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Biomedical subjects

B Singer

Publications and source records attributed to B Singer.

At least 127 records · Page 7Linked to original sources
PubMed1986

Replication and transcription of polynucleotides containing ethenocytosine, ethenoadenine and their hydrated intermediates.

Replication with Escherichia coli DNA polymerase I (Pol I) and transcription with DNA-dependent RNA polymerase from Escherichia coli or calf thymus, using as templates synthesized ribo- or deoxyribopolynucleotides containing 1,N6-ethenoadenine (epsilon A) or 3,N4-ethenocytosine (epsilon C), showed that only epsilon C could direct significant misincorporation. The hydrated intermediate of epsilon C caused errors only upon transcription, but not upon replication. epsilon A was a very poor mutagen as assessed by replication with Pol I. Transcription of polynucleotides containing epsilon A under error-prone conditions caused frequent A misincorporation which could not be detected in replication assays. It is concluded that epsilon C may lead to point mutations, specifically directing the misincorporation of thymine. The analogous derivative, epsilon A, is bulky and is likely to be bypassed rather than read. This mechanism could cause frameshift mutation, as generally found for other bulky adducts.

Adenosine↗
PubMed1986

Neutral reactions of haloacetaldehydes with polynucleotides: mechanisms, monomer and polymer products.

The generally accepted mechanism for the formation of etheno derivatives upon reaction of adenosine or cytidine with haloacetaldehydes involves two intermediates. The first, a primary addition to the exocyclic amino group, has not been experimentally verified. The second, a cyclic form of the first intermediate, has been described in monomers but presumed to be too unstable to exist in polynucleotides since such derivatives would be readily dehydrated to other derivatives at pHs below neutrality. We have found that the cyclic intermediates of adenosine and cytidine are the predominant products in polynucleotides, even upon extensive reaction with chloroacetaldehyde at neutrality. The hydrated compounds have half-lives at pH 7, 37 degrees C, of 1.4 h and 13 h for adenosine and cytidine, respectively. Two types of evidence are presented for the existence of the first intermediate, a (1-hydroxy-2-chloroethyl)-substituted exocyclic amino group. Firstly, poly d[A-T] cannot form etheno derivatives (except when denatured) and the observed cross-linking is therefore attributed to alkylation by the chlorinated sidechain of the adenine residue (A), acting on the N6 of A on the opposite strand. Secondly, our results show that blocking of the acceptor nitrogen, needed for cyclization, leads to the formation of relatively stable derivatives of adenosine and cytidine. Guanosine, as a monomer, is modified extensively, but in synthetic polymers no reaction was detected, possibly due to secondary structure.

Acetaldehyde↗
PubMed1985

In vivo formation and persistence of modified nucleosides resulting from alkylating agents.

Alkylating agents are ubiquitous in the human environment and are continuously synthesized in vivo. Although many classes exist, interest has been focused on the N-nitroso compounds, since many are mutagens for bacteria, phage, and cells, and carcinogens for mammals. In contrast to aromatic amines and polyaromatic hydrocarbons which can react at carbons, simple alkylating agents react with nitrogens and oxygens: 13 sites are possible, including the internucleotide phosphodiester. However, only the N-nitroso compounds react extensively with oxygens. In vivo, most possible derivatives have been found after administration of methyl and ethyl nitroso compounds. The ethylating agents are more reactive toward oxygens than are the methylating agents and are more carcinogenic in terms of total alkylation. This is true regardless of whether or not the compounds require metabolic activation. It has been hypothesized that the level and persistence of specific derivatives in a "target" cell correlates with oncogenesis. However, no single derivative can be solely responsible for this complex process, since correlations cannot be made for even a single carcinogen acting on various species or cell types. Some derivatives are chemically unstable, and the glycosyl bond is broken (3- and 7-alkylpurines), leaving apurinic sites which may be mutagenic. These, as well as most adducts, are recognized by different enzymatic activities which remove/repair at various rates and efficiencies depending on the number of alkyl derivatives, as well as enzyme content in the cell and recognition of the enzyme. Evaluation of human exposure requires early and sensitive methods to detect the initial damage and the extent of repair of each of the many promutagenic adducts.

Alkylating Agents↗
PubMed1984

Comparison of repair of methylated pyrimidines in poly(dT) by extracts from rat liver and Escherichia coli.

Partially purified preparations of O6-alkylguanine-DNA alkyltransferase from rat liver and E. coli were tested for their ability to repair O4-methylthymine in a methylated poly(dT) X poly(dA) substrate. The bacterial preparation readily carried out this reaction, but no loss of O4-methylthymine was obtained with the rat liver protein. These results indicate a significant difference in specificity between the mammalian and bacterial proteins which could have important consequences for carcinogenesis and mutagenesis by alkylating agents in mammalian cells.

Animals↗
PubMed1984

N4-Methoxydeoxycytidine triphosphate is in the imino tautomeric form and substitutes for deoxythymidine triphosphate in primed poly d[A-T] synthesis with E. coli DNA polymerase I.

N4- Methoxydeoxycytidine triphosphate ( mo4dCTP ) substitutes for dTTP in poly d[A-T] synthesis with E. coli DNA polymerase I (Pol I). In parallel experiments using as template-primer, poly d[G-C], no incorporation of [14C] mo4dC was detected. This indicates that this deoxy derivative acts as the imino tautomer, as previously found for the riboderivative . Nearest neighbor analysis of transcripts of poly d[A-T] containing mo4dC shows that the derivative substitutes for only one base. In replication, singlestranded mo4dC -containing polymers gave little misincorporation, including that of dATP which can hydrogen-bond to mo4dC in the imino form, if the methoxy group is anti to the N-3. It is therefore assumed that the methoxy group is constrained anti in a polymer such as d[A-T], but can be in the syn form in singlestranded polymers and not recognized by DNA polymerase. mo4dC destabilizes the poly d[A-T] helix, as indicated by a lowered and less cooperative melting. Steric factors such as adjacent base displacement were invoked for similar findings with the doublestranded r( U61 , mo4C39 ) X r(A).

Base Sequence↗
PubMed1984

Assessment of mutagenic efficiency of two carcinogen-modified nucleosides, 1,N6-ethenodeoxyadenosine and O4-methyldeoxythymidine, using polymerases of varying fidelity.

Terminal deoxynucleotidyl transferase (TdT) was used to prepare copolymers of dA and 1,N6-ethenodeoxyadenosine (epsilon dA). When used as templates for Escherichia coli DNA polymerase I (Pol I) and compared with poly (dA), normal dTTP incorporation was not significantly affected by the presence of 7% epsilon dA. dGTP misincorporation was only slightly increased and occurred about once for every 500 epsilon dA residues. The error-prone polymerase from avian myeloblastosis virus (AMV reverse transcriptase) increased this error rate 5- to 20-fold to a maximum of 1 dG/25 epsilon dA. No dCTP misincorporation was detected with either polymerase. In transcription with E. coli DNA-dependent RNA polymerase, no errors were revealed by nearest neighbor analysis. Poly (dA) treated with chloroacetaldehyde under conditions producing the same proportion of epsilon dA (without the hydrated form) as the synthesized template behaved in the same manner with a similar low level of misincorporation of dG. Such treatment of alternating poly d(A-T) caused structural changes indicative of crosslinks but did not alter its template properties. Increasing the amount of epsilon dA in either synthesized or modified polymers greatly decreased the template activity without increasing the error rate. It is suggested that epsilon dA generally does not prevent dT incorporation but behaves as a bulky lesion which is bypassed. In contrast to the low mutagenic efficiency of epsilon dA, O4-methyldeoxythymidine (m4dT), in copolymers with dA, directed the misincorporation of 1 dG/12 m4dT with Pol I and 1 dG/3 m4dT with reverse transcriptase. Nearest neighbor analysis of transcripts showed the incorporation of 1 dG/12 m4dT. These data are in agreement with the previous reported mutagenicity of m4dT in alternating poly d(A-T, m4T).

Acetaldehyde↗
PubMed1984

Alkylation of the O6 of guanine is only one of many chemical events that may initiate carcinogenesis.

The original hypothesis that chemical alteration of DNA can ultimately lead to carcinogenesis had been extended to a concept that the presence and persistence (lack of removal) of O6-alkyl G in an organ or cell population is the important requirement for tumorigenesis by alkylating agents. There are, however, many examples given in the text in which the organ specificity does not correlate with the amount of O6-alkyl G, and indeed, in some instances, no tumors result even though it can be shown that the DNA of many organs contains O6-alkyl G and that cell proliferation occurs. In some cases, there are clearly genetic factors. For example, the brain tumor incidence in two mouse strains differ but O6-alkyl G persistence is the same. Differing amounts or repair capability of O6-alkyl G in species, organs, or cells is not sufficient to explain variations in tumor incidence. Consideration must be given to other alkyl derivatives formed by alkylating carcinogens since at least six derivatives can lead to mispairing. Additionally, depurination has profound biological effects and evidence is emerging that bulky carcinogens such as aflatoxin and N-hydroxy-acetylaminofluorene cause rapid depurination (42). The role of phosphotriesters is as yet unknown. In attempting to come to a conclusion concerning the mode of tumor initiation by alkylating agents, we must not ignore the differences between alkyl groups. Dr. Pegg's paper focuses mainly on methylation, while my arguments stress ethylation. When the number of O6-methylguanines greatly exceeds that of O-methyl-pyrimidines, the former is more likely to be the initiating event. However, ethylation is generally more carcinogenic than methylation, if one considers that much less total alkylation is necessary for tumor development. The O-ethylpyrimidines produced by N-nitroso ethylating agents are more numerous than the O6-ethylguanines and they appear to be more persistent; that is, poorly repaired. Table 3 in Dr. Pegg's paper gives strong support to the potential initiation efficiency of the O4-ethylthymine and perhaps even to the idea that some yet-unidentified event is responsible for the carcinogenicity of diethylnitrosamine.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylating Agents↗
PubMed1983

Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis.

O2-and O4-alkyldeoxythymidine are among the four O-alkyl base-modified derivatives produced by the reaction of N-nitroso alkylating agents with nucleic acids in vitro and in vivo. We find that both O2- and O4-methyl-dTTP can substitute for dTTP in alternating poly(dA-dT)-primed DNA synthesis. Up to 22% of the pyrimidines in the newly synthesized polymer were found by HPLC analysis to be O-methyldeoxythymidine. Little polymer synthesis was observed in the absence of dTTP. However, the O-methyl-dTTPs did not inhibit polymerization of dATP and dTTP. Polymers containing O2- or O4-methyldeoxythymidine were obtained in good yield, retaining the secondary structure of alternating poly(dA-dT). This was shown by the data for thermal transition under different conditions. In contrast, poly(dA-dT).poly(dA-dT) methylated or ethylated to less than 4% total modification by alkylnitrosoureas had a distinctly less stable structure. Neither O2- nor O4-methyldeoxythymidine can form more than one hydrogen bond with adenosine. The unchanged secondary structure of polymers containing these modified thymidines indicates that stacking interactions must play a major role in helix stabilization. O-Alkyldeoxythymidine may be formed by N-nitroso carcinogens that react intracellularly. We have shown that the triphosphates can be utilized by Escherichia coli DNA polymerase I as dTTP. The incorporated O4-methyl-dT causes misincorporation of G, both in transcription and synthesis. When O2-methyl-dT is present, less, but definite, misincorporation results.

DNA Polymerase I↗
PubMed1983

In vitro discrimination of replicases acting on carcinogen-modified polynucleotide templates.

Three different poly(dC)s with modifications that block the N-3 of deoxycytidine were used as templates for polymer synthesis by Escherichia coli DNA polymerase I (EC 2.7.7.7). In contrast to previously reported results with transcriptases, the hydrated form of 3,N(4)-ethenodeoxycytidine (epsilondC.H(2)O) did not mispair. Both 3,N(4)-ethenodeoxycytidine (epsilondC) and 3-methyldeoxycytidine (m(3)dC) led to dTMP misincorporation: 1/20 epsilondC and 1/80 m(3)dC. No other misincorporations appeared to be significant in amount. Thus, both qualitatively and quantitatively, replication errors resulting from carcinogen-modified bases are less frequent than errors in transcription of the same deoxypolynucleotides. Replication of comparable ribopolynucleotide templates by cucumber RNA-dependent RNA polymerase (EC 2.7.7.48) was strongly inhibited by epsilonrC.H(2)O and epsilonrC, so that the fidelity of this enzyme could not be assessed. However, both poly(dC) and poly(rC) containing dU or rU led to incorporation of rA. The presence of even small amounts of purines in poly(rC) greatly depressed synthesis, but the complementary base was incorporated. The finding that an RNA replicase can utilize a deoxypolynucleotide template is a further indication that, at least in vitro, the specificity of the relationship of enzymes and their natural templates is not absolute.

Carcinogens↗
PubMed1982

Chloroacetaldehyde-treated ribo- and deoxyribopolynucleotides. 1. Reaction products.

The in vitro reaction of the vinyl chloride metabolite chloroacetaldehyde (CAA) with cytosine and adenine residues in ribo- and deoxyribopolynucleotides leads to the formation of the relatively stable hydrated etheno derivatives 3,N4-(N4-alpha-hydroxyethene)adenine (epsilon C . H2O) and 1,N6-(N6-alpha-hydroxyethene)cytosine (epsilon A . H2O). Under physiological conditions the hydrates are slowly converted to 3,N4-ethenocytosine (epsilon C) and 1,N6-ethenoadenine (epsilon A). The half-life at pH 7.25 of epsilon C . H2O in poly(rC) is 4.9 h at 50 degrees C and of epsilon A . H2O in poly(rA) is 1.4 h at 37 degrees C. These dehydration rates in polymers are similar to those for hydrates in monomers. The reactivity of A and C residues is greatly suppressed in double-stranded polymers. Adenine residues are about 10 times less reactive in poly(rA) . poly(rU) than A in single-stranded polymers. Under similar reaction conditions no reaction of C residues in poly(rC) . poly(rG) was detected. In vinyl chloride exposed cells, where CAA is formed, the cyclic etheno derivatives of A and C are likely to occur preferentially in single-stranded regions of nucleic acids, with the hydrate forming a major proportion of the modification.

Acetaldehyde↗
PubMed1982

Chloroacetaldehyde-treated ribo- and deoxyribopolynucleotides. 2. Errors in transcription by different polymerases resulting from ethenocytosine and its hydrated intermediate.

Chloroacetaldehyde-modified poly(rC) or poly(dC) was prepared containing either 8-36% 3,N4-ethenocytidine (epsilon C) or 8-36% of a mixture of epsilon C and the hydrated epsilon C (epsilon C . H2O), with the hydrate greatly predominating (greater than 90%). These ribo- and deoxyribonucleotide templates were transcribed with DNA-dependent RNA polymerases from Escherichia coli and calf thymus, in the presence of either Mn2+ or Mg2+ and all four ribonucleoside triphosphates. All the polymers tested were transcribed with either cation present. In an earlier report from this laboratory [Spengler, S., & Singer, B. (1981) Nucleic Acids Res. 9. 365], transcriptional ambiguities resulting from epsilon C residues in enzymatically synthesized poly(rC, epsilon rC) were studied with E. coli DNA-dependent RNA polymerase in the presence of Mn2+. The misincorporations there reported were confirmed when poly(rC, epsilon rC) and poly(dC, epsilon dC), prepared by reaction of poly(rC) and poly(dC) with CAA, were transcribed in the presence of either Mn2+ or Mg2+. We now report that the presence of hydrated epsilon C in polymers also leads to misincorporations but with reproducible differences from those found with epsilon C alone. Nearest-neighbor analysis of the transcription products showed that the hydrate caused misincorporation of A greater than U much greater than C while epsilon C caused misincorporation of U greater than A much greater than C. The extent of misincorporation in transcription was less with Mg2+ than with Mn2+, but the pattern of ambiguity was the same with both cations and with both ribo- and deoxyribocytidylate polymers. Calf thymus DNA-dependent RNA polymerase IIB was also used to transcribe deoxyribocytidine polymers with Mn2+ as the cation. epsilon C and epsilon C . H2O both caused a high level of misincorporation of U , A, and C, but the preferred misincorporations differed slightly from those found with E. coli DNA-dependent RNA polymerase. For both prokaryotic and eukaryotic enzymes, the type of misincorporation resulting from the loss of hydrogen bonding by modification of the N-3 of C not only differed between epsilon C and the hydrated intermediate but also both differed from the transcriptional errors resulting from the presence of 3-methylcytidine in poly(dC) or poly(rC). We conclude that the errors made by these polymerases during transcription do not result primarily from the conditions used (cation, ribo- or deoxyribotemplate) but must be at least in part attributed to the enzyme recognizing some facet of the modified base other than the lack of normal hydrogen bonding.

Acetaldehyde↗
PubMed1982

Reconstitution of rods from tobacco mosaic virus protein and RNA modified with bulky carcinogens.

Tobacco mosaic virus (TMV) RNA was treated with radioactive N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF) and (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BaP diol epoxide) to obtain 3-25 adducts per molecule. Modified full length 30S RNAs and unmodified RNA were reconstituted for various time periods with TMV protein. The particulate products were separated by ultracentrifugation, and the amounts of virus-like material were quantitated by UV spectrophotometry. The length distribution and general appearance of the virus-like rods were studied by electron microscopy. Neither type of carcinogen prevented typical rod formation, but the rate of formation and the maximal yield of reconstituted particles diminished with increasing modification by both agents. The rod length distribution also showed progressively lesser numbers of full-length virus rods. The particulate material contained approximately the same number of adducts as the modified RNA. Thus, it appears that these carcinogen modifications of guanine residues at the N-2 or C-8 atoms did not prevent orderly protein assembly on the RNA but instead slowed up this process and frequently stopped it, possibly at sites where adducts happen to be clustered.

2-Acetylaminofluorene↗