Reaction of acid-activated mitomycin C with calf thymus DNA and model guanines: elucidation of the base-catalyzed degradation of N7-alkylguanine nucleosides

Mitomycin C (MC, 1) forms covalent adducts under acidic activating conditions (pH approximately 4) with deoxyguanosine, d(GpC), and guanine residues of calf thymus DNA. In the case of deoxyguanosine, five adducts arise from a common precursor, N7-(2' beta, 7'-diaminomitosen-1'-yl)-2'-deoxyguanosine (10a; not isolated), which hydrolyzes spontaneously via two pathways: scission of the glycosidic bond to form N7-(2' beta, 7'-diaminomitosen-1' alpha-yl)guanine (5) and its 1' beta-isomer (6) and imidazolium ring opening to generate three 2,6-diamino-4-hydroxy-5-(N-formyl-2' beta, 7'-diaminomitosen-1' beta-yl)pyrimidine (FAPyr) derivatives that are substituted at N6 by isomeric 2'-deoxyribose units [i.e., 1' beta-furanose (7), 1' alpha-furanose (8), and 1' beta-pyranose (9)]. The structures of 5-9 were determined by spectroscopic methods. The same five adducts were obtained from d(GpC), but only the guanine adducts 5 and 6 were formed in DNA. Adducts 7-9 interconvert during high-performance liquid chromatography (HPLC). The unexpected isomerization of the deoxyribose moiety of the initially formed 1' beta-furanose adduct 7 to those of 8 and 9 occurs upon imidazolium ring opening, as discerned by the course of imidazolium cleavage of the simple models N7-ethyl- and N7-methylguanosine and N7-methyl-2'-deoxyguanosine. All ring-opened N7-alkylguanosine derivatives studied here exist as a mixture of distinct N-formyl rotamers, manifested by multiple interconverting peaks on HPLC and in the 1H NMR spectra. In the UV spectra of such derivatives, a new and diagnostic maximum at 218 nm (at pH 7) is observed. Acid-activated MC is found to alkylate preferentially the Gua-N7 position in deoxyguanosine or d(GpC), in contrast to reductively activated MC, which preferentially alkylates the Gua-N2 position. This finding is explained by the different electronic structures of acid- and reduction-activated MC. In DNA, the N7 specificity of acid-activated MC is partially offset by steric factors.     

Differences in sequence selectivity of DNA alkylation by isomeric intercalating aniline mustards

Two DNA-targeted mustard derivatives, N,N-bis(2-chloroethyl)-4-(5-[9-acridinylamino]-pentamido)aniline and 4-(9-[acridinylamino]butyl 4-(N,N-bis[2-chloroethyl]-aminobenzamide, which are isomeric compounds where the mustard is linked to the DNA-binding 9-aminoacridine moiety by either a -CONH- or a -NHCO- group, show significant differences in the sequence selectivity of their alkylation of DNA. The CONH isomer is a more efficient alxylating agent than the NHCO compound by an order of magnitude, consistent with the larger electron release of the CONH group to the aniline ring. However, the pattern of alkylation by the two compounds is also very different, with the CONH isomer preferring alkylation of guanines adjacent to 3'- or 5'-adenines and cytosines (for example those in sequences 5'-CGC, 5'-AGC, 5'-CGG and 5'-AGA) while the isomeric NHCO compound shows preference for guanines in runs of Gs. In addition, both isomers alkylate 3'-adenines in runs of adenines. Both compounds also show completely different patterns of alkylation to their untargeted mustard counterparts, since 4-MeCONH-aniline mustard alkylates all guanines and adenines in runs of adenines, while 4-Me2NCO-aniline mustard fails to alkylate DNA at all. These differences in alkylation patterns between the CONH- and its isomeric NHCO- compounds and their relationships between the alkylation patterns of the isomers and their biological activities are discussed.     

Ground squirrel hepatitis virus DNA: molecular cloning and comparison with hepatitis B virus DNA

Ground squirrel hepatitis virus (GSHV) shares many ultrastructural antigenic, molecular, and biological features with hepatitis B virus (HBV) of humans, indicating that they are members of the same virus group. Both viruses contain small circular DNA molecules which are partially single stranded. Here, we ligated an endonuclease EcoRI digest of GSHV DNA with EcoRI-cleaved plasmid vector pBR322 and cloned recombinant plasmids in Escherichia coli C600. Two cloned recombinants were characterized. One (pGS2) was found to contain only part of the GSHV genome, and the other (pGS11) was found to contain the entire viral DNA. A restriction endonuclease cleavage map of the GSHV insert in pGS11 and the locations of certain physical features of the virion DNA were determined. The relative positions of the single-stranded region, the unique 5' end of the short DNA strand, and the unique nick in the long DNA strand in GSHV DNA were found to be the same as those previously described for HBV DNA. Hybridization with an HBV [32P]DNA probe containing the apparent coding sequence for the major polypeptide of HBV surface antigen and a probe containing the putative coding sequence for the major polypeptide of the HBV core revealed specific homology with different restriction fragments of GSHV DNA. The two homologous regions had approximately the same locations relative to the single-stranded region, the 5' end of the short strand, and the nick in the long strand in the two viral DNAs. These results suggest that in both viruses the genes for the major HBV surface antigen and core polypeptides have the same locations relative to unique physical features of the viral DNAs.     

Mechanisms of DNA damage by chromium(V) carcinogens.

Reactions of bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) with pUC19 DNA, single-stranded calf thymus DNA (ss-ctDNA), a synthetic oligonucleotide, 5'-GATCTATGGACTTACTTCAAGGCCGGGTAATGCTA-3' (35mer), deoxyguanosine and guanine were carried out in Bis-Tris buffer at pH 7.0. The plasmid DNA was only nicked, whereas the single-stranded DNA suffered extensive damage due to oxidation of the ribose moiety. The primary oxidation product was characterized as 5-methylene-2-furanone. Although all four bases (A, C, G and T) were released during the oxidation process, the concentration of guanine exceeds the other three. Orthophosphate and 3'-phosphates were also detected in this reaction. Likewise, the synthetic oliogomer exhibits cleavage at all bases with a higher frequecncy at G sites. This increased cleavage at G sites was more apparent after treating the primary oxidation products with piperidine, which may indicate base oxidation as well. DNA oxidation is shown to proceed through a Cr(V)-DNA intermediate in which chromium(V) is coordinated through the phosphodiester moiety. Two alternative mechanisms for DNA oxidation by oxochromate(V) are proposed to account for formation of 5-methylene-2-furanone, based on hydrogen abstraction or hydride transfer from the C1' site of the ribose followed by hydration and two successive beta-eliminations. It appears that phosphate coordination is a prerequisite for DNA oxidation, since no reactions between chromium(V) and deoxyguanosine or guanine were observed. Two other additional pathways, hydrogen abstraction from C4' and guanine base oxidation, are also discussed.     

Adduction of catechol estrogens to nucleosides

We report the formation, detection, quantitation and structural characterization of products resulting from the adduction of deoxynucleosides (deoxyadenosine, deoxyguanosine, deoxycytidine and 5-methyldeoxycytidine) to the catechol estrogens (CE) of estrone, estradiol-17beta and estradiol-17 alpha. The crude products are obtained in a one-pot synthesis through oxidation of catechols to quinones and subsequent Michael-type reaction with the deoxynucleosides in acidic medium.In all experiments, adducts are detected by electrospray ionization mass spectrometry analysis after HPLC separation (LC/ESI/MS(n)). The two pyrimidines deoxycytidine and 5-methyldeoxycytidine yield only CE adducts to deoxynucleosides, which correspond to stable adducts on DNA. For purines, the results depend on the CE (2,3- or 3,4-catechols) used, the function and configuration on carbon 17 (ketone for estrone, alcohol for alpha and beta isomers of estradiol), and on the purine itself (deoxyadenosine or deoxyguanosine). Both stable adducts and deglycosylated adducts are formed, and therefore formation of stable adducts on DNA as well as the loss of purines from the DNA strands could be possible. MS(2) and MS(3) experiments prove to be relevant for further structural determinations, enabling in some cases the elucidation of the regiochemistry of adduction on the A and B rings of the steroid moiety.     

Specificity of proofreading by the 3'----5' exonuclease of the DNA polymerase-primase of Drosophila melanogaster

The DNA polymerase-primase from Drosophila melanogaster contains a cryptic 3'----5' exonuclease that can be detected after separation of the 182-kDa polymerase subunit from the four-subunit enzyme. To determine the specificity of excision of mispaired nucleotides by the exonuclease, we have utilized primed phi X174am3 single-stranded DNA containing a noncomplementary nucleotide at the 3'-primer terminus, opposite deoxyadenosine at position 587 in the amber3 codon of the template strand. In the absence of polymerization, the preference for excision of the mispaired nucleotide from the primer is C greater than A much greater than G. Excision under these conditions is inhibited by the addition of deoxyguanosine monophosphate. Under conditions of concomitant DNA synthesis, the preference for excision at this site becomes A = G much greater than C, and excision is insensitive to deoxyguanosine monophosphate. The high fidelity of DNA synthesis exhibited by the isolated 182-kDa polymerase subunit is not reduced by concentrations of deoxyguanosine monophosphate or adenosine monophosphate that inhibit proofreading by prokaryotic DNA polymerases. Thus, the 3'----5' exonuclease of the Drosophila DNA polymerase-primase participates in exonucleolytic proofreading by excising noncomplementary nucleotides prior to extension of the primer by polymerase action. The deoxynucleoside triphosphate analogs N2-(p-butylphenyl)deoxyguanosine triphosphate and N2-(p-butylphenyl)deoxyadenosine triphosphate are potent inhibitors of DNA polymerase alpha. Like calf thymus DNA polymerase delta, recently determined to have proofreading capability, DNA synthesis by the isolated Drosophila 182-kDa polymerase subunit was not inhibited by the two analogs. In contrast, DNA synthesis by the intact Drosophila polymerase-primase complex was inhibited greater than 95% by these analogs.     

Rapid quantification of global DNA methylation by isocratic cation exchange high-performance liquid chromatography

The DNA of many eukaryotes is methylated at specific cytosine residues in connection with gene regulation. Here we report a method for the quantification of global cytosine methylation based on enzymatic hydrolysis of DNA, dephosphorylation, and subsequent high-performance cation exchange chromatography. Nucleosides are separated in less than 3 min under isocratic conditions on a benzenesulfonic acid-modified silica phase and detected by UV absorption. As little as 1 microg of DNA is sufficient to measure 5-methyldeoxycytosine levels with a typical relative standard deviation of less than 3%. As a proof of concept, the method was applied for analysis of DNA from several Arabidopsis thaliana mutants affected in DNA methylation and from Medicago sativa seedlings treated with the environmental pollutant chromium(VI).     

New dye-labeled terminators for improved DNA sequencing patterns.

We have used two new dye sets for automated dye-labeled terminator DNA sequencing. One set consists of four, 4,7-dichlororhodamine dyes (d-rhodamines). The second set consists of energy-transfer dyes that use the 5-carboxy-d-rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein as the donor dye. Both dye sets utilize a new linker between the dye and the nucleotide, and both provide more even peak heights in terminator sequencing than the dye-terminators consisting of unsubstituted rhodamine dyes. The unsubstituted rhodamine terminators produced electropherograms in which weak G peaks are observed after A peaks and occasionally C peaks. The number of weak G peaks has been reduced or eliminated with the new dye terminators. The general improvement in peak evenness improves accuracy for the automated base-calling software. The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA.     

Depurination and imidazole ring-opening in nucleosides and DNA alkylated by styrene oxide

Styrene-7,8-oxide was reacted with guanosine and deoxyguanosine and four isomeric 7-alkylguanosines were isolated, two of each being substitutions through the alpha and beta carbon of styrene oxide. The diastereomeric adducts imidazole ring-opened at an identical rate but the alpha- and beta-adducts differed (half-lives 90 and 56 min, respectively, pH 10, 24 degrees C). The 7-beta alkyl-deoxyguanosine derivatives ring-opened at a six times slower rate, which was similar to 7-methyldeoxyguanosine. The diastereomeric guanosine products also depurinated at the same rate but the beta-derivatives depurinated faster than the alpha-derivatives (t1/2 35 vs. 79 min, respectively, pH 1, 70 degrees C). The differences in the ring-opening and depurination of the alpha- and beta-isomers corresponded to their respective pK alpha values (7.31-7.32 vs. 7.16-7.19). The 7-alkyldeoxyguanosine derivatives of styrene oxide depurinated equally fast as 7-methyldeoxyguanosine. By contrast, the depurination of 7-alkylguanine was 15 times slower in the single-stranded DNA and 55 times slower in the double-stranded DNA.     

Translesion synthesis past acrolein-derived DNA adduct,gamma -hydroxypropanodeoxyguanosine,by yeast and human DNA polymerase eta

gamma-Hydroxy-1,N(2)-propano-2'deoxyguanosine (gamma-HOPdG) is a major deoxyguanosine adduct derived from acrolein, a known mutagen. In vitro, this adduct has previously been shown to pose a severe block to translesion synthesis by a number of polymerases (pol). Here we show that both yeast and human pol eta can incorporate a C opposite gamma-HOPdG at approximately 190- and approximately 100-fold lower efficiency relative to the control deoxyguanosine and extend from a C paired with the adduct at approximately 8- and approximately 19-fold lower efficiency. Although DNA synthesis past gamma-HOPdG by yeast pol eta was relatively accurate, the human enzyme misincorporated nucleotides opposite the lesion with frequencies of approximately 10(-1) to 10(-2). Because gamma-HOPdG can adopt both ring closed and ring opened conformations, comparative replicative bypass studies were also performed with two model adducts, propanodeoxyguanosine and reduced gamma-HOPdG. For both yeast and human pol eta, the ring open reduced gamma-HOPdG adduct was less blocking than gamma-HOPdG, whereas the ring closed propanodeoxyguanosine adduct was a very strong block. Replication of DNAs containing gamma-HOPdG in wild type and xeroderma pigmentosum variant cells revealed a somewhat decreased mutation frequency in xeroderma pigmentosum variant cells. Collectively, the data suggest that pol eta might potentially contribute to both error-free and mutagenic bypass of gamma-HOPdG.     

A model for intramolecular recombination of herpesvirus DNA leading to the formation of circular, circular-linear and fragmented genomes.

A model is presented for intramolecular recombination of herpesvirus DNA. It is proposed that the terminal repeat sequences of the viral DNA contain insertion sequences which may integrate with homologous repeat sequences between the long (L) and short (S) components. In class 2 herpes-virus DNA (as defined by Honess &; Watson, 1977) in which the repeat sequences flank the S component only, circular-linear DNA molecules can be formed as an intermediate step. Reorientation of the S component leads to the formation of two DNA isomers. In class 3 herpesvirus DNA in which repeat sequences flank both the L and S components, either circular-linear or 8-shaped DNA molecules are proposed as intermediates leading to the formation of four DNA isomers. Fragmentation of the S component could lead to the formation of small circular DNA molecules.     

Identification of Genomic Regions Required for DNA Replication during Drosophila Embryogenesis

A collection of Drosophila deficiency stocks was examined by bromodeoxyuridine (BrdU) labeling of embryos to analyze the DNA replication patterns in late embryogenesis. This permitted us to screen 34% of the genome for genes that when absent in homozygous deficiencies affect the cell cycle or DNA replication. We found three genomic intervals that when deleted result in cessation of DNA replication in the embryo, 39D2-3;E2-F1, 51E and 75C5-7;F1. Embryos deleted for the 75C5-7;F1 region stop DNA replication at the time in embryogenesis when a G(1) phase is added to the mitotic cell cycle and the larval tissues begin to become polytene. Thus, this interval may contain a gene controlling these cell cycle transitions. DNA replication arrests earlier in embryos homozygous for deletions for the other two regions. Analysis of the effects of deletions in the 39D2-3;E2-F1 region on DNA replication showed that the block to DNA replication correlates with deletion of the histone genes. We were able to identify a single, lethal complementation group in 51E, l(2)51Ec, that is responsible for the cessation of replication observed in this interval. Deficiencies that removed one of the Drosophila cdc2 genes and the cyclin A gene had no effect on replication during embryogenesis. Additionally, our analysis identified a gene, pimples, that is required for the proper completion of mitosis in the post-blastoderm divisions of the embryo.     

Characterization of adducts produced in DNA by isomeric 1,2-diaminocyclohexaneplatinum(II) complexes

The cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP) is active as a result of its bifunctional reactions with DNA. Many other platinum complexes also have therapeutic activity. Of current interest are complexes containing 1,2-diaminocyclohexane (DACH). The DACH ligand exists in three isomeric forms with reported differences in therapeutic activity in the order R,R greater than S,S greater than R,S-DACH-Pt. The reaction of the sulphate form of each of these three isomers with DNA has been characterized as a possible explanation for the apparent differences in antitumor activity. These reactions have been characterized by platinating pure DNA followed by enzyme digestion, HPLC separation and analysis by atomic absorption and nuclear magnetic resonance. The spectrum of adducts produced was similar for each isomer and similar to that reported for cis-DDP with adduction at d(GpG), d(ApG) and (dG)2. The R,S-isomer additionally demonstrated isomeric adducts at d(GpG) and d(ApG). The kinetics of formation of the various adducts was the same for each isomer; total platination of DNA was complete in 15 min as were bifunctional adducts at d(GpG) and (dG)2. However, rearrangement to bifunctional adducts took several hours in the case of adducts at d(ApG) sequences. These results did not provide a reason for the different activities of the isomers. It is suggested that the interaction of these adducts with metabolic processes such as DNA repair might explain these differences.     

Double-hairpin elements in the mitochondrial DNA of allomyces: evidence for mobility

The mitochondrial DNA (mtDNA) of the chytridiomycete fungus Allomyces macrogynus contains 81 G+C-rich sequence elements that are 26-79 bases long and can be folded into a unique secondary structure consisting of two stem-loops. At the primary sequence level, the conservation of these double-hairpin elements (DHEs) is variable, ranging from marginal to complete identity. Forty of these DHEs are inserted in intergenic regions, 35 in introns, and 6 in variable regions of rRNA genes. Ten DHEs are inserted into other DHE elements (twins); two even form triplets. A comparison of DHE sequences shows that loop regions contain more sequence variation than helical regions and that the latter often contain compensatory base changes. This suggests a functional importance of the DHE secondary structure. We further identified nine DHEs in a 4-kb region of Allomyces arbusculus, a close relative of A. macrogynus. Eight of these DHEs are highly similar in sequence (90%-100%) to those in A. macrogynus, but only five are inserted at the same positions as in A. macrogynus. Interestingly, DHEs are also found in the mtDNAs of other chytridiomycetes, as well as certain zygomycete and ascomycete fungi. The overall distribution pattern of DHEs in fungal mtDNAs suggests that they are mobile elements.     

Acyclovir triphosphate is a suicide inactivator of the herpes simplex virus DNA polymerase

The triphosphate form of 9-[(2-hydroxyethoxy)-methyl]guanine (acyclovir), ACVTP, inactivates the herpes simplex virus type 1 DNA polymerase. ACVTP does not innately inactivate resting polymerase, but becomes an inactivator only while being processed as an alternative substrate. Pseudo first-order rates of inactivation were measured at varying concentrations of ACVTP and fixed concentrations of the natural substrate, deoxyguanosine triphosphate. These studies indicated that a reversible enzyme-ACVTP (Michaelis-type) complex is formed at the active site prior to inactivation. The formation of this complex was competitively retarded by deoxyguanosine triphosphate. An apparent dissociation constant (KD) of 3.6 +/- 0.2 (S.D.) nM was determined for ACVTP from this reversible complex. A second method for the estimation of the KD which used the extrapolated initial velocities produced a value of 5.9 +/- 0.4 (S.D.) nM. The rate of conversion of the reversible complex to the inactivated complex, at saturating ACVTP, was calculated to be 0.24 min-1. No reactivation of enzyme activity was detected following isolation of the inactivated complex by rapid desalting on Sephadex G-25. Under these conditions, an overall reactivation rate of 1.5 X 10(-5) min-1 could have been easily detected. Therefore, the overall inhibition constant must have been less than 3 pM. In contrast, when host DNA polymerase alpha was incubated with 14 microM ACVTP, only 60% inhibition of enzyme activity was observed, but inactivation was not detected. These data indicate that ACVTP functions as a suicide inactivator of the herpes simplex virus type 1 DNA polymerase, and is only a weak reversible inhibitor of DNA polymerase alpha.     

Polynucleotide block polymers consisting of a DNA.RNA hybrid joined to a DNA.DNA duplex. Synthesis and characterization of dGn.rCidCk duplexes.

The synthesis of several nucleic acid block polymers of the general type dGn.rCidCk is described. The key steps in this procedure were the joining of dCk oligomers, protected at the 3'-OH with an acetyl group, to rCi oligomers by T4 DNA ligase and the purification of the products by RPC-5 column chromatography. The block polymers were characterized by 20% polyacrylamide gel electrophoresis, UV and CD spectra, analytical Cs2SO4 buoyant density analyses, helix-coil transitions and S1 nuclease studies. NMR studies on one member of this series, dGn.rC11dC16, were reported recently (Selsing, E., Wells, R.D., Early, T.A., and Kearns, D.R. (1978) Nature 275, 249-250). The NMR studies and the results described herein indicate that these block polymers are linear duplexes with two adjoining conformations yet are hydrogen-bonded and base-stacked throughout with minimal disruption of the helix at the junction of the two conformations. Computer model building studies described in the following paper (Selsing, E., Wells, R.D., Alden, C.J., and Arnott, S. (1979) J. Biol. Chem. 254, 5417-5422) predict that these nucleic acids contain a bend at the junction region.     

Formation of alkali labile linkages in DNA by hedamycin and use of hedamycin as a probe of protein-DNA complexes.

Hedamycin forms a stable complex with DNA and introduces alkali labile linkages in the DNA. These labile linkages are located at deoxyguanosine residues and are cleaved by the treatment used for breakage at bases alkylated by dimethyl sulfate. The reaction of hedamycin with all G residues in the chain is not uniform, and certain positions, particularily those in TG tracts, are especially reactive. The reaction of hedamycin with DNA can be inhibited by ethidium bromide, suggesting that intercalation is important in positioning the reactive group of hedamycin near to the base which is modified. The low amount of hedamycin needed to produce observable breakage, its specificity for reaction with DNA and its ability to react with DNA under mild conditions make it suitable for use as a probe of protein-DNA complexes. This was shown by the ability of lac repressor and RNA polymerase to block reaction of hedamycin with the DNA of the lac regulatory region.     

Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the alpha 47 gene.

The herpes simplex virus genome consists of two components, L and S, that invert relative to each other to yield four isomeric arrangements, prototype (P), inversion of the S component (Is), inversion of the L component (Il), and inversion of both components (Isl). Previous studies have shown that the 500-base-pair a sequences flanking the two components contain a cis-acting site for inversion. In an attempt to insert a third copy of the alpha 4 gene, the major regulatory gene mapping in the repeats flanking the S component, a fragment containing the alpha 4 gene and an origin of DNA synthesis, was recombined into the thymidine kinase gene mapping in the unique sequences of the L component. The resulting recombinants showed massive rearrangements and deletions mapping in the S component and in the junction between the L and S components. One recombinant (R7023) yielded two isomeric DNA arrangements, a major component consisting of Is and a minor component consisting of Isl. In these arrangements, the genome lacked the gene specifying glycoprotein E and all contiguous genes located between it and the alpha 0 gene in the inverted repeats of the L component. Among the deleted sequences were those encoding an origin of viral DNA synthesis, the alpha 47 gene, and the a sequences located at the junction between the L and S-components. The recombinant grew well in rabbit skin, 143TK-, and Vero cell lines. We conclude that the four unique genes deleted in R7023 are not essential for the growth of herpes simplex virus, at least in the cell lines tested, and that the b sequence of the inverted repeats of the L component also contains cis-acting sites for the inversion of herpes simplex virus DNA sequences.