Misincorporation in DNA synthesis after modification of template or polymerase by MNNG, MMS and UV radiation

The synthetic DNA polymers, poly(dG-dC), poly(dC), poly(dA-dT), poly(dA) and poly(dT), were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and UV irradiation. The modified polymers were used as templates to examine the incorporation of non-complementary nucleotides by E. coli DNA polymerase I. Methylation of poly(dG-dC) by MNNG predominantly induced the misincorporation of dTMP, whereas methylation by MMS induced that of dAMP. Treatment of poly(dT) with MNNG caused the misincorporation of dGMP to a considerable extent, but MMS did not enhance the error on poly(dT). The misincorporation of dAMP on poly(dC) and that of dGMP on poly(dA) were also increased by these chemicals. UV irradiation of poly(dT) and poly(dC) induced the error of dGMP and dAMP, respectively. These data on MNNG and MMS in vitro were in fair agreement with the directions of mutation in vivo. But the predominant induction of transitions by UV in vitro did not agree with the UV-induced transversions in E. coli. This inconsistency suggested the participation of other factors than direct mispairing in UV-induced transversion. Modification of DNA polymerase I by MNNG changed the ratio of polymerase to 3' leads to 5' exonuclease activity altering the fidelity of this enzyme, whereas MMS and UV-irradiation did not alter the fidelity of the enzyme.     

Inhibitory effects of various 3'-deoxyribonucleotides on DNA polymerase alpha 2-primase from developing cherry salmon (Oncorhynchus masou) testes

DNA polymerase alpha 2-primase has been purified 2750 fold from developing cherry salmon (Oncorhynchus masou) testes by the following purification steps: fractional extraction, phosphocellulose (1st), ammonium sulfate fractionation, DEAE-cellulose, phosphocellulose (2nd), hydroxylapatite and single-stranded DNA-cellulose column chromatographies. Final preparation of this enzyme has a specific activity of 107,000 units/mg protein (activated salmon sperm DNA as template-primer). DNA primase activity (rGTP dependent incorporation of labelled dGMP into poly (dC) or rNTP dependent incorporation of dNMP into M13 single-stranded DNA) was tightly associated with DNA polymerase alpha activity during all stage of this purification process. Inhibition of DNA primase activity by six kinds of 3'-deoxyribonucleotides was studied by using rNTP dependent DNA synthesis on M13 DNA as template. The inhibition constants (Ki) were larger than those of DNA-dependent RNA polymerases I and II. However, Ki/Km values were very close.     

Factor C from rabbit liver. A new poly(dC) and poly[d(G-C)] template-selective stimulatory protein of DNA polymerases

We have undertaken a search for mammalian DNA-binding proteins that enhance the activity of DNA polymerases in a template sequence-specific fashion. In this paper, we report the extensive purification and characterization of a new DNA-binding protein from rabbit liver that selectively stimulates DNA polymerases to copy synthetic poly[d(G-C)] and the poly(dC) strand of poly(dC).poly(dG) as well as single-stranded natural DNA that contains stretches of oligo(dC). The enhancing protein, a polypeptide of 65 kDa designated factor C, stimulates the copying of the two synthetic templates by Escherichia coli DNA polymerase I, Micrococcus luteus polymerase, and eukaryotic DNA polymerases alpha and beta, but not by avian myeloblastosis virus polymerase. Factor C, however, does not affect utilization by these polymerases of the poly(dG) strand of poly(dC).poly(dG), of poly(dC) primed by oligo(dG), or of poly(dA).poly(dT) and poly[d(A-T)]. With polymerase I, Michaelis constants (Km) of poly[d(G-C)] and of the poly(dC) strand of poly(dC).poly(dG) are decreased by factor C 37- and 4.7-fold, respectively, whereas maximum velocity (Vmax) remains unchanged. By contrast, neither the Km value of the poly(dG) strand of poly(dC).poly(dG) nor the Vmax value with this template is altered by factor C. Rates of copying of activated DNA, denatured DNA, or singly primed M13 DNA are not affected significantly by factor C. However, primer extension analysis of the copying of recombinant M13N4 DNA that contains runs of oligo(dC) within an inserted thymidine kinase gene shows that factor C increases processivity by specifically augmenting the efficiency at which polymerase I traverses the oligo(dC) stretches. Direct binding of factor C to denatured DNA is indicated by retention of the protein-DNA complex on columns of DEAE-cellulose. Binding of factor C to poly[d(G-C)] is demonstrated by the specific adsorption of the enhancing protein to columns of poly[d(G-C)]-Sepharose. We propose that by binding to poly[d(G-C)] and to poly(dC).poly(dG), factor C enables tighter binding of some DNA polymerases to these templates and facilitates enzymatic activity.     

Mode of Action and Specificity of a Nuclease Preferential for Thymidine Residue from Neurospora crassa Mycelia

A nuclease from N. crassa mycelia was found to attack both heat-denatured and native DNA in endonucleolytic manner. The products of exhaustive degradation of heat-denatured DNA were mainly di- to pentanucleotides bearing 5′-phosphoryl groups. 5′-Mononucleotides amounted to 4.4% of the total products and the base distribution was in the following order: dTMP > dCMP > dGMP > dAMP. Analysis of the residues at 5′- and 3′-termini of the oligonucleotides showed that thymidine was predominant at both termini, especially at 3′- termini. Also the analysis of terminal residues produced by limited digestion (27% and 55.5 % of the substrate were rendered acid soluble, respectively) gave the same results as above. Therefore, it was suggested that N. crassa nuclease has some preference for thymidine residue to hydrolyze the sequence of ?T ↓ pT? or ?T ↓ pX-predominantly. The activity toward synthetic polymers was in the following order; poly d(A-T) ? poly dA poly dT > poly d(G-C) > poly dGpoly dC. The correlation between GC-contents and the activity was also investigated.     

Studies on the inhibition of highly purified calf thymus 8S and 7.3S DNA polymerase alpha by aphidicolin.

On activated DNA aphidicolin competitively inhibits the incorporation of dCMP by both calf thymus DNA polymerase alpha A2 and C enzymes and inhibits the incorporation of the other three deoxynucleoside monophosphates apparently non-competitively. However, aphidicolin does not inhibit the incorporation of dAMP into poly(dT) . oligo(A)10 nor does it inhibit the incorporation of dGMP into poly(dC) . oligo(dG)10, but, it does competitively inhibit the incorporation of dTMP into poly(dA) . oligo(dT)10.     

Studies on the miscoding properties of 1,N6-ethenoadenine and 3,N4-ethenocytosine, DNA reaction products of vinyl chloride metabolites, during in vitro DNA synthesis.

1,N6-Ethenoadenine (epsilon A) and 3,N4-ethenocytosine (epsilon C) are formed when electrophilic vinyl chloride (VC) metabolites, chloroethylene oxide (CEO) or chloroacetaldehyde (CAA) react with adenine and cytosine residues in DNA. They were assayed for their miscoding properties in an in vitro system using Escherichia coli DNA polymerase I and synthetic templates prepared by reaction of poly(dA) and poly(dC) with increasing concentrations of CEO or CAA. Following the introduction of etheno groups, an increasing inhibition of DNA synthesis was observed. dGMP was misincorporated on CAA- or CEO-treated poly(dA) templates and dTMP was misincorporated on CAA- or CEO-treated poly(dC) templates, suggesting that epsilon A and epsilon C may miscode. The error rates augmented with the extent of reaction of CEO or CAA with the templates. Base-pairing models are proposed for the epsilon A.G. and epsilon C.T pairs. The potentially miscoding properties of epsilon A and epsilon C may explain why metabolically-activated VC and its reactive metabolites specifically induce base-pair substitution mutations in Salmonella typhimurium. Promutagenic lesions may represent one of the initial steps in VC- or CEO-induced carcinogenesis.     

Deoxyribonucleic acid of Cancer pagurus. II. Template activity for a DNA-dependent DNA polymerase of eukaryotic cells

The template activity of Cancer pagurus DNA and its two components (poly d(A-T) and main component) in response to a DNA polymerase purified from regenerating rat liver has been studied and compared to the results previously obtained with synthetic templates. In the double-stranded native state, whole crab DNA and the main component were poor templates. Their replication was increased by thermal denaturation and inhibited by actinomycin. Like the synthetic copolymer poly[d(A-T)·d(T-A)], native crab poly d(A-T) could be copied and its duplication was not inhibited by actinomycin. The structural difference between native poly d(A-T) Form I, isolated on a density gradient, and partially renatured poly d(A-T) Form II, isolated on hydroxylapatite, resulted in a modification of their template activity. The kinetic studies of [³H] dGMP and [³H] dAMP incorporation confirmed the importance of single-stranded regions (particulary dC regions) in the initiation of the in vitro duplication.     

Conformational transitions of polynucleotides in the presence of rhodium complexes

We studied the effects of hexammine and tris(ethylene diamine) complexes of rhodium on the conformation of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) using spectroscopic techniques and an enzyme immunoassay. Circular dichroism spectroscopic measurements showed that Rh(NH3)6(3+) provoked a B-DNA----Z-DNA----psi-DNA conformational transition in poly(dG-dC).poly(dG-dC). Using the enzyme immunoassay technique with a monoclonal anti-Z-DNA antibody, we found that the left-handedness of the polynucleotide was maintained in the psi-DNA form. In addition, we compared the efficacy of Rh(NH3)6(3+) and Rh(en)3(3+) to provoke the Z-DNA conformation in poly(dG-dC).poly(dG-dC) and poly(dG-m5dC.poly(dG-m5dC). The concentrations of Rh(NH3)6(3+) and Rh(en)3(3+) at the midpoint B-DNA----Z-DNA transition of poly(dG-dC).poly(dG-dC) were 48 +/- 2 and 238 +/- 2 microM, respectively. The psi-DNA form of poly(dG-dC).poly(dG-dC) was stabilized at 500 microM Rh(NH3)6(3+). With poly(dG-m5dC).poly(dg-m5dC), both counterions provoked the Z-DNA form at approximately 5 microM and stabilized the polynucleotide in this form up to 1000 microM concentration. These results show that trivalent complexes of Rh have a profound influence on the conformation of poly(dG-dC).poly(dG-dC) and its methylated derivative. Furthermore, the Rh complexes are capable of maintaining the Z-DNA form at concentration ranges far higher than that of other trivalent complexes. Our results also demonstrate that the efficacy of trivalent inorganic complexes to induce the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) is dependent on the nature of the ligand as well as the polynucleotide modification. Differences in charge density and hydration levels of counterions or base sequence- and counterion-dependent specific interactions between DNA and metal complexes might be possible mechanisms for the observed effects.     

Conformational peculiarities of polynucleotides with a nonrandom base sequence according to the 1H----3H exchange rate in C8H groups of purinic residues

We have determined the 1H----3H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT).poly(dA-dT), poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT) as well as homopolynucleotides poly(dA).poly(dT) and poly(dG).poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4-6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25 degrees C. The rate constants for adenine (kA) and guanine (kG) of polynucleotides were compared with corresponding constants for E. coli DNA. dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution. Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT).poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating "wrinkled" DNA model. The conformations of poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT), according to the exchange data obtained are within the B form. For homopolynucleotides in 0.15 M NaCl, the KA value for poly(dA).poly(dT) is nearly the same as kA for B-DNA, which indicates the similarity of their conformations, whereas the kG value for poly(dG).poly(dC) is 1.7-fold lower in comparison with the kG value in B-DNA. This seems to be connected with the existence of B = A conformation equilibrium for poly(dG).poly(dC) in solution. The increase of NaCl concentration to 3 M results in a B----Z transition in the case of poly(dG-dC).poly(dG-dC) and in the shift of B = A equilibrium towards the A-form in the case of poly(dG).poly(dC) as is evidenced by alterations of their KG values. Poly(dA-dT).poly(dA-dT) in 6 M CsF and poly(dA-dC).poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the "X-type" CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA).poly(dT) in 6 M CsF corresponds to the "heteronomous" DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.     

Interactions of nuclear estrogen receptor with DNA and RNA

The interaction of the nuclear estrogen receptor from hen oviduct with nucleic acids were studied by competition assay using DNa-cellulose centrifugation. We demonstrated that the estradiol-receptor complex binds similarly well to poly(A) RNA and denatured DNA. The estrogen receptor was found to interact more strongly with poly(G), poly(U) than with poly(A), poly(C). The receptor complex binds similarly to poly(A) and poly(dA), and to poly(U) and poly(dU). However, the receptor complex shows stronger binding to poly(G) than to poly(dG) and to poly(C) than to poly(dC). Studies with heteropolyribonucleotides indicated that poly(U1G1) is more effective in competing for the estrogen receptor, and poly(AC) and poly(AUG) are moderately effective, whereas poly(ACU) is least effective. GMP and dGMP showed some competition for the nuclear receptor at 300-fold higher nucleotide concentrations than that of the synthetic poly(G). Observations that the nuclear estrogen receptor binds to poly(A) RNA and interacts selectively with polyribonucleotides suggest that the estrogen receptor-RNA interaction may play a role for the function of estrogens in gene regulation.     

Conversion of dNTP to dNMP dependent on DNA synthesis in isolated Yoshida sarcoma nuclei

Nuclei isolated from Yoshida sarcoma cells had activity for conversion of dGTP to dGMP dependent on DNA synthesis. The ratio of nucleotide generation/generation + incorporation was 0.4 ± 0.1, indicating that approx. 40% of the incorporated dGMP was excised. Two lines of evidence indicated the dependence of this activity on DNA synthesis. (1) The activity was observed only in the presence of ATP, which is essential for nuclear DNA synthesis. (2) Inhibitors of DNA synthesis, such as N-ethylmaleimide, aphidicolin, spermine and KCl, also inhibited ATP- or DNA synthesis-dependent dGMP generation. Although nuclei contain nucleoside triphosphatase (N-nucleotidase), this enzyme was not involved appreciably in DNA synthesis-dependent dGMP generation. The reason for this was explained by the following findings. (a) Inhibitors did not decrease dGMP production in the complete absence of DNA synthesis. (b) Inhibitors did not inactivate N-nucleotidase to the same degree as they inhibited DNA synthesis-dependent dGMP generation. (c) Addition of ATP reduced dGTP hydrolysis catalyzed by N-nucleotidase. (d) GDP had no appreciable effect on DNA synthesis-dependent dGMP generation, but had a diluting effect on dGMP production catalyzed by N-nucleotidase. These results show that the pathway of dGMP generation in isolated nuclei was switched on addition of ATP from a N-nucleotidase-catalyzed one to a DNA polymerase-exonuclease-catalyzed one.     

Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.     

DNA polymerase alpha associated primase from rat liver: physiological variations

A primase activity associated to DNA polymerase alpha from rat liver is described. Both activities were absent in normal adult rat liver but were concomitantly induced after partial hepatectomy. As previously shown for polymerase alpha and DNA topoisomerase II, primase activity reached a maximum value 40-43 h after the partial removal of the liver. Primase activity was shown to catalyze dNMP incorporation on unprimed single-stranded DNA template (M13 DNA) in the presence of rNTP. The activity was not detectable on poly(dA) or poly(dG) but was efficient on poly(dT) or poly(dC). However, the reliability of the primase assay in the presence of poly(dC) was dependent upon the degree of purification of the enzyme. The ribo primers were about 10 nucleotides long, and the reaction was completely independent of alpha-amanitin, a strong inhibitor of RNA polymerases II and III. Primase and polymerase were found tightly associated. A cosedimentation on a 5-20% sucrose gradient was always obtained, independent of the ionic strength. There was also a close coincidence between alpha-polymerase and primase activities during phosphocellulose, hydroxylapatite, and single-stranded DNA Ultrogel chromatography. It has been previously demonstrated by us and others that primase and alpha-polymerase are on separated polypeptides. The association of two activities in the replication complex and the conditions allowing their separation are discussed.     

Characterization of DNA primase separated from DNA polymerase alpha-DNA primase complex of calf thymus

It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus (Yoshida, S. et al. (1983) Biochim. Biophys. Acta 741, 348-357). In the present study, the primase activity was separated from DNA polymerase alpha by treating purified 10S DNA polymerase alpha with 3.4 M urea followed by a fast column chromatography (Pharmacia FPLC, Mono Q column equilibrated with 2 M urea). Ten to 20 % of the primase activity was separated from 10S DNA polymerase alpha by this procedure but 80-90% remained in the complex. The separated primase activity sedimented at 5.6S through a gradient of glycerol. The separated primase was strongly inhibited by araATP (Ki = 10 microM) and was also sensitive to salts such as KCl (50% inhibition at 30 mM). The primase used poly(dT) or poly(dC) as templates efficiently, but showed little activity with poly(dA) or poly(dI). These properties agree well with those of the primase activity in the DNA polymerase alpha-primase complex (10S DNA polymerase alpha). These results indicate that the calf thymus primase may be a part of the 10S DNA polymerase alpha and its enzymological characters are preserved after separation from the complex.     

Purification and characterization of varicella-zoster virus-induced DNA polymerase.

Infection of WI-38 human fibroblasts with varicella-zoster virus led to the stimulation of host cell DNA polymerase synthesis and induction of a new virus-specific DNA polymerase. This virus-induced DNA polymerase was partially purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. This virus-induced enzyme could be distinguished from host cell enzyme by its chromatographic behavior, template specificity, and its requirement of salt for maximal activity. The enzyme could efficiently use poly(dC).oligo(dG)12-18 as well as poly(dA).oligo(dT)12-18 as template-primers. It required Mg2+ for maximal polymerization activity and was sensitive to phosphonoacetic acid, to which host alpha- and beta-DNA polymerase were relatively resistant. In addition, this induced DNA polymerase activity was enhanced by adding 60 mM (NH4)2SO4 to the reaction mixture.     

The mode of inhibitory action by aphidicolin on eukaryotic DNA polymerase alpha.

The mode of action by aphidicolin on DNA polymerase alpha from the nuclear fraction of sea-urchin blastulae was studied. The inhibition of DNA polymerase alpha by aphidicolin was uncompetive with activated DNA and competitive with the four deoxynucleoside triphosphates using activated DNA as a template-primer. For truncated (residual or limited) DNA synthesis with only three deoxynucleoside triphosphates, aphidicolin inhibited the residual synthesis more strongly in the absence of dCTP than in the absence of each of the other three deoxynucleoside triphosphates. The inhibition was reversed with excess dCTP but not with the other three deoxynucleoside triphosphates. That is, aphidicolin inhibited DNA polymerase alpha by competing with dCTP with a Ki value of 0.5 microgram/ml and by not competing with the other three deoxynucleoside triphosphates. dTMP incorporation with the activated DNA was more sensitive to aphidicolin than dGMP or dTMP incorporation with poly(dC). (dG)12-18 or poly(dA) . (dT)12-18. Similar results were obtained for DNA polymerase alpha (B form) from mouse myeloma MOPC 104E.     

Factor D is a selective single-stranded oligodeoxythymidine binding protein.

Factor D, a protein purified from rabbit liver that selectively enhances traversal of template oligodeoxythymidine tracts by diverse DNA polymerases, was examined for the sequence specificity of its binding to DNA. Terminally [32P]-labeled oligomers with the sequence 5'-d[AATTC(N)16G]-3', N being dT, dA, dG, or dC, were interacted with purified factor D and examined for the formation of protein-DNA complexes that exhibit retarded electrophoretic mobility under nondenaturing conditions. Whereas significant binding of factor D to 5'-d[AATTC(T)16G]-3' is detected, there is no discernable association between this protein and oligomers that contain 16 contiguous moieties of dG, dA, or dC. Furthermore, factor D does not form detectable complexes with the duplexes oligo(dA).oligo(dT) or poly(dA).poly(dT). The preferential interaction of factor D with single-stranded poly(dT) is confirmed by experiments in which the polymerase-enhancing activity of this protein is protected by poly(dT) against heat inactivation two- and four-fold more efficiently than by poly(dA) or poly(dA).poly(dT), respectively.     

Conformational Peculiarities of Polynucleotides with a Nonrandom Base Sequence According to the 1H→ 3H Exchange Rate in C8H Groups of Purinic Residues

Abstract

We have determined the ¹H→³H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT)·poly(dA-dT), poly(dG-dC)·poly(dG- dC) and poly(dA-dC)·poly(dG-dT) as well as homopolynucleotides poly(dA)·poly(dT) and poly(dG)·poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4–6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25°C. The rate constants for adenine (k_A) and guanine (k_G) of polynucleotides were compared with corresponding constants for E.coli DNA, dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution.

Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT)·poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating “wrinkled” DNA model. The conformations of poly(dG-dC)·poly(dG-dC) and poly(dA-dC)·poly(dG-dT), according to the exchange data obtained, are within the B form. For homopolynucleotides in 0.15 M NaCl, the k_A value for poly(dA)·poly(dT) is nearly the same as k_A for B-DNA, which indicates the similarity of their conformations, whereas the k_G value for poly(dG)·poly(dC) is 1.7-fold lower in comparison with the k_G value in B-DNA. This seems to be connected with the existence of B? A conformation equilibrium for poly(dG)·poly(dC) in solution.

The increase of NaCl concentration to 3 M results in a B→Z transition in the case of poly(dG-dC)·poly(dG-dC) and in the shift of B?A equilibrium towards the A-form in the case of poly(dG)·poly(dC), as is evidenced by alterations of their K_G values. Poly(dA-dT)·poly(dA-dT) in 6 M CsF and poly(dA-dC)·poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the “X-type” CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA)·poly(dT) in 6 M CsF corresponds to the “heteronomous” DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.     

Reaction of cis-diamminedichloroplatinum (II) and DNA in B or Z conformation

The nature of the adducts and the conformational changes produced in poly(dG-m5dC).poly(dG-m5dC) by cis-diamminedichloroplatinum(II) (cisPt) have been studied. In the reaction of cisPt and B-DNA, the main adduct is bidentate and arises from an intrastrand cross-link between two guanine residues separated by a cytosine. This was deduced from the study of the compounds by t.l.c. after acid hydrolysis of the polymer. The platinated polymer is not digested by S1 nuclease. The antibodies to Z-DNA bind to the platinated polymer with a smaller affinity than to poly (dG-br5dC).poly(dG-br5dC). The c.d. spectrum differs from that of poly(dG-br5dC).poly(dG-br5dC) or poly(dG-m5dC).poly-(dG-m5dC) in Z conformation. It is concluded that the bidentate adduct induces a conformational change from the B form towards a distorted Z form. In the reaction of cisPt and Z-DNA, a monodentate adduct is formed. This adduct stabilizes the Z conformation as shown by c.d. and binding to the anti-Z-DNA antibodies. At room temperature, the second function of the drug can still react with small ligands such as NH4HCO3. By heating, the second function reacts with a guanine residue. A bidentate adduct is formed as in the reaction of cisPt and B-DNA and it induces a transition from the Z form to the distorted Z form.     

The Escherichia coli O6-methylguanine-DNA methyltransferase does not repair promutagenic O6-methylguanine residues when present in Z-DNA

The repair of O6-methylguanine present in N-methylnitrosourea (MNU)-treated alternating polynucleotides MNU-poly(dG-dC) X poly(dG-dC) and MNU-poly(dG-me5dC) X poly(dG-me5dC] was investigated using O6-methylguanine-DNA methyltransferase purified from Escherichia coli. Both modified polynucleotides are equally good substrates for the DNA methyltransferase when they are in the B-form. The substrate properties of the MNU-treated polynucleotides do not differ from those of MNU-treated DNA. One of these modified polynucleotides, MNU-poly(dG-me5dC) X (dG-me5dC), can adopt the Z-conformation under physiological conditions. The conformational transition of the poly(dG-me5dC) X poly(dG-me5dC) from the B-form to the Z-form was monitored by the modification of its spectroscopic properties and by the specific binding of antibodies raised against Z-DNA. The O6-methylguanine residues are repaired in MNU-poly(dG-me5dC) X poly(dG-me5dC) in B-form. At variance, the conversion of this template to the Z-form completely inhibits the repair of the O6-methylguanine residues. The cooperative transition from the Z- to the B-form of MNU-poly(dG-me5dC) X poly(dG-me5dC), mediated by intercalating drugs such as ethidium bromide, restores the ability of MNU-poly(dG-me5dC) X poly(dG-me5dC) to be substrate for the transferase. These results imply that the promutagenic DNA lesion O6-methylguanine persists in Z-DNA fragments and suggest that DNA conformation modulates the extent of DNA repair and, as a result, plays an important role in determining the mutagenic potency of chemical carcinogens.