Enzyme DNase Research Articles

Oxygen Free Radical Damage to DNA: TRANSLESION SYNTHESIS BY HUMAN DNA POLYMERASE η AND RESISTANCE TO EXONUCLEASE ACTION AT CYCLOPURINE DEOXYNUCLEOSIDE RESIDUES

Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.

Antibacterial Effects of Different Food-Related Phosphates Using Aeromonas hydrophila

Aeromonas hydrophila is considered to be an emergent food-related bacterium. Phosphates are used as additives, mainly in meat products, to improve the quality of these foods. The antibacterial properties of phosphates are also well known. In this work, two A. hydrophila strains in early exponential phase were used: (A) A. hydrophila ATCC 7965 and (B) A. hydrophila derived from food, isolated in our laboratory. MIC and MBC studies were performed to assess the antibacterial effects of four phosphates assayed in brain heart infusion broth (BHI) and modified complete defined synthetic medium (mCDS) as compared to cooked ground meat medium (CM). The MBC values of the phosphates in CM were significantly higher than MIC values in BHI broth and mCDS medium (P < 0.05). In the two latter media, the growth of both A. hydrophila strains was totally inhibited by concentrations between 0.5 and 3.0%. Although all the assayed phosphates proved to have bactericidal effects on A. hydrophila, 0.5% sodium acid pyrophosphate (SAPP) exhibited greater effects in both strains and was selected for subsequent experiments. The bacteriolytic effect of SAPP was spectrophotometrically determined (260nm of absorbance) by means of the leakage of intracellular nucleotides and microscopically confirmed by the presence of massive gelatinous aggregates. These were identified by enzymes (RNase, DNase, and proteinase) that hydrolyzed the nucleotides and proteins released during cellular lysis in the presence of SAPP. It was concluded that 0.5% SAPP can have bactericidal and bacteriolytic effects in early exponential phase A. hydrophila cells.

Induction of Anti-DNA Antibodies in Preautoimmune NZB×NZW F1 Mice by Immunization with a DNA-DNase I Complex

Recent studies suggest that anti-DNA antibodies may arise from the immune response to a complex of DNA and a DNA-binding protein. One of the protein targets frequently recognized by anti-DNA antibodies is the enzyme DNase I. To investigate the possible role of DNase I in the induction of anti-DNA antibodies, we immunized preautoimmune NZB×NZW F1 mice with a complex of DNA and DNase I emulsified in complete Freund's adjuvant. Control mice received DNA or DNase in adjuvant. IgG anti-dsDNA antibodies were induced in 50% of the mice immunized with DNA-DNase, in 25% of the mice immunized with DNase and in 6% of the mice immunized with DNA. However, immunized mice that produced anti-DNA antibodies did not develop renal disease. These data show that a DNA-binding protein like DNase may act as carrier in the immune response that leads to anti-DNA antibodies production in an autoimmune strain, but the induced anti-dsDNA antibodies have a low pathogenic potential.

Molecular, biochemical and immunological studies of hen pancreatic deoxyribonuclease I

Deoxyribonuclease I (DNase I) was purified from the hen pancreas to electrophoretic homogeneity using six-step column chromatography. The purified enzyme showed a molecular mass of about 33 kDa and maximum activity at pH 7.0. It required divalent cations, Mg 2+ and Ca 2+, for its activity and was inhibited by EDTA, EGTA and an antibody specific to the purified enzyme but not by G-actin. A 1066-bp cDNA encoding hen DNase I was constructed from the total RNA of a hen pancreas using a combination of the reverse transcriptase–polymerase chain reaction and rapid amplification of cDNA ends methods, followed by sequencing. The cDNA was expressed in Escherichia coli, and the recombinant polypeptide exhibited significant enzyme activity. The mature hen DNase I protein was found to consist of 262 amino acids. In human and bovine DNase I four amino acid residues, Glu-13, Tyr-65, Val-67 and Ala-114 are involved in actin binding, whereas in the hen DNase I these positions were occupied by Asp, Phe, Ser and Phe, respectively. A survey of the DNase I distribution in 15 hen tissues showed that the pancreas had the highest levels of both DNase I enzyme activity and DNase I gene expression. The results of our phylogenetic and immunological analyses indicate that the hen DNase I is not closely related to the mammalian enzymes. This is the first report in which has been described the results of molecular, biochemical and immunological analyses on hen DNase I.

Development from actions of bacterial phospholipases C on eucaryotic plasma membranes to molecular biology of GPI-anchored proteins

Bacterial phospholipases C are known to act on biomembranes, since they can cleave the phosphodiester linkage between the polar head and the hydrophobic moiety of each phospholipid in these membranes. These enzymes have been classified into three groups; phosphatidylcholine (PC)-, sphingomyelin (SM)- and phosphatidylinositol (PI)-degrading phospholipases C. Enzymatic properties and toxicities of these phospholipases C are reviewed, in relation to author's research. Studies on the hemolytic phospholipases of Clostridium sp., Bacillus cereus etc., revealed that hydrolysis of choline-containing phospholipids such as PC and SM was responsible for the hemolysis of mammalian erythrocytes by these enzymes in the presence of Ca2+ and/or Mg2+. Also, the studies on a structure-activity correlation of SM-hydrolyzing phospholipase C from B. cereus disclosed the similarity of active sites between this enzyme and bovine pancreatic DNase I. By action of PI-degrading phospholipases C, several membrane proteins such as alkaline phosphatase, 5'-nucleotidase, VSG (protozoal surface glycoprotein) etc., were shown to be released from the plasma membranes of eucaryotic cells. From structural analysis, these proteins have been revealed to be glycosylphosphatidylinositol (GPI)-anchored proteins bound to the plasma membranes with carboxyl terminal-attached glycolipid. Biochemistry and molecular biology of GPI-anchored proteins, including the structures and biosynthetic routes of GPI glycolipids as well as the process of GPI attachment to proteins, requirements of C-terminal signal peptide for the protein modification by GPI, and distribution of GPI-anchored proteins in living world, are described in relation to our studies.

4-hydroxy-17-methylincisterol, an inhibitor of DNA polymerase-α activity and the growth of human cancer cells in vitro

An ergosterol derivative, 4-hydroxy-17-methylincisterol (HMI), was found to be an inhibitor of mammalian DNA polymerases in vitro. HMI inhibited the activity of calf thymus DNA polymerase α (pol. α). Among the polymerases tested, pol. α was the most sensitive to inhibition by HMI, and the inhibition was concentration dependent. The inhibitory effect of HMI on pol. α was almost the same as that shown by aphidicolin, a well-known potent pol. α inhibitor. HMI had relatively less effect on rat DNA pol. β, human immunodeficiency virus type 1 reverse transcriptase (HIV-RT), and calf thymus terminal deoxynucleotidyl transferase (TdT) in vitro, and did not influence the activities of prokaryotic DNA polymerases such as Klenow Fragment of DNA polymerase I, or the DNA-metabolic enzyme DNase I. HMI was found to be able to prevent the growth of human cancer cell lines originating from patients with leukemia or various solid tumors; its ic 50 values ranged from 7.5 to 12 μM. We also synthesized other ergosterol derivatives and tested them, and found that two compounds, 17-methylincisterol and 4-acetyl-17-methylincisterol, have similar inhibitory effects.

Studies on inhibitors of mammalian DNA polymerase α and β: Sulfolipids from a pteridophyte, Athyrium niponicum

Three sulfolipid compounds, 1, 2 and 3, have been isolated from a higher plant, a pteridophyte, Athyrium niponicum, as potent inhibitors of the activities of calf DNA polymerase α and rat DNA polymerase β. The inhibition by the sulfolipids was concentration dependent, and almost complete inhibition of DNA polymerase α and DNA polymerase β was achieved at 6 and 8 μg/mL, respectively. The compounds did not influence the activities of calf thymus terminal deoxynucleotidyl transferase, prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase and Taq polymerase, the DNA metabolic enzyme DNase I, and even a DNA polymerase from a higher plant, cauliflower. Similarly, the compounds did not inhibit the activity of the human immunodeficiency virus type 1 reverse transcriptase. The kinetic studies of the compounds showed that DNA polymerase α was inhibited non-competitively with respect to the DNA template and substrate, whereas DNA polymerase β was inhibited competitively with both the DNA template and substrate. The binding to DNA polymerase β could be stopped with non-ionic detergent, but the binding to DNA polymerase α could not.

Expression analysis of recombinant herpes simplex virus type 1 DNase.

Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus propagation, by using mammalian cell expression systems based on vaccinia virus and on Semliki Forest virus (SFV)1. Although the establishing of recombinant vaccinia virus failed due to the apparent toxicity of the herpesviral enzyme, soluble and functional HSV-1 DNase was efficiently expressed in BHK-21 cells by the vaccinia virus/T7 RNA polymerase hybrid system as well as by recombinant Semliki Forest virus. Using rabbit antiserum ExoC, directed against the C-terminal residues 503-626, or mouse monoclonal antibody (MAb) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the identical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 DNase coincided with the overproduction of a single major 85-kDa protein reaching an optimum between 16 h and 36 h after infection. At later times of infection the enzymatic activity vanished. Thus, recombinant SFV may be an appropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infection does not exceed 24 h. Due to the limitations of transient expression systems, the vaccinia/T7 RNA polymerase hybrid system is suited for expression analysis on a small scale, and for studying intracellular interactions of the enzyme as demonstrated by immunofluorescence microscopy studies. Using vector pTM1, recombinant HSV-1 DNase was efficiently overproduced in BHK-21 cells at 6 h after transfection and was shown to colocalize with the cellular chromatin at sites apparently distinct from the bulk of the herpesviral replication sites the way it is observed for the enzyme of lytically infected cells. The deleting of the 123 C-terminal amino acid residues did not alter this nuclear localization of HSV-1 DNase, suggesting that the latter sequences and other herpesviral factors are not required for the chromatin association.

Ethidium bromide intercalation and chromatin structure: A thermal analysis

Differential Scanning Calorimetry has been performed in the temperature range 310 K-410 K on intact thymocytes and physiologically isolated chromatin following Ethidium bromide intercalation. Native thymocytes exhibited four main thermal transitions (at 339 K, 347 K, 362 K and 375 K) that were assigned to the melting of different cellular components. At increasing dye concentrations an enthalpy redistribution became evident between the thermal transition at 362 K related to the melting of nucleosome organized in the 10 nm filament, and the transition at 375 K related to the melting of nucleosome organized in the 30 nm (or more) fiber. In correlation with increasing concentrations of Ethidium bromide, the disappearance and the subsequent reappearance of the highest temperature transition seem to be related to the unwrapping and subsequent wrapping of the chromatin fiber. Under similar condition, free DNA and digested chromatin do not show any enthalpy redistribution in their calorimetric profiles following Ethidium bromide intercalation. On the contrary, physiologically isolated chromatin displayed similar enthalpy redistribution between transitions assigned to chromatin DNA melting. An interesting difference appeared in the calorimetric profile of isolated chromatin with respect to the in situ material after chromatin extraction. In fact, a transition at 354 K, probably related to the melting of linker DNA became apparent (the transition at 362 K was assigned to the melting of DNA around the core particle). Selective digestions with different enzymes (micrococcal nuclease, proteinase K and DNase I) were carried out on thymocytes to verify the assignment of the main thermal transitions. In order to clarify the nature of the high temperature transitions native thymocytes were treated with topoisomerase I that removes superhelical turns from topologically closed DNA molecules. A comparison of calorimetric data with thermal denaturation profiles obtained by spectropolarimetric measurements on physiologically isolated chromatin gave further confirmation to the peak assignment by distinguishing the thermal transitions related to protein denaturation from the ones assigned to chromatin-DNA.

Flow cytometric analysis of in vivo polyamine deprivation in Lewis lung carcinoma (3LL) cells using the monoclonal antibody SPM8-2

It has previously been shown that the monoclonal antibody SPM8-2 recognizes free spermine and spermidine as well as polyamines bound by an amide bond. In the present work it is demonstrated that this antibody also interacts with spermidine, spermine, and to a lesser extent N1- and N8-acetyl spermidine in an ELISA test where the polyamines are bound by reaction with formaldehyde. 3LL Lewis lung carcinoma cells from tumor-grafted mice were labeled with fluorescein-conjugated monoclonal antibody SPM8-2 and analyzed by flow cytometry. Both viable cells and formaldehyde-fixed and subsequently permeabilized cells showed fluorescent staining. However, most polyamines present in the cells are not directly available for antibody binding. Treatment of fixed cells with DNase or RNase greatly increased fluorescent staining, suggesting that some polyamines are co-localized with DNA and RNA. Antibody labeling of the cells was prevented by addition of free spermine. 3LL cells from tumors of mice treated by a polyamine depleting regimen had decreased intracellular spermidine levels and bound less antibody when compared to untreated controls. After digestion with RNase, the cells from treated mice bound considerably less fluorescent antibody than tumor cells from untreated mice, while their RNA content was similar. In contrast, fluorescent staining after DNase digestion was only slightly affected by the treatment with a polyamine depleting regimen. This suggests that the pools of polyamines which are co-localized with RNA are depleted more readily than those associated with DNA. Since only a small proportion of the intracellular polyamines is accessible to the bulky antibodies, treatment with hydrolytic enzymes (DNase, RNase) is necessary to reveal specific compartments of the polyamines and to demonstrate qualitative and semi-quantitative differences of their distribution within cells.

Characterization of Monoclonal Antibodies to the Zta and DNase Proteins of Epstein-Barr Virus.

Two monoclonal antibodies (mAb) were derived and designated 4F10 and 311H. 4F10 was against the Epstein-Barr virus (EBV) Zta protein and 311H specifically recognized EBV DNase enzyme. Using mAb 4F10 as a probe, the Zta protein could be detected as a 36-kD molecule in L5 cells and as a 38-kD molecule in B95-8 cells, reflecting the fact reported by other laboratories, using rabbit polyclonal antisera, that the Zta protein was variously modified in different host cells. 311H mAb was generated using antigens purified from one-step His-Bind column chromatography. The antigenic epitope recognized by this mAb was mapped within the residues 1-152 of EBV DNase by reacting the mAb with three distinct truncated mutants. Also, using 311H as a reagent to trace the kinetic expression of EBV DNase proteins in EBV-infected Akata cells, the Western blotting results indicated that DNase antigen could be detected at 12 h postactivation. The feasibility of applying these two mAb in the investigation of EBV biology is discussed. Copyright 1997 S. Karger AG, Basel

Recombinant human deoxyribonuclease for hematopoietic stem cell processing.

Numerous reagents are used in the collection processing and storage of hematopoietic progenitor cells for transplantation. To decrease potential variations in the final component for transplantation, these reagents should be uniform in safety, potency, and efficacy. Pharmaceutical-grade reagents are ideal but often are not available. Recently, recombinant human deoxyribonuclease (DNase) was approved for the treatment of patients suffering from the pulmonary complications of cystic fibrosis. We tested this pharmaceutical for toxicity to hematopoietic progenitor cells. These cells were exposed to a range of incubation concentrations for both the recombinant enzyme and bovine DNase previously used in this laboratory. No loss of nucleated cells or hematopoietic progenitors was observed after short-term incubation (1 h) or with direct addition to the culture medium. No incremental toxicity was observed in using recombinant enzyme with murine anti-B cell antibodies and rabbit complement in an immunologic purge technique. A variable effect on cell recovery after thawing of cryopreserved bone marrow cells was observed for both enzyme sources. These data suggest that the pharmaceutical-grade, recombinant human DNase may substitute for previously used reagent-grade protein from animal sources.

EPR Spin-trapping studies of radical damage to DNA

EPR spectroscopy has been employed, in conjunction with spin-trapping [using 2-methyl-2-nitrosopropane dimer (MNP)] to study the attack of ˙OH (generated by the Fe2+–EDTA/H2O2 couple) on deoxyribonucleic acid (DNA) and its components. The results confirm that for pyrimidine deoxyribonucleosides and deoxyribonucleotides the alkenic C5–C6 double bond of the base moiety is the predominant site of attack (as with the pyrimidine bases, nucleosides and nucleotides) though weak signals from sugar–derived radicals are also observed. Both sugar and base radicals have also been detected for purines (both ribose and deoxyribose derivatives), providing evidence that rapid transfer occurs from the initially formed base radicals to the sugarphosphate backbone.The spectra of the adducts from double-stranded (ds) and single-stranded (ss) DNA are found to vary with pH and ˙OH flux. With dsDNA at pH 7, an anisotropic spectrum due to a slowly tumbling adduct of a high molecular weight polymer radical-adduct is accompanied by a more isotropic signal due to a mobile, low molecular weight radical-adduct, the relative yield of which increases as the pH is lowered. Close similarities are observed between the spectra of dsDNA at pH 2 and ssDNA at pH 7, with a greater occurrence of strand-breakage under these conditions. Treatment of the partially-immobilised DNA adducts formed at pH 6–7 either with the enzyme DNase or with acid releases more mobile adducts enabling information to be obtained about sites of initial attack (at pyrimidine bases) and mechanisms of transfer of damage.

Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic 'mini-genes'.

Several gene transfer techniques that employ 'naked DNA' molecules have recently been developed and numerous gene therapy protocols that make use of 'naked-DNA' have been proposed. We studied the possibility of enhancing the stability of 'naked DNA vectors' and thus also gene transfer and expression efficiencies, by constructing phosphorothioate (PS-) double strand DNA molecules and functional transcription units. We first synthesized short PS-double strand DNA molecules by the annealing of two complementary, 35 nt long, oligonucleotides. The accessibility of DNA modifying enzymes to this molecule was significantly decreased: T4-ligase and kinase activity were respectively reduced up to 1/2 and to 1/6, as compared to the normal phosphodiester molecule. Nucleolytic stability was increased either to purified enzymes (DNase I and Bal31) or to incubations in fresh serum, cell culture medium or in muscle protein extract. Phosphorothioate end-capped complete eukaryotic transcription units (obtained by Taq polymerase amplification with PS-primers) were not significantly protected from nucleolytic attack. On the contrary, synthetic transcription units, 'mini genes', obtained by Taq amplification with 1, 2 or 3 PS-dNTP substitutions, were resistant to DNase I and Bal31 nucleolytic activity. Transcription efficiency, driven by the T7 promoter, was 96.5, 95 and 33.5% (respectively with 1, 2 or 3 substitutions), as compared to the normal phosphodiester molecules.

Nuclear scaffold organization in the X-ray sensitive Chinese hamster mutant cell line, xrs-5.

Nuclear organization was probed in the radiation-sensitive Chinese hamster ovary CHO) cell line, xrs-5, and compared with parental CHO K1 cells using the resinless section technique and DNase I digestions. The resinless section data showed no gross morphological differences in core filaments from the nuclear scaffolds of unirradiated CHO K1 and xrs-5 cells. However, the nuclear scaffolds of irradiated xrs-5 cells (1 Gy) had significantly increased ground substance. Irradiated and unirradiated CHO K1 cell nuclear scaffolds were morphologically identical. These data suggest that both CHO K1 and xrs-5 cell nuclear scaffolds had internal nuclear scaffolding networks that could provide DNA attachment sites. The rate of DNase I digestion of unirradiated CHO K1 and xrs-5 was not significantly different, but the extent of digestion was greater in unirradiated CHO K1 cells that in xrs-5 cells suggesting that less xrs-5 cell chromatin at DNA attachment points is accessible to the enzyme DNase I. The extents of digestion in irradiated (1 Gy) CHO K1 and xrs-5 cell nuclei also differed but the relationship was reversed. The irradiated xrs-5 cell samples were digested to a greater extent compared with CHO K1 cells. These chromatin digestion data suggest that the matrix attachment regions in xrs-5 cells are different from CHO K1 cells. The different DNA attachment organization in the xrs-5 cells may play a role in modulating radiation sensitivity.

Direct potentiometric membrane electrode measurements of heparin binding to macromolecules

AbstractThe use of a newly developed heparin responsive polymeric membrane electrode for directly monitoring the binding of heparin to a variety of other biological macromolecules is examined. Potentiometric titrations of protamine, low‐density lipoprotein (LDL), polybrene, DNase enzyme, and polylysine with either porcine or beef lung heparin can be followed conveniently by the electrode at various solution pHs. Such titrations yield endpoints that are used to determine the stoichiometry for the interaction of the different heparins with these macromolecules. Based on prior heparin calibration curves, the titration data can be recast in Scatchard form to obtain binding constants for these solution‐phase heparin‐macromolecular reactions. The speed and simplicity of the proposed method make it an attractive alternative to classical solid‐phase binding methods or newer homogeneous fluorescent polarization schemes for studying the solution‐phase interaction of heparin with other macromolecules.

Survival of Staphylococcus aureus in Oral Administration Liquid Medicaments and Influence of Count Medium on Survival

The survival of Staphylococcus aureus was studied in 30 oral administration liquid medicaments (15 syrups and 15 solutions) to determine the effectiveness of the preservatives, the influence of the culture medium used in the enumeration of the surviving microorganisms, and the loss of the enzyme coagulase, phosphatase, DNase (deoxyribonuclease), and thermonuclease. Samples were inoculated with 6.3–.5 × 10s viable cells per milliliter and were stored at room temperature for 60 days. Aliquots were taken for analysis at 0,15,22,30, and 60 days after samples were inoculated. The enumeration of S. aureus was made by most probable number method (MPN) with six liquid culture media: triptone soy (TS), TS with 10% NaCI (TSS), TS and TSS with 0.2% catalase, Mannitol salt, and Tellurite-mannitol-glycine. The survival of S. aureus was lower in solutions than in syrups, decreased with the storage time, and depended on the culture medium utilized in the enumeration. Nonselective media were more sensitive than selective ones; that is, a better percentage of recovery was achieved with TS and the catalase medium. The preservative was effective in 93.3% of the samples. Coagulase was the most stable enzyme and phosphatase, DNase, and thermonuclease disappeared during the storage period.

Enzymes and germination of spores of the yeast Saccharomyces cerevisiae

Enzymes in spores of the yeast Saccharomyces cerevisiae were determined and compared with those in vegetative cells. The activities of phosphatase, oxidase and enzymes in the glycolytic bypass were high (about 3–10 fold) in spores, whereas those of dipeptidase, protease, DNase, RNase and TCA cycle enzymes were low in spores, being only 50% of the activities in vegetative cells. Enzymes in the glycolysis, glutathione and acetate metabolic pathways remained unchanged before and after sporulation. Germination of the yeast spores was repressed in the presence of d-aspartate, d-glutamate, d,l-methionine, d,l-cysteine, l-isoleucine, l-histidine and l-threonine, or bivalent metal ions such as Ni 2+, Co 2+ and Zn 2+.

Coupled kinetic analysis of cleavage of DNA by esperamicin and calicheamicin

A coupled kinetic analysis of esperamicin, calicheamicin, and DNase I cleavage of covalently closed circular PM2 DNA has been carried out. Analysis of the optical density data derived from agarose gel electrophoresis experiments shows that esperamicin A,, like the hydrolytic enzyme DNase I, produces mainly single-strand breaks in DNA. These agents cause covalently closed circular form I DNA to be initially converted to nicked circular form I1 DNA. However, the ratio of the rate constant for this process (k,') to that associated with conversion of form I1 to linear form I11 DNA ( k i ) is not consistent with completely random nicking, and some double-strand cleavage may occur. The values of k , ' / k i found for DNase I and esperamicin A, were 5.4 nd 3.9, respectively. The behavior of these agents sharply contrasts with that of esperamicin C and calicheamicin, for which double-strand cleavage of DNA is deduced from the analysis. Although the rate constant for introducing the first break in DNA for calicheamicin is lower than the corresponding rate constant for esperamicin C, the second break (in the opposing strand) is fast for calicheamicin, making it the better double-strand cleaving agent. These drugs are unique among antitumor agents in that a single activation event on the warhead portion produces a double-strand break in DNA without the need to posttreat the DNA with other agents in order to induce a cleavage. The cleavage kinetics are discussed in terms of the structural differences in these unusual anticancer drugs. The newly discovered anticancer drugs esperamicin1*2 and calicheamicin3*' (Figure 1) exhibit high potency against murine tumor lines. These compounds possess sugar groups appended to an unusual 1,5-diyn-3-ene core structure referred to as the uwarheadn. the presence of reducing agents and D N A the warhead undergoes a rearrangement to create a phenylene diradical that causw strand breaks by attacking the sugar groups (1) Golik, J.; Clardy, J.; Dubay, G.; Groenewold, G.; Kawaguchi, H.; Konishi, M.; Krishnan, B.; Ohkuma, H.; Saitoh, K.4.; Doyle, T. W. J. Am. Chcm. Soc. 1987. 109. 3461-3462. Golik. J.: Dubav. G.: Groenwold. 0.: Kawaguchi, H.; Konishi, M.; Krishnan. B.; Ohhma, H.i Saitoh, K.4.; Doyle; *TO whom correspondence should be a d d r d . T. W. Ibid. 1987, 109, 3461-3462. 0002-7863/91/1513-5434$02.50/0 CP 1991 American Chemical Society DNA Cleavage In J . Am. Chem. Soc., Vol. 113. No. 14, I991 5435

A method for determination of iododeoxyuridine substitution of thymidine using reversed-phase high-performance liquid chromatography

An assay for thymidine substitution by iododeoxyuridine (IdUrd) using reversed-phase high-performance liquid chromatography (HPLC) has been developed. Three principal steps in this procedure are: extraction of DNA from cell or tissues, hydrolysis of DNA into deoxynucleosides and separation using HPLC. Approximately 1 microgram of DNA was recovered from 10(5) cells by phenol extraction, and subjected to hydrolysis into deoxynucleosides which required a three-stage DNA digestion using enzymes DNAse I. phosphodiesterase I and alkaline phosphatase. The deoxynucleosides were separated on the Microsorb C18 column with isocratic elution; 90-100% of the DNA was recovered as deoxynucleosides on the column. The method was used to determine quantitatively the percent IdUrd substitution of thymidine in Chinese hamster lung cells in vitro and BA1112 rhabdomyosarcoma in WAG/Rij rats perfused with IdUrd. It was possible to determine the thymidine substitution by IdUrd as small as 1% using a few micrograms of DNA. The close correspondence between the percent substitutions determined by HPLC and those determined by radioactive assay using [125I]-labelled IdUrd, confirmed the accuracy of our HPLC method. The HPLC analysis is especially suitable for the determination of percent IdUrd substitution of thymidine in tissue biopsies from animals used in in vivo experiments or humans undergoing radiation treatment.