Mechanism of bleomycin: evidence for a rate-determining 4'-hydrogen abstraction from poly(dA-dU) associated with the formation of both free base and base propenal

When poly(dA-[4'-3H]dU) was degraded by activated bleomycin under a variety of conditions, 50 +/- 10% of the deoxyuridine residues were converted to uracil and uracil propenal, paralleling observations made with DNA. By manipulation of the concentration of O2 in solution, the relative ratio of uracil propenal to uracil could be varied between 0.03 for anaerobic activation and 7.0 for activation at 3 atm of O2. Tritium selection effects on 4'-hydrogen abstraction were also measured under these conditions and found to range from 7.2 to 12.5. These results strongly suggest that the formation of both uracil and uracil propenal is the consequence of a rate-determining 4'-carbon-hydrogen bond cleavage and of an O2-dependent partitioning of the intermediate produced by this cleavage.     

Mechanism of bleomycin: evidence for 4'-ketone formation in poly(dA-dU) associated exclusively with free base release

Incubation of poly(dA-[3'-3H]dU), poly(dA-[5'-3H]dU), or poly(dA-[5'-3H]dT) under a variety of conditions with activated bleomycin resulted in the production of free nucleic acid base, base propenal, and a small amount of 3H2O. Adjustment of the terminated reaction mixture to pH 10 and incubation at 95 degrees C resulted in a time-dependent increase in 3H2O to an amount equal to the amount of free base. If the terminated reaction mixture was incubated with NaBH4 prior to the heat and alkaline treatment, the release of 3H2O was significantly inhibited. These results are consistent with the generation by activated bleomycin of a 4'-ketone yielding free base, with the exchange of the 3'- and 5'-hydrogens by enolization and with the alkaline-induced strand scission occurring from this intermediate.     

The mechanism of free base formation from DNA by bleomycin. A proposal based on site specific tritium release from Poly(dA.dU)

Poly(dA.dU), which is specifically tritiated at the 1'-, 2'- (ribo configuration), 3'-, or 4'-position of deoxyuridine, has been synthesized and the fate of the tritium has been determined upon degradation of the polymer by bleomycin, Fe(II), and O2. No tritium is labilized from the 1'-3H-labeled polymer as 3H2O; however, the resulting 3-(uridin-1'-yl)-2-propenal (uracil propenal) has the expected specific activity. The 2'-3H-labeled polymer affords 3H2O and no label in the uracil propenal. This result and the lack of solvent incorporation into the uracil propenal suggest that proton abstraction from C-2' to afford the trans-propenal is highly stereospecific. For the 3'-3H-labeled polymer, 3H2O is formed and the specific activity of the uracil propenal is identical to that of the deoxyuridine. This suggests that the labilization of the 3'-H is exclusively associated with free uracil formation. 3H2O is also formed from the 4'-3H-labeled polymer. These findings along with previous studies are consistent with the formation of uracil propenal and free uracil by the trapping of the initially formed 4'-radical species by O2 or by a monooxygen species, respectively.     

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C-1' chemistry.

Incubation of Fe(II) bleomycin and O2 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm O2 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to determine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1'-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1'-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H2O nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1'-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-1' chemistry, but a direct consequence of C-4' chemistry.     

UV-induced pyrimidine hydrates in DNA are repaired by bacterial and mammalian DNA glycosylase activities

Escherichia coli endonuclease III and mammalian repair enzymes cleave UV-irradiated DNA at AP sites formed by the removal of cytosine photoproducts by the DNA glycosylase activity of these enzymes. Poly(dG-[3H]dC) was UV irradiated and incubated with purified endonuclease III. 3H-Containing material was released in a fashion consistent with Michaelis-Menten kinetics. This 3H material was determined to be cytosine by chromatography in two independent systems and microderivatization. 3H-Containing material was not released from nonirradiated copolymer. When poly(dA-[3H]dU) was UV irradiated, endonuclease III released 3H-containing material that coeluted with uracil hydrate (6-hydroxy-5,6-dihydrouracil). Similar results are obtained by using extracts of HeLa cells. There results indicate that the modified cytosine residue recognized by endonuclease III and the mammalian enzyme is cytosine hydrate (6-hydroxy-5,6-dihydrocytosine). Once released from DNA through DNA-glycosylase action, the compound eliminates water, reverting to cytosine. This is consistent with the known instability of cytosine hydrate. The repairability of cytosine hydrate in DNA suggests that it is stable in DNA and potentially genotoxic.     

Ultraviolet-induced thymine hydrates in DNA are excised by bacterial and human DNA glycosylase activities

Ultraviolet irradiation of DNA results in various pyrimidine modifications. We studied the excision of an ultraviolet thymine photoproduct by Escherichia coli endonuclease III and by a preparation of human WI-38 cells. These enzymes cleave UV-irradiated DNA at apyrimidinic sites formed by glycosylic removal of the photoproduct. Poly(dA-[3H]dT).poly(dA-[3H]dT) was UV irradiated and incubated with purified E. coli endonuclease III. 3H-Containing material was released in a manner consistent with Michaelis-Menten kinetics. This 3H-labeled material was determined to be a mixture of thymine hydrates (6-hydroxy-5,6-dihydrothymine), separable from unmodified thymine by chromatography in three independent systems. Both cis-thymine hydrate and trans-thymine hydrate were chemically and photochemically synthesized. These coeluted with the enzyme-released 3H-containing material. No thymine glycol was released from the UV-irradiated polymer. Similar results were obtained with extracts of WI-38 cells as the enzyme source. The release of thymine hydrates by both glycosylase activities was directly proportional to the amount of enzyme and the irradiation dose to the DNA substrate. These results demonstrate the modified thymine residues recognized and excised by endonuclease III and the human enzyme to be a mixture of cis-thymine hydrate and trans-thymine hydrate. The reparability of these thymine hydrates suggests that they are stable in DNA and therefore potentially genotoxic.     

Photochemical demonstration of stacked C.C+ base pairs in a novel DNA secondary structure

The secondary structure of the alternating polydeoxynucleotide sequence poly[d(C-T)] was studied as a function of pH by ultraviolet absorbance and circular dichroism spectroscopy and by the analysis of UV-induced photoproducts. As the pH was lowered, poly[d(C-T)] underwent a conformational transition that was characterized by changes in the long-wavelength region (280-320 nm) of the CD spectrum. These changes have previously been interpreted as evidence for the formation of a core of stacked, protonated C X C+ base pairs in a double-helical complex of poly[d(C-T)], with the thymidyl residues being looped out into the solvent [Gray, D. M., Vaughan, M., Ratliff, R. L., & Hayes, F. N. (1980) Nucleic Acids Res. 8, 3695-3707]. In the present work, poly[d(C-T)] was labeled with [U-14C]cytosine and [methyl-3H]thymine and irradiated at pH values both above and below the conformational transition point (monitored by CD spectroscopy). The distribution of radioactivity in uracil means value of uracil dimers, uracil means value of thymine dimers (the deamination products of cytosine means value of cytosine and cytosine means value of thymine dimers, respectively), and thymine-means value of thymine dimers was then determined. As the pH was decreased, we found an increase in the yield of uracil means value of uracil dimers and a decrease in the yield of uracil means value of thymine dimers, which occurred concomitantly with the change in the CD spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Definitive characterization of human thymine glycol N-glycosylase activity

An N-glycosylase activity that released cis-[3H]-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol, TG) from chemically oxidized poly(dA-[3H]dT) was unambiguously characterized both in extracts of HeLa cells and in purified Escherichia coli endonuclease III. This was accomplished by use of microderivatization procedure that quantitatively converted cis-TG to 5-hydroxy-5-methylhydantoin (HMH). The reaction products were analyzed by high-pressure liquid chromatography before and after derivatization by using cis-[14C]TG and [14C]HMH, which had been independently synthesized, as reference compounds. This technique facilitated construction of a v/[E]t plot for the enzyme activity in HeLa cells, permitting estimation of its specific activity. The results obtained prove the existence of both human and bacterial N-glycosylase activities that effect removal of TG from DNA.     

Synthesis of Cyclobutane Nucleosides 2-Preparation of Thymine and Uracil Analogues

1-(2-Oxocyclobutyl-4-benzoyloxymethyl)-2,4(1H,3H)-pyrimidinedione and 1-(2-oxocyclobutyl-4-benzoyloxymethyl)-5-methyl-2,4(1H,3H)-pyrimidinedione can be prepared by reaction of uracil and thymine, respectively, with 3-benzoyloxymethyl-2-bromocyclobutanone. The N-alkylation gave both cis and trans isomers with the trans isomer predominating for uracil whereas the trans isomer was the only product which could be isolated for thymine. Both series were subjected to borohydride reduction followed by transesterification with methoxide giving the corresponding uracil and thymine nucleoside analogues. The uracil derivative 1-(2-oxocyclobutyl-4-benzoyloxymethyl)-2,4(1H,3H)-pyrimidinedione was irradiated in aqueous acetonitrile to generate isonucleoside analogues.     

Optically detected zero field magnetic resonance characterization of a bromine atom-containing polynucleotide, poly(dA-br5dU)

Phosphorescence and optically detected zero field magnetic resonance ( ODMR ) spectra are reported for a bromine atom-containing polynucleotide, poly(dA- br5dU ). The triplet state luminescence of poly(dA- br5dU ) is dominated by the phosphorescence of the bromouracil base which possesses sub-millisecond triplet lifetimes. Characteristic multiple slow passage ODMR transitions, which are observed in both br5dUrd and poly(dA- br5dU ), are assigned to the triplet state of bromouracil. In addition, an abnormally-perturbed adenine triplet state, which is not apparent in the phosphorescence spectrum of poly(dA- br5dU ), is detected and identified by its slow passage ODMR and amplitude-modulated phosphorescence microwave double resonance spectra. It is proposed that the perturbed adenine is a minor component of the polynucleotide structure which is present in regions of altered stacking induced by the high polarizability of the Br atom.     

Uracil in deoxyribonucleotide polymers reduces their template-primer activity for E. coli DNA polymerase I.

The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA). poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT). The polymerizing and the primer terminus adding reactions of a homogenous E. coli DNA polymerase I preparation, as measured by incorporation of [3H]dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates. Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer.     

Local effect for [5-3H]cytosine decays: Production of a chemical product with possible mutagenic consequences

Purified bacterial DNA containing [2-¹⁴C, 5-³H]cytosine was stored at ?196 °C to accumulate tritium decays. At various storage times samples were analyzed by two-dimensional thin-layer chromatography, following hydrolysis of the DNA. The major product detected as a function of an increasing number of tritium decays was uracil, which was formed with an efficiency of 28% per tritium decay. Uracil possesses the genetic coding properties of thymine and, therefore, would account for the high efficiency of C → T transitions previously reported in mutagenesis studies employing [5-³H]cytosine. A possible reaction mechanism leading to the formation of uracil from [5-³H]cytosine decay is presented.     

Metabolism of thymine nucleotides synthesized via the ''de novo'' mechanism in normal, megaloblastic and methotrexate-treated human cells and in a lymphoblastoid cell line.

Human bone-marrow cells and lymphocytes were incubated with [3H]deoxyuridine (dU) to study the metabolism of thymine nucleotides labelled via the thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) step of the 'de novo' biosynthetic pathway. (1) Continuous labelling with [3H]dU was used to compare incorporation of label into DNA with the specific radioactivities of thymine nucleotides separated by paper chromatography. (2) Cells were also labelled with [3H]dU at 13 degrees C, and 'chased' in unlabelled medium at 37 degrees C in order to quantify the proportion of thymine nucleotides incorporated into DNA and the proportion degraded. Only 40% of labelled thymine nucleotides were incorporated into lymphocyte DNA during a 'chase', whereas 100% were incorporated by MOLT 4 cells (a lymphoblastoid cell line of thymic origin, Thy-ALL line). Unincorporated nucleotides were rapidly degraded in lymphocytes, but degradative activity was very low in MOLT 4 cells. The results described here reinforce our previous conclusions [Taheri, Wickremasinghe & Hoffbrand (1981) Biochem. J. 194, 451-461] that there is a single thymine nucleotide compartment in Thy-ALL cells, but at least two pools in lymphocytes and bone-marrow cells. This compartmentation of nucleotides in human cells is consistent with a model which proposes that deoxyribonucleotides are localized near replication forks by the activity of multienzyme complexes [Mathews, North & Reddy (1978) Adv. Enz. Regul. 17, 133-156]. Our results also suggest that thymine nucleotides derived by the 'de novo' mechanism may be more highly localized than those derived by salvage. In cells from patients with megaloblastic anaemia owing to deficiency of vitamin B12 or folate or in normal cells treated with methotrexate, there was a massive accumulation of labelled dUMP and decreased incorporation of label into DNA. There was no measurable incorporation of labelled deoxyuridine residues into DNA of megaloblastic cells, but deoxyuridine residues were detected in DNA of cells treated with methotrexate.     

Deglyco-bleomycin. Degradation of DNA and formation of a structurally unique Fe(II) . CO complex

In analogy with bleomycin, deglyco-bleomycin B2 has been found to form a stable, diamagnetic complex with Fe(II) and CO. Although the stoichiometry of this complex appeared to be the same as that formed with bleomycin, the geometry of the deglyco-bleomycin complex was fundamentally different, especially as regards orientation of the beta-aminoalanine moiety. In the presence of Fe(II) and O2, deglyco-bleomycin A2 and deglyco-bleomycin B2 were found to release [3H]thymine from radiolabeled PM-2 DNA; when employed at limiting concentrations, deglyco-bleomycin A2 and B2 gave about half as much [3H]thymine release as the respective bleomycins. In view of the spectral evidence (Burger, R. M., Horwitz, S. B., Peisach, J., and Wittenberg, J. B. (1979) J. Biol. Chem. 254, 12299-12302) that Fe(II) . bleomycin . CO has the same geometry as the complex formed by initial association of bleomycin, Fe(II), and O2, the accumulated data suggest strongly that all metal complexes of bleomycin (derivatives) capable of DNA degradation need not have the same geometry.     

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA)

To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.     

Examination of the mechanism of sucrose synthetase by positional isotope exchange

The mechanism of the sucrose synthetase reaction has been probed by the technique of positional isotope exchange. [beta-18O2, alpha beta-18O]UDP-Glc has been synthesized starting from oxygen-18-labeled phosphate and the combined activities of carbamate kinase, hexokinase, phosphoglucomutase, and uridine diphosphoglucose pyrophosphorylase. The oxygen-18 at the alpha beta-bridge position of the labeled UDP-Glc has been shown to cause a 0.014 ppm upfield chemical shift in the 31P NMR spectrum of both the alpha- and beta-phosphorus atoms in UDP-Glc relative to the unlabeled compound. The chemical shift induced by each of the beta-nonbridge oxygen-18 atoms was 0.030 ppm. Incubation of [beta-18O2, alpha beta-18O]UDP-Glc with sucrose synthetase in the presence and absence of 2,5-anhydromannitol did not result in any significant exchange of an oxygen-18 from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. It can thus be concluded that either sucrose synthetase does not catalyze the cleavage of the scissile carbon-oxygen bond of UDP-Glc in the absence of fructose or, alternatively, the beta-phosphoryl group of the newly formed UDP is rotationally immobilized.     

On the recognition and cleavage mechanism of Escherichia coli endodeoxyribonuclease V, a possible DNA repair enzyme

Escherichia coli endodeoxyribonuclease V acts at many sites of damage in duplex DNA, including apurinic/apyrimidinic sites, lesions induced by ultraviolet light which are not pyrimidine dimers, adducts of 7-bromomethylbenz[a]anthracene, and, as demonstrated earlier (Gates, F. T., and Linn, S. (1977a) J. Biol. Chem. 252. 1647-1653), it degrades uracil-containing duplex DNA most efficiently. The cleavage rate increases with increasing substitution of uracil for thymine in T5 DNA, with a replacement of one-eight of thymine generating the apparent maximum cleavage rate. However, the apparent reaction limit with DNA containing 3.8% of thymine replaced by uracil corresponds to cleavage at only 6% of the dUMP residues. Evidently, the enzyme recognizes some peculiarities of abnormal DNA structure, but not simply distortions, since some lesions, including pyrimidine dimers, are not substrates. Endonuclease V generates double strand breaks in a constant ratio to single strand nicks, regardless of the substrate. It degrades DNA processively, completing the digestion of one substrate molecule before proceeding to the next. The enzyme also appears to act cooperatively. Cleavage at methylbenz[a]anthracene adducts is usually or always 5' to the lesion. Endonuclease V seems well suited to act as a DNA repair enzyme, surveying the genome for structural distortions generated by lesions for which specific repair systems might not exist.     

Oxidative damage to 5-methylcytosine in DNA.

Exposure of pyrimidines of DNA to ionizing radiation under aerobic conditions or oxidizing agents results in attack on the 5,6 double bond of the pyrimidine ring or on the exocyclic 5-methyl group. The primary product of oxidation of the 5,6 double bond of thymine is thymine glycol, while oxidation of the 5-methyl group yields 5-hydroxymethyluracil. Oxidation of the 5,6 double bond of cytosine yields cytosine glycol, which decomposes to 5-hydroxycytosine, 5-hydroxyuracil and uracil glycol, all of which are repaired in DNA by Escherichia coli endonuclease III. We now describe the products of oxidation of 5-methylcytosine in DNA. Poly(dG-[3H]dmC) was gamma-irradiated or oxidized with hydrogen peroxide in the presence of Fe3+ and ascorbic acid. The oxidized co-polymer was incubated with endonuclease III or 5-hydroxymethyluracil-DNA glycosylase, to determine whether repairable products were formed, or digested to 2'-deoxyribonucleosides, to determine the total complement of oxidative products. Oxidative attack on 5-methylcytosine resulted primarily in formation of thymine glycol. The radiogenic yield of thymine glycol in poly(dG-dmC) was the same as that in poly(dA-dT), demonstrating that 5-methylcytosine residues in DNA were equally susceptible to radiation-induced oxidation as were thymine residues.     

Streospecific substitution of oxygen-18 for sulfur in nucleoside phosphorothioates

Reaction of nucleoside phosphorothioates with N-bromosuccinimide in dioxane and H218O leads to the exchange of sulfur for oxygen-18. Using the Sp-isomers of adenosine 5'-O-(1-thiodiphosphate) and adenosine 3',5'-cyclic phosphorothioate, it can be shown by 31P NMR spectroscopy that this reaction proceeds with inversion of configuration yielding the Rp-isomers of [alpha-18O]ADP and [18O]cAMP, respectively. Adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate) are likewise converted to [beta-18O]ATP and [gamma-18O]ATP although the stereochemistry of the former reaction has yet to be evaluated. With very slight modifications this reaction is applicable to all the common bases.     

Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate

In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled [gamma-17O,18O]ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[17O,18O]phosphate proceeds with inversion of configuration. Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb [Webb, M. R. (1982) Methods Enzymol. 87, 301-316]. We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O. These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate.