Fluorescence properties of a benzo(a)pyrene 7,8 dihydrodiol 9,10-oxide-DNA adduct. Conformation and effects of intermolecular DNA interactions

The pyrene-like fluorescence of the covalent benzo(a)pyrene diol-epoxide-DNA complex prepared by reacting 7,8,-dihydrodiol 9,10-epoxy benzo(a)pyrene (BPDE) with DNA in aqueous solution $\text{in vitro}$, has been investigated. It is shown that this fluorescence is $\text{sensitive}$ to molecular oxygen, to the concentration of $\text{native}$ DNA and to the ionic strength (KCl concentration), but is $\text{insensitive}$ to the concentration of $\text{denatured}$ DNA. These effects are related to the conformation of the pyrene-like chromophore of BPDE. Most of the fluorescence of a $\text{dilute}$ solution of the DNA-bound benzo(a)pyrene derivative originates from binding sites in which the pyrene moiety is not intercalated between the DNA base pairs, but is located on the outside of the DNA double helix.     

Lack of mutagenicity and metabolic inactivation of aphidicolin by rat liver microsomes

In view of the possible utilization of aphidicolin, a specific inhibitor of DNA polymerase α, in the treatment of neoplastic diseases, it seemed important to assess the mutagenic effect of the drug and the possible modification induced by metabolic activation in the liver. This paper shows that aphidicolin lacks mutagenicity in the Ames' $\text{Salmonella}$-microsome test in agreement with our previous observation that it does not induce DNA repair synthesis in HeLa cells. During the studies of mutagenicity we have observed that aphidicolin is converted to inactive derivative(s) by rat liver microsomal oxidases. The reaction is dependent on time and temperature and requires NADP⁺ and glucose-6-P. The metabolites are not mutagenic and they do not induce DNA repair synthesis in HeLa cells. Therefore the possible anti-cancer use of aphidicolin is not hampered by its partial metabolic inactivation in liver. Our results suggest however that aphidicolin will possibly be clinically useful at concentrations higher than those expected from our studies with human DNA polymerase $\text{α}\text{in vitro}$ and human neoplastic cell lines $\text{in vivo}$. The metabolic derivative(s) of aphidicolin is inactive both against cellular DNA polymerase α and Herpes simplex viral DNA polymerase.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

Identification of a phi X174 coded protein involved in the inhibition of beta-galactosidase synthesis in Escherichia coli

The synthesis of β-galactosidase (EC 3.2.1.23: β-D-galactoside galactohydrolase) in $\text{Escherichia}\text{coli}$ is repressed as a result of infection with single-stranded DNA phage ØX174. An $\text{amber}$ mutant in ØX174 cistron A, which codes for two proteins, does not inhibit the enzyme synthesis while $\text{amber}$ mutants in all other genes do cause repression. A mutant near the amino-terminal end of cistron A, which produces the small 35,000 molecular weight cistron A polypeptide, also inhibits the synthesis of β-galactosidase. Inhibition is also observed in an $\text{Escherichia}\text{coli}\text{rep}$ mutant which does not support the replication of replicative-form DNA. Exogenous nucleotide bases and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) do not have any effect on the degree of repression.     

The effect of 5-azacytidine on E. coli DNA methylase.

5-Azacytidine, when added to growing $\text{E.}$$\text{coli}$ K12, causes a decrease in DNA methylation assayed $\text{in}$$\text{vitro}$. This decrease is greater when $\text{E.}$$\text{coli}$ DNA is used as substrate than when calf thymus DNA is used. The decrease in activity is not due to the inhibition of protein synthesis caused by this drug, since neither chloramphenicol nor rifampin causes a decrease in enzyme activity. The effect is specific for the DNA(cytosine-5)methylase; the methylation of adenine is not affected. The concentration of drug that inhibits the DNA methylase by 50% is the same concentration that inhibits cell growth by 50%.     

Electron microscopy study of viral DNA packaging with lambda head mutants

In a previous study, various intermediates in λ DNA packaging were visualized after lysis of λ-infected cells with osmotic shock and sedimentation through a sucrose formalin cushion onto electron microscope grids. Along this line, a systematic screening for intermediates accumulated in all head mutants available was performed. λA^?-infected cells accumulate only empty spherical protein shells (petit λ) bound at an intermediate point along the DNA thread. In situ digestion experiments with restriction endonuclease EcoRI show that the petit λ-DNA complexes are formed at a fixed point on the DNA concatemer. In $\text{λNu1}{}^{\mathrm{?}}\text{-}\text{infected}$ cells, however, most petit λ was not bound to DNA. In Fec^? cells, which are defective in formation of concatemers but normal in head protein synthesis, most petit λ did not sediment onto the carbon film of the grid. In $\text{D}{}^{\mathrm{?}}$ mutant, petit λ, partially full heads and empty heads with released DNA were observed. λFI^?-infected cells also accumulate petit λ and partially full heads. The present studies suggest that protein pNu1 is required for complex formation between head precursors and DNA concatemers, $\text{p}\text{A}$ for the initiation of DNA packaging, $\text{p}\text{D}$ and $\text{p}\text{F}$I for the promotion of DNA packaging, and $\text{p}\text{D}$ for stabilization of head structures. The results obtained with other head mutants involved in formation of mature proheads and head completion confirm earlier results obtained by different techniques.     

DNA postreplication repair gap filling in temperature-sensitive dnaG, dnaC, dnaA mutants of Escherichiacoli

Gaps in daughter-strand deoxyribonucleic acid (DNA) synthesized after exposure of wild-type $\text{Escherichia}\text{coli}$ to ultraviolet light are filled during reincubation. In this study the $\text{dnaG}\text{,}\text{dnaC}$, and $\text{dnaA}$ gene products have been examined for their role in postreplication repair. These gene products are unique in their specific control of certain types of DNA synthesis: initiation of rounds of replication and chain propagation. Initiation of rounds of replication is not essential to gap filling; however, chain propagation by short DNA piece initiation appears to be essential for gap filling.     

Transformability of DNA polymerase-deficient mutants of Bacillus subtilis

The effect of a deficiency in DNA polymerase on recombination in $\text{Bacillus}$$\text{subtilis}$ has been studied. It is concluded that the major DNA polymerase of $\text{B.}$$\text{subtilis}$ is not required for recombination, and that the recombination deficiency of a previously described DNA polymerase-deficient mutant is actually due to a $\text{rec}$ mutation. Genetic crosses imply that this recombination deficiency is not $\text{recA}$ or $\text{recB}$.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Iron(II)-bleomycin. Biochemical and spectral properties in the presence of radical scavengers

Consistent with a recent literature report (Repine, J. E. $\text{et}$$\text{al.}$ (1981) $\text{Proc.}$$\text{Nat.}$$\text{Acad.}$$\text{Sci.}$$\text{USA}$$\text{7}\text{?}$$\text{8}\text{?}$, 1001–1003), the release of [³H]-thymine from PM-2 DNA by Fe(II)-H₂O₂-generated ·OH was suppressed by dimethyl sulfoxide. In contrast, DMSO did not affect [³H]-thymine release mediated by Fe(II)-bleomycin. Under aerobic conditions in the presence of $\text{t}$-butyl phenylnitrone, Fe(II)-BLM produces an epr signal that has been presumed to arise by transfer of ·OH or $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ from the “active complex” of bleomycin to the spin trap. Remarkably, high concentrations (80 mM) of PBN had no effect on the ability of Fe(II)-BLM to solubilize [³H]-thymine, although the ability of authentic ·OH to degrade DNA was completely suppressed under these condition. The suproxide dismutase catalyst tetrakis(4-N-methylpyridyl)porphineiron(III) also failed to suppress BLM-mediated DNA degradation. Moreover, the epr signal observed with 1.6 mM Fe(II)-BLM in the presence of 80 mM PBN was found to be much less intense than that produced by 1.6 mM Fe(II) and 290 mM H₂O₂, but equivalent in intensity to that obtained with 45 mM Fe(II) and exoess H₂O₂. We conclude that the fragmentation of DNA produced by Fe(II)-BLM can be due neither to free ·OH nor to $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$. We suggest that DNA degradation is initiated by an “active complex” consisting of BLM, metal and oxygen that functions by abstracting H· from susceptible sites on DNA.     

Kinetics of hydrolysis to tetraols and binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-oxide and its tetraol derivatives to DNA. Conformation of adducts

When the major reactive metabolite of benzo(a)pyrene, $\text{trans}$ -7,8-dihydroxy - $\text{anti}$-9,10-epoxy -7,8,9,10-tetrahydrobenzo(a)pyrene ($\text{anti}$-BPDE) is incubated with DNA in aqueous solution at 25°C, both covalent binding and hydrolysis of $\text{anti}$-BPDE to its tetraols occur. Using fluorescence and absorption spectroscopy it is shown that hydrolysis of $\text{anti}$-BPDE is markedly accelerated by DNA. In the presence of 5A₂₆₀ units of DNA per ml in cacodylate buffer solution, at an initial concentration of DNA phosphate/$\text{anti}$-BPDE ratio of 100, both the extent of covalent binding to DNA ( < 7% of the total $\text{anti}$-BPDE initially present) and hydrolysis of $\text{anti}$-BPDE reach their maximum levels within less than five minutes after mixing. Absorption and electric linear dichroism spectra indicate that the tetraols bind non-covalently to DNA by an intercalation mechanism, whereas the covalent product displays the characteristics of an externally bound complex.     

Inhibition of exonuclease V after infection of E. coli by bacteriophage T7

Exonuclease V ($\text{recBC}$ DNase) is inactivated in $\text{E. coli}$ between 4 and 7 min after infection by T7. This process requires protein sythesis. The inactivation does not occur when T7 is deficient for its RNA polymerase and thus does not express the genes involved in DNA replication and phage maturation. Some implications of this new function of T7 are discussed with respect to the processes of infection and DNA replication.     

A spectrofluorimetric investigation of calf thymus DNA modified by BP diolepoxide and 1-pyrenyloxirane

7β,8α-Dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP diolepoxide, $\text{1}$) and 1-pyrenyloxirane ($\text{2}$) bind chemically to calf thymus DNA. The fluorescence efficiency of pyrenyl groups in mutagen modified DNA varies appreciably with its conformation and decreases in the order: pyrenees, modified denatured DNA and modified native DNA. A particularly interesting observation is that the fluorescence efficiency of mutagen modified DNA intensifies substantially upon denaturation. Our results suggest that the pyrenyl groups in mutagen modified DNA are intercalated between the base pairs of DNA. Since both $\text{1}$ and $\text{2}$ are powerful frame-shifting mutagens for S. typhimurium TA-98, the intercalative covalent binding of these compounds to DNA may provide a molecular basis for their mutagenic activity.     

Template specificity changes of DNA-dependent RNA polymerase in B. subtilis during sporulation

Previous work has indicated that loss of ability of DNA dependent RNA polymerase, from stationary phase cultures of $\text{B. subtilis}$, to transcribe phage øe DNA was a $\text{sine qua non}$ for sporulation. To ascertain if this change in template specificity was sporulation-specific, we repeated these experiments using a defined sporulation medium. The changes observed previously did not occur in the defined medium although sporulation was normal. The ability of the enzyme to transcribe other DNA templates was also examined. Similar studies were carried out using a polymerase from a rifamycin-resistant, sporulation conditional mutant. The significance of these findings with regard to the regulation of sporulation in $\text{B. subtilis}$ is discussed.     

Inactivation of SV40 replication by derivatives of benzo[a]pyrene.

When African green monkey kidney cell lines, infected with simian virus 40, were exposed to benzo[a]pyrene-7,8-dihydrodiol or $\text{anti}$-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, inhibition of progeny virus formation was observed. Alkylation of SV40 DNA with $\text{anti}$-BPDE inhibits the infectivity of this viral DNA; however, the inactivation does not follow a single-hit mechanism. Studies on [³H]thymidine incorporation indicate that SV40 DNA synthesis is markedly impaired for the first 12 hours following BPDE treatment; 24 to 36 hours later, however, SV40 DNA synthesis is almost normal. These data suggest that the inhibition of SV40 DNA synthesis by BP derivatives is reversible and that the observed reduction in viral titer requires some other explanation.     

Sequences of the 1.672 g/cm3 satellite DNA of Drosophila melanogaster.

The 1.672 g/cm³ satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin $\text{D}\text{CsCl}$ gradient, and a netropsin sulfate/CsCl gradient. The resulting DNA was homogeneous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucleotide sequence analysis indicates the presence of two different DNA species in this satellite, poly $\text{A-A-T-A-T}\text{T-T-A-T-A}$ and poly$\text{A-A-T-A-T-A-T}\text{T-T-A-T-A-T-A}$. Further physical, chemical and template properties of the isolated complementary strands demonstrate that these two repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. These results further suggest that the sequence heterogeneity observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or chromosomal region.     

Specificity of DNA uptake in genetic transformation of gonococci

Genetic transformation of gonococci to streptomycin resistance was inhibited by homologous DNA or by DNA from related $\text{Neisseriae}$, but not by high concentrations of heterologous DNAs. Gonococci were capable of adsorbing large quantities (up to about 50 μg per 10⁸ cells) of both homologous and heterologous DNA, which could not be eluted by strong shearing forces. Treatment with externally added DNase removed virtually all the heterologous DNA while a small fraction of the homologous DNA, not influenced by the presence of excess heterologous DNA, remained cell-bound in a form resistant to nuclease treatment. Competing homologous DNA suppressed nuclease-resistant binding. These findings suggest that gonococci have two types of DNA binding components at their surface. Competence of gonococci for genetic transformation undergoes a rapid decay if the cells are incubated with homologous (but $\text{not}$ with heterologous) DNA.     

Altered DNA sedimentation by the presence of erythrocytes in alkaline sucrose gradient centrifugation

The presence of a relatively small number of red cells was found to affect DNA sedimentation profile of normal lymphocytes and acute leukemia cells, as observed by the alkaline sucrose gradient centrifugation technique coupled with the fluorometric measurement of DNA. Significant alteration was observed at a nucleated cell/erythrocyte ratio of $\text{20}\text{1}$ to $\text{0.2}\text{1}$, resulting in retardation of the $\text{S}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value and the entire sedimentation profile. This effect seemed to be rather specific to erythrocyte lysate, since corresponding amounts of erythrocyte ghost, IgM, bovine serum albumin, and an increased number of nucleated cells did not influence the profile to an appreciable degree.     

Transposon (Tn5)-mediated suppressive integration of ColE1 derivatives into the chromosome of Escherichia coli K12 (dnaA)

While integration of ColE1 had not been observed previously by ordinary suppressive integration, a $\text{dnaA}$ (Ts) $\text{E. coli}$ strain with Tn$\text{5}$ at various sites of the chromosome and ColE1 or its mini-derivative, pAO3, but not pSC101, inserted by the same transposon produced integratively suppressed strains depending on the RecA function. In contrast to Hfr strains made with a stringently controlled plasmid, they contained the plasmid not only in an integrated but in an autonomous state at an amount comparable to the strain containing the plasmid only autonomously. Introduction of a RecA-deficient mutation to the strain with an integrated ColE1 derivative through conjugation failed. This is likely to be due to lethality of such a strain without RecA-dependent excision of the integrated high copy number plasmid or to quantitative deficiency of DNA polymerase I in addition to the $\text{recA}$ mutation.     

Cyclopenta(f)isoquinoline derivatives designed to bind specifically to native deoxyribonucleic acid. 3. Interaction of 6-carbamylmethyl-8-methyl-7H-cyclopenta(f)isoquinolin-3(2H)-one with deoxyribonucleic acids and polydeoxyribonucleotides

By the use of space-filling models, a novel compound, 6-carbamylmethyl-8-methyl-7$\text{H}$-cyclopenta[f]isoquinolin-3(2$\text{H}$)-one ($\text{1}$) was devised which would be expected to hydrogen bond specifically to GC pairs in the major groove of the double helix such that (i) the amino group of the cytosine molecule donates a hydrogen bond to the C-3 carbonyl of the isoquinoline moiety and (ii) the amide proton of the side chain donates a hydrogen bond to the N-7 of guanine. From difference spectra studies it was found that $\text{1}$ binds to native calf thymus DNA better than to denatured DNA; $\text{1}$ inhibited RNA synthesis by a DNA-dependent RNA polymerase; and equilibrium dialysis experiments revealed that $\text{1}$ binds to poly(dG).poly(dC), whereas no such binding to poly(dA).poly(dT) was observed.