Synthesis and incorporation of pyrrole carboxamide nucleoside triphosphates by DNA polymerases

We have synthesised and examined the enzymatic incorporation properties of the 5'-triphosphates of 2'-deoxyribosyl pyrrole 3-monocarboxamide (dMTP) and 2'-deoxyribosyl pyrrole 3,4-dicarboxamide (dDTP). These analogues we had hoped would behave as ambivalent base analogues in that they can present two alternative hydrogen-bonding faces either by rotation about the carboxamide group or about the glycosidic bond. The two pyrrole derivatives, dMTP and dDTP, exhibit a preference for incorporation with Klenow polymerase. They are preferentially incorporated as either A or C.     

Cleavage of DNA by viologen related compounds and their incorporation into oligodeoxyribonucleotides.

The DNA cleavage reaction by viologen and related compound such as 2,7-diazapyrenium salt was investigated. These viologen analogues were successfully incorporated into the oligothymidylate in the form of covalent bonding at the site of the phosphorous backbone through the linker arm.     

DNA base flipping by a base pair-mimic nucleoside

On the basis of non-covalent bond interactions in nucleic acids, we synthesized the deoxyadenosine derivatives tethering a phenyl group (X) and a naphthyl group (Z) by an amide linker, which mimic a Watson-Crick base pair. Circular dichroism spectra indicated that the duplexes containing X and Z formed a similar conformation regardless of the opposite nucleotide species (A, G, C, T and an abasic site analogue F), which was not observed for the natural duplexes. The values among the natural duplexes containing the A/A, A/G, A/C, A/T and A/F pairs differed by 5.2 kcal mol(-1) while that among the duplexes containing X or Z in place of the adenine differed by only 1.9 or 2.8 kcal mol(-1), respectively. Fluorescence quenching experiments confirmed that 2-amino purine opposite X adopted an unstacked conformation. The structural and thermodynamic analyses suggest that the aromatic hydrocarbon group of X and Z intercalates into a double helix, resulting in the opposite nucleotide base flipping into an unstacked position regardless of the nucleotide species. This observation implies that modifications at the aromatic hydrocarbon group and the amide linker may expand the application of the base pair-mimic nucleosides for molecular biology and biotechnology.     

Synthesis and fluorescence characterization of pteridine adenosine nucleoside analogs for DNA incorporation.

Two fluorescent adenosine analogs, 4-amino-6-methyl-8-(2-deoxy-beta-d-ribofuranosyl)-7(8H)-pteridone (6MAP) and 4-amino-2,6-dimethyl-8-(2'-deoxy-beta-d-ribofuranosyl)-7(8H)-pteridone (DMAP), have been synthesized as phosphoramidites. These probes are site-selectively incorporated into oligonucleotides using automated DNA synthesis. Relative quantum yields are 0.39 for 6MAP and 0.48 for DMAP as monomers and range from >0.01 to 0.11 in oligonucleotides. Excitation maxima are 310 (6MAP) and 330 nm (DMAP) and the emission maximum for each is 430 nm. Fluorescence decay curves of each are monoexponential exhibiting lifetimes of 3.8 and 4.8 ns for 6MAP and DMAP, respectively. When these probes are incorporated into oligonucleotides they display quenching of fluorescence intensity, increases in the complexity of decay curves, and decreases in mean lifetimes. Because these changes are apparently mediated by interactions with neighboring bases, spectral changes that occur as probe-containing oligonucleotides meet and react with other molecules provide a means of monitoring these interactions in real time. These probes are minimally disruptive to DNA structure as evidenced by melting temperatures of probe-containing oligonucleotides that are very similar to those of controls. Digestion of probe-containing oligonucleotides with P1 nuclease confirms probe stability as fluorescence levels are restored to those expected for each monomer. These adenosine analog probes are capable of providing information on DNA structure as it responds to binding or catalysis through interaction with other molecules.     

Cleavage of DNA by mammalian DNA topoisomerase II

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.     

Effective anomerisation of 2'-deoxyadenosine derivatives during disaccharide nucleoside synthesis

The formation of a disaccharide nucleoside (11) by O3'-glycosylation of 5'-O-protected 2'-deoxyadenosine or its N6-benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding alpha-anomeric product (12). The latter reaction can be explained by instability of the N-glycosidic bond of purine 2'-deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2'-deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3'-O-beta-D-ribofuranosyl-2'deoxyadenosines and its alpha-anomer.     

Inhibition of pyrimidine incorporation without inhibition of DNA synthesis.

A putative lymphocytic chalone was tested measuring the incorporation of purine and pyrimidine nucleosides and by cytophotometry. The pyrimidine precursors were inhibited but not the purines. Thymidine and deoxycytidine incorporation even performed simultaneously with cytophotometry can be misleading in the analysis of the inhibition of cell division.     

Cleavage of a model DNA replication fork by a methyl-specific endonuclease

Epigenetic DNA methylation is involved in many biological processes. An epigenetic status can be altered by gain or loss of a DNA methyltransferase gene or its activity. Repair of DNA damage can also remove DNA methylation. In response to such alterations, DNA endonucleases that sense DNA methylation can act and may cause cell death. Here, we explored the possibility that McrBC, a methylation-dependent DNase of Escherichia coli, cleaves DNA at a replication fork. First, we found that in vivo restriction by McrBC of bacteriophage carrying a foreign DNA methyltransferase gene is increased in the absence of homologous recombination. This suggests that some cleavage events are repaired by recombination and must take place during or after replication. Next, we demonstrated that the enzyme can cleave a model DNA replication fork in vitro. Cleavage of a fork required methylation on both arms and removed one, the other or both of the arms. Most cleavage events removed the methylated sites from the fork. This result suggests that acquisition of even rarely occurring modification patterns will be recognized and rejected efficiently by modification-dependent restriction systems that recognize two sites. This process might serve to maintain an epigenetic status along the genome through programmed cell death.     

Photo-induced DNA interstrand cross-links formed by a coumarin-modified nucleoside

Coumarins are a class of naturally occurring compounds that have been shown to form photochemical DNA interstrand cross-links (ICLs). However, study of a coumarin base has not been explored. Using nucleophilic substitution and phosphoramidite chemistry, we synthesized a coumarin base-containing oligonucleotide. Upon exposure to long-wave ultraviolet light, the coumarin-modified oligonucleotide formed ICLs with complementary oligonucleotide containing dT and dC opposite the coumarin base, presumably through a [2?+?2] cycloaddition mechanism. Moderate yields with both bases were observed; though, dT has a higher reactivity than dC. Overall, this work provides new means for biochemical characterization of ICLs formed by coumarins.     

Cleavage of DNA by brown algal polyphenols

Extracts of marine algae have been tested to determine their ability to cleave DNA. The species producing positive results wereAscophyllum nodosum, Fucus serratus, F. spiralis, F. vesiculosus, Halidrys siliquosa andHimanthalia elongata. Partial purification of each extract by dialysis against water revealed that the active compounds in each species were high molecular weight polyphenols.     

Cleavage of phosphorothioate-substituted DNA by restriction endonucleases

M13 RF DNA was synthesized in vitro in the presence of various single deoxynucleoside 5'-O-(1-thiotriphosphate) phosphorothioate analogues, and the three other appropriate deoxynucleoside triphosphates using a M13 (+)-single-stranded template, Escherichia coli DNA polymerase I and T4 DNA ligase. The resulting DNAs contained various restriction endonuclease recognition sequences which had been modified at their cleavage points in the (-)-strand by phosphorothioate substitution. The behavior of the restriction enzymes AvaI, BamHI, EcoRI, HindIII, and SalI towards these substituted DNAs was investigated. EcoRI, BamHI, and HindIII were found to cleave appropriate phosphorothioate-substituted DNA at a reduced rate compared to normal M13 RF DNA, and by a two-step process in which all of the DNA is converted to an isolable intermediate nicked molecule containing a specific discontinuity at the respective recognition site presumably in the (+)-strand. By contrast, SalI cleaved substituted DNA effectively without the intermediacy of a nicked form. AvaI, however, is only capable of cleaving the unsubstituted (+)-strand in appropriately modified DNA.     

Synthesis and recognition by DNA polymerases of a reactive nucleoside for DNA diversification

The synthesis of 1-(2-deoxy-beta-D-erythro-pentofuranosyl)imidazole-4-hydrazide having the features of an ambigous base is reported. The recognition of the analogue by DNA polymerases as an incoming triphosphate as well as a template base was investigated. The mutagenic properties was evaluated by PCR. The potential of this new monomer for DNA diversification is illustrated by the reactivity of the nucleobase towards various aldehydes.     

Cleavage of abasic sites in DNA by intercalator-amines

Anthraquinone and naphthalene diimide intercalators with amine-containing side chains cleave plasmid DNA at abasic sites (apurinic or apyrimidinic (AP) sites). The intercalator-amine is substantially more effective than the amine itself; many intercalators with diamine side chains cleave most of the abasic sites at micromolar concentration (30 min at 37 degrees C). Intercalators with two amino moieties in the side chain are more efficient than those with one, arguing for a role for each of two amines in the cleavage mechanism. Side chains ending in tertiary amines are somewhat more effective than those ending in primary amines, indicating that imine formation is not required for cleavage at the abasic site. We also report a systematic study of abasic site cleavage by polyamines, including piperidine, spermine, spermidine and 12 other di-, tri- and tetra-amines. For polyamines as well as intercalator-amines, examples with three carbon atoms between neighboring nitrogens atoms cleave most efficiently. This may reflect a particularly favorable geometry for proton abstraction for these species. The effect of nitrogen-nitrogen spacing on the pKa values of the nitrogens may contribute as well. Overall, cleavage of plasmid DNA at adventitious abasic sites by intercalator-amines bearing two nitrogens in a single side chain occurs readily.     

Cleavage of cellular DNA by calicheamicin gamma1

It is assumed that the efficient antitumor activity of calicheamicin gamma1 is mediated by its ability to introduce DNA double-strand breaks in cellular DNA. To test this assumption we have compared calicheamicin gamma1-mediated cleavage of cellular DNA and purified plasmid DNA. Cleavage of purified plasmid DNA was not inhibited by excess tRNA or protein indicating that calicheamicin gamma1 specifically targets DNA. Cleavage of plasmid DNA was not affected by incubation temperature. In contrast, cleavage of cellular DNA was 45-fold less efficient at 0 degrees C as compared to 37 degrees due to poor cell permeability at low temperatures. The ratio of DNA double-strand breaks (DSB) to single-stranded breaks (SSB) in cellular DNA was 1:3, close to the 1:2 ratio observed when calicheamicin gamma1 cleaved purified plasmid DNA. DNA strand breaks introduced by calicheamicin gamma1 were evenly distributed in the cell population as measured by the comet assay. Calicheamicin gamma1-induced DSBs were repaired slowly but completely and resulted in high levels of H2AX phosphorylation and efficient cell cycle arrest. In addition, the DSB-repair deficient cell line Mo59J was hyper sensitive to calicheamicin gamma. The data indicate that DSBs is the crucial damage after calicheamicin gamma1 and that calicheamicin gamma1-induced DSBs are recognized normally. The high DSB:SSB ratio, specificity for DNA and the even damage distribution makes calicheamicin gamma1 a superior drug for studies of the DSB-response and emphasizes its usefulness in treatment of malignant disease.     

Cleavage of a model DNA replication fork by a Type I restriction endonuclease

Cleavage of a DNA replication fork leads to fork restoration by recombination repair. In prokaryote cells carrying restriction–modification systems, fork passage reduces genome methylation by the modification enzyme and exposes the chromosome to attack by the restriction enzyme. Various observations have suggested a relationship between the fork and Type I restriction enzymes, which cleave DNA at a distance from a recognition sequence. Here, we demonstrate that a Type I restriction enzyme preparation cleaves a model replication fork at its branch. The enzyme probably tracks along the DNA from an unmethylated recognition site on the daughter DNA and cuts the fork upon encountering the branch point. Our finding suggests that these restriction–modification systems contribute to genome maintenance through cell death and indicates that DNA replication fork cleavage represents a critical point in genome maintenance to choose between the restoration pathway and the destruction pathway.     

Cleavage of lambda DNA by a site-specific endonuclease from Serratia marcescens.

Three sites recognized by SmaI endonuclease, purified from Serratia marcescens SB, have been located on lambda DNA at 0.406, 0.656, and 0.825 fractional lengths from the left end of the DNA molecule.