base pair flanking the enzyme cleavage sites.     

Crosslinking of DNA to nuclear lamina proteins by UV irradiation in vivo

We have been able to demonstrate that a fraction of DNA becomes crosslinked to nuclear lamina shells isolated from Ehrlich ascites tumour cells irradiated with UV light. Terminal labeling of short DNA fragments covalently attached to proteins reveals that DNA has become crosslinked to all three lamins and to a protein comigrating with vimentin.     

Neocarzinostatin-induced DNA base release accompanied by staggered oxidative cleavage of the complementary strand

Treatment of an end-labeled DNA restriction fragment with the nonprotein chromophore of neocarzinostatin induced lesions which, after treatment with endonuclease IV or putrescine, were expressed as site-specific double-strand breaks. Analysis of the termini at cleavage sites in each strand showed that the neocarzinostatin-induced lesions consisted of an apurinic/apyrimidinic site plus a closely opposed break in the complementary strand. The break always occurred opposite the base two positions upstream from the apurinic/apyrimidinic site and had the 3'-phosphate and 5'-aldehyde termini characteristic of neocarzinostatin-induced breaks. This positioning suggests that neocarzinostatin simultaneously attacks two DNA sugars on opposite edges of the minor groove. The sequence specificity for formation of apurinic/apyrimidinic sites with closely opposed breaks reflected that of neocarzinostatin-induced mutagenesis. The potent mutagenicity of these lesions may be attributable to the presence of closely opposed damage in both DNA strands.     

Chemically and photochemically initiated DNA cleavage by an insulin-mimeticbisperoxovanadium complex

Chemically and photochemically induced cleavage of DNA by the insulin-mimetic compound NH₄[VO(O₂)₂-(1,10-phenanthroline)], bpV(phen), have been studied.⁵¹V NMR and absorption indicate that photoirradiation with low energy UV light of aqueous solutions containing bpV(phen) leads to the conversion of the compound to simple vanadates. Photoillumination of the compound in the presence of supercoiled pBR322 DNA results in cutting of the plasmid to produce nicked circular and linear DNA. Quantitative analysis of agarose gel data shows that bpV(phen) is a single strand nicking agent exhibiting sequence and/or base specificity.