Formation Of Guanine Research Articles

Probing interaction of a trilysine peptide with DNA underlying formation of guanine–lysine cross-links: insights from molecular dynamics

DNA-protein cross-links constitute bulky DNA lesions that interfere with the cellular machinery. Amongst these stable covalently tethered adducts, the efficient nucleophilic addition of the free amino group of lysines onto the guanine radical cation has been evidenced. In vitro addition of a trilysine peptide onto a guanine radical cation generated in a TGT oligonucleotide is so efficient that competitive addition of a water molecule, giving rise to 8-oxo-7,8-dihydroguanine, is not observed. This suggests a spatial proximity between guanine and lysine for the stabilization of the prereactive complex. We report all-atom microsecond scale molecular dynamics simulations that probe the structure and interactions of the trilysine peptide (KKK) with two oligonucleotides. Our simulations reveal a strong, electrostatically driven yet dynamic interaction, spanning several association modes. Furthermore, the presence of neighbouring cytosines has been identified as a factor favoring KKK binding. Relying on ab initio molecular dynamics on a model system constituted of guanine and methylammonium, we also corroborate a mechanistic pathway involving fast deprotonation of the guanine radical cation followed by hydrogen transfer from ammonium leaving as a result a nitrogen reactive species that can subsequently cross-link with guanine. Our study sheds new light on a ubiquitous mechanism for DNA-protein cross-links also stressing out possible sequence dependences.

Singlet O2 Oxidation of a Deprotonated Guanine-Cytosine Base Pair and Its Entangling with Intra-Base-Pair Proton Transfer.

We report an experimental and computational study on the 1 O2 oxidation of gas-phase deprotonated guanine-cytosine base pair [G ⋅ C-H]- that is composed of 9HG ⋅ [C-H]- and 7HG ⋅ [C-H]- (pairing 9H- or 7H-guanine with N1-deprotonated cytosine), and 9HG ⋅ [C-H]- _PT and 7HG ⋅ [C-H]- _PT (formed by intra-base-pair proton transfer from the N1 of guanine to the N3 of [C-H]- ). The conformer-averaged reaction product ions and cross section were measured over a center-of-mass collision energy range from 0.1 to 0.5 eV using a guided-ion-beam tandem mass spectrometer. To explore conformation-specific reactivity, collision dynamics of 1 O2 with each of the four [G ⋅ C-H]- conformers was simulated at B3LYP/6-31G(d). Trajectories showed that the 1 O2 oxidation of the base pair entangles with intra-base-pair proton transfer, and prefers to occur in a collision when the base pair adopts a proton-transferred structure; trajectories also indicate that the 9HG-containing base pair favors stepwise formation of 4,8-endoperoxide of guanine, whereas the 7HG-containing base pair prefers concerted formation of guanine 5,8-endoperoxide. Using trajectory results as a guide, potential energy surfaces (PESs) along all possible reaction pathways were established using the approximately spin-projected ωB97XD/6-311++G(d,p)//B3LYP/6-311++G(d,p) method. PESs have not only rationalized trajectory findings but provided more accurate energetics and indicated that the proton-transferred base-pair conformers have lower activation barriers for oxidation than their non-proton-transferred counterparts.

Computational Study of Oxidation of Guanine by Singlet Oxygen (1 Δg ) and Formation of Guanine:Lysine Cross-Links.

Oxidation of guanine in the presence of lysine can lead to guanine-lysine cross-links. The ratio of the C4, C5 and C8 crosslinks depends on the manner of oxidation. Type II photosensitizers such as Rose Bengal and methylene blue can generate singlet oxygen, which leads to a different ratio of products than oxidation by type I photosensitizers or by one electron oxidants. Modeling reactions of singlet oxygen can be quite challenging. Reactions have been explored using CASSCF, NEVPT2, DFT, CCSD(T), and BD(T) calculations with SMD implicit solvation. The spin contamination in open-shell calculations were corrected by Yamaguchi's approximate spin projection method. The addition of singlet oxygen to guanine to form guanine endo- peroxide proceeds step-wise via a zwitterionic peroxyl intermediate. The subsequent barrier for ring closure is smaller than the initial barrier for singlet oxygen addition. Ring opening of the endoperoxide by protonation at C4-O is followed by loss of a proton from C8 and dehydration to produce 8-oxoGox . The addition of lysine (modelled by methylamine) or water across the C5=N7 double bond of 8-oxoGox is followed by acyl migration to form the final spiro products. The barrier for methylamine addition is significantly lower than for water addition and should be the dominant reaction channel. These results are in good agreement with the experimental results for the formation of guanine-lysine cross-links by oxidation by type II photosensitizers.

Ion-selective formation of a guanine quadruplex on DNA origami structures.

DNA origami nanostructures are a versatile tool that can be used to arrange functionalities with high local control to study molecular processes at a single-molecule level. Here, we demonstrate that DNA origami substrates can be used to suppress the formation of specific guanine (G) quadruplex structures from telomeric DNA. The folding of telomeres into G-quadruplex structures in the presence of monovalent cations (e.g. Na(+) and K(+)) is currently used for the detection of K(+) ions, however, with insufficient selectivity towards Na(+). By means of FRET between two suitable dyes attached to the 3'- and 5'-ends of telomeric DNA we demonstrate that the formation of G-quadruplexes on DNA origami templates in the presence of sodium ions is suppressed due to steric hindrance. Hence, telomeric DNA attached to DNA origami structures represents a highly sensitive and selective detection tool for potassium ions even in the presence of high concentrations of sodium ions.

A Biophysical Study of the G-Quadruplex-Insulin Interaction

The formation of guanine (G)-quadruplex structures in the guanine-rich tandem repeats of the insulin-linked polymorphic region (ILPR) have potential effects on transcription of the insulin gene. Recent studies demonstrate that the ILPR G-quadruplexes can bind to insulin. The energetics of the binding between insulin and the G-quadruplexes formed by the most common ILPR repeat sequence have also been characterized in prior work. We have studied the proton transfer involved in the interaction between insulin and this DNA sequence by conducting isothermal titration calorimetry (ITC) experiments in various buffers and analyzing the observed enthalpy change. The transcriptional activity of a number of ILPR repeat sequences, including the consensus sequence, have been measured previously. Transcriptional activity for less common repeats is significantly lower than that of the consensus sequence but can be increased substantially by varying only one or two nucleotides. To determine the potential role of G-quadruplex formation and stability in regulating transcription, we have studied the second and third most common ILPR repeats as well as their variant sequences that exhibit increased transcriptional activity. ITC was used to characterize the energetics of the binding interaction between insulin and each of the four ILPR repeat sequences. The bulk thermodynamic measurements performed at various temperatures from 20 - 37 degrees Celsius provide insight into these biomolecular interactions.

Characterizing the Biomolecular Interactions between Insulin and G-Quadruplex DNA

The formation of guanine (G)-quadruplex structures in the guanine-rich tandem repeats of the insulin-linked polymorphic region (ILPR) is linked to transcriptional effects on the insulin gene. Recent studies demonstrate that the ILPR G-quadruplexes can bind to insulin while the energetics of this interaction with the most common ILPR repeat sequences have been characterized. Studies have also measured the transcriptional activity of less common ILPR repeats. Transcriptional activity for these repeats is significantly lower than that of the consensus sequence but can be increased substantially by varying only one or two nucleotides. To determine the potential role of G-quadruplex formation and stability in regulating transcription, we have studied the second and third most common ILPR repeats as well as their variant sequences that exhibit increased transcriptional activity. Circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC) have been used to characterize the binding interaction between insulin and each of the four ILPR repeat sequences. The bulk thermodynamic measurements performed at various temperatures from 20 - 37 degrees Celsius provide insight into these biomolecular interactions. Correction et al.Biophysical JournalApril 16, 2013In Brief2013. Characterizing the Biomolecular Interactions between Insulin and G-Quadruplex DNA. Nicole L. Michmerhuizen, Christine M. Timmer, Kumar Sinniah. Biophys. J. 104(2)s: 419a. Full-Text PDF Open Archive

The Binding of G‐Quadruplex DNA to Insulin: From Single Molecule to Bulk‐Methods

The formation of guanine (G)‐quadruplex structures in the guanine‐rich tandem repeats of the insulin‐linked polymorphic region (ILPR) is linked to transcriptional effects on the insulin gene. Recent studies demonstrate that the ILPR G‐quadruplexes can bind to insulin, and while this may impact the transcription of insulin, little is known about the binding mechanism. We have performed single molecule atomic force microscopy (AFM), differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and fluorescence quenching spectroscopy to characterize the binding interaction between G‐quadruplex DNA and insulin. In this presentation, I will describe the bulk thermodynamic measurements performed at various temperatures from 10 to 37 °C. The experimental results based on KD demonstrate a moderate affinity by the ILPR consensus sequence to insulin. In addition to thermodynamic measurements, kinetic off‐rates were measured by single molecule force‐pulling measurements and these results will be discussed in relation to kinetic off‐rates measured from the unfolding of G‐quadruplex DNA.

Characterizing the Binding of G-Quadruplex DNA to Insulin by Single Molecule and Bulk Ensemble Methods

The formation of guanine (G)-quadruplex structures in the guanine-rich tandem repeats of the insulin-linked polymorphic region (ILPR) is linked to transcriptional effects on the insulin gene. Recent studies demonstrate that the ILPR G-quadruplexes can bind to insulin, and while this may impact the transcription of insulin, little is known about the binding mechanism. We have performed single molecule atomic force microscopy (AFM), differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and fluorescence quenching spectroscopy measurements to characterize the binding interaction between G-quadruplex DNA and insulin.

Stability of compounded thioguanine oral suspensions

PURPOSE. Updated information on the stability of compounded thioguanine oral suspensions prepared with currently available ingredients, as well as results of testing to determine if the addition of an antioxidant could extend shelf life by inhibiting formation of guanine, are presented. METHODS. Using triturated thioguanine tablets, three compounded suspensions were prepared: (1) a reference formulation containing methylcellulose and simple syrup, (2) an equivalent formulation using Ora-Plus and Ora-Sweet, and (3) an antioxidant-containing formulation prepared by adding ascorbic acid to the equivalent formulation. The compounded batches were stored at room temperature (19-23 °C). The chemical stability of the suspensions was evaluated immediately after compounding and at weekly intervals by a validated liquid chromatography-mass spectrometry (LCMS) assay method; physical stability was evaluated by regular visual checks and weekly pH testing. RESULTS. As demonstrated by serial LCMS testing, mean thioguanine levels in sampled batches of all three suspensions remained above accepted standards and mean guanine formation remained within acceptable limits for up to 63 days. The addition of ascorbic acid appeared to slow guanine formation but did not significantly extend the shelf life of the suspension. CONCLUSION. Compounded oral suspensions of thioguanine 20 mg/mL exhibited acceptable chemical and physical stability for up to nine weeks at 19-23 °C. The addition of ascorbic acid at a concentration of 0.1% to the suspension was not effective in consistently increasing the shelf life of the thioguanine suspensions.

Investigating the Binding of Insulin to G-Quadruplex DNA by Differential Scanning Calorimetry

The formation of guanine (G)-quadruplex in the guanine-rich tandem repeats of the insulin-linked polymorphic region (ILPR) is linked to transcriptional effects on the insulin gene. Recent studies demonstrate that these G-quadruplexes can bind insulin, and while this may impact the transcription of insulin, little is known about the binding mechanism. We have performed differential scanning calorimetry to characterize the binding interaction between insulin and G-quadruplex DNA. The consensus quadruplex ILPR sequence shows an unfolding temperature at 93.2 degrees celsius, while this peak is absent in a scrambled version of this sequence. The unfolding temperature of an insulin-G-quadruplex mix showed ∼ 3 degrees celsius change in the Tm for the insulin peak in comparison with the unfolding of insulin alone, and a ∼1 degree celsius change from insulin-scrambled DNA mix. These results provide additional support for the binding of insulin to G-quadruplex DNA. Experiments are currently in progress to measure the binding constant of this interaction by isothermal titration calorimetry.

A Single Molecule Force Spectroscopy Study of the Binding Interaction Between Insulin and G-Quadruplex DNA

The formation of guanine (G)-quadruplex in the guanine-rich tandem repeats of the insulin-linked polymorphic region (ILPR) is linked to transcriptional effects on the insulin gene. Recent studies demonstrate that these G-quadruplexes can bind insulin, and while this may impact the transcription of insulin, little is known about the binding mechanism. A single molecule force spectroscopy study was performed to examine the selective binding of insulin to the ILPR G-quadruplex DNA. In this study, the insulin was covalently attached to a flat gold surface while the quadruplex DNA was attached to an AFM probe. The rupture force between insulin and quadruplex DNA was measured at various force loading rates. To confirm the specificity of the binding, control studies were performed by blocking the tethered G-quadruplex with “free insulin” in solution. Additional control studies were performed with a shorter DNA sequence incapable of forming a G-quadruplex on the AFM tip and a scrambled DNA sequence of the ILPR sequence. Results from the dynamic force-pulling studies are described based on the Bell-Evans and Dudko-Hummer models.

Part II. Mechanism of Formation of Guanine as one of the Major Products in the 254 nm Photolysis of Guanine Derivatives: Concentration and pH Effects

The quantum yield of formation of guanine (Gua), one of the major products formed in the 254 nm steady-state photolysis of the following Gua derivatives (9-ethylguanine [9Et-Gua], deoxyguanosine [dGuo], guanosine [Guo], guanosine 5′-monophosphate [GMP]) was determined under different conditions. The formation yield increases in the following order: 9Et-Gua < dGuo ∼ Guo < GMP. Electron scavengers or triplet quenchers were incorporated into the irradiated solutions with the purpose of reacting specifically with postulated or previously identified intermediates in the photolysis of Gua derivatives. A decrease in the yield of formation of Gua is observed with increasing electron scavenger concentration or with pH. These results suggest a major contribution from Gua-derivative electron adducts on the process of N(9)–R bond breakage. At an acidic pH a tautomer of the radical cation (E) of Guo is proposed as the precursor for Gua formation. The relative efficiency of the radical cation for initiating the release of free Gua depends on the pH of the solution, being less than 39% in neutral pH. Reactions from OH radicals may also result in base release as shown using N2O as additive. Finally, the formation of aggregates by the bases at concentrations used, plays an important role in the deactivation of the excited states and also in the probability of formation of the free base.

Adenine-containing DNA-DNA cross-links of antitumor nitrogen mustards.

Nitrogen mustards (NMs) are useful chemotherapeutic agents in the treatment of lymphoma, leukemia, multiple myeloma, and ovarian carcinoma. The antitumor activity of NMs has been attributed to their ability to cross-link the twin strands of DNA. The resulting bifunctional lesions, if not repaired, can inhibit DNA replication and transcription, eventually leading to cell cycle arrest, apoptosis, and the inhibition of tumor growth. The predominant bifunctional DNA lesions of NM have been reported to involve the distal guanine bases in the opposite strands of 5'-GNC sequences. In the present work, the formation of guanine-adenine and adenine-adenine adducts of N,N-bis(2-chloroethyl)methylamine (mechlorethamine) in double-stranded DNA is demonstrated. Guanine-adenine cross-links of mechlorethamine were identified as N-(2-[N3-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine (N3A-N7G-EMA), N-(2-[N1-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine, and N-(2-[N(6)-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine. All three adducts were produced interstrand, while N3A-N7G-EMA was the dominant intrastrand G-A cross-link. The prevalent adenine-adenine mechlorethamine lesions have the structure of N,N-bis(2-[N3-adenyl]ethyl)methylamine (bis-N3A-EMA). DNA-derived lesions have the same HPLC retention times, UV spectra, and MS/MS fragmentation patterns as the authentic standards prepared independently. bis-N3A-EMA lesions were produced in a concentration-dependent manner in calf thymus DNA treated with increasing amounts of mechlorethamine. Furthermore, HPLC-ESI-MS/MS analysis was used to demonstrate the formation of analogous N3-N3 adenine lesions in DNA treated with aromatic nitrogen mustards, N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid and L-phenylalanine mustard. The presence of cross-linked adenine-adenine lesions may explain the enhanced cytotoxicity and mutagenicity of NMs in cells deficient in N3-alkyladenine glycosylase.

Modified guanines representing O(6)-alkylation by the cyclophosphamide metabolites acrolein and chloroacetaldehyde: synthesis, stability, and ab initio studies.

Alkylation of DNA by acrolein and/or chloroacetaldehyde may result in the mutations that lead to the therapy-induced leukemia associated with cyclophosphamide (and ifosfamide) treatment. O(6)-(n-Propanalyl)guanine (O(6)-PAG) and O(6)-(ethanalyl)guanine (O(6)-EAG) were synthesized for use as authentic standards in investigations of DNA alkylation by acrolein and chloroacetaldehyde, respectively. Preparation of the O-methyl oximes of these aldehydes aided in confirming the structural assignments of O(6)-PAG and O(6)-EAG. HPLC was used to study the stability of O(6)-PAG under a variety of conditions. The decomposition of O(6)-PAG was attributed to an alpha,beta-elimination reaction resulting in the formation of guanine and acrolein. In 0.1 M phosphate-DMSO (9:1), O(6)-PAG (1-10 mM) had a half-life of approximately 1 h (pH 7.4, 37 degrees C). In 0.05 M Tris-DMSO (9:1), the apparent half-life of O(6)-PAG (1-10 mM) was approximately 16 h (pH 7.4, 37 degrees C). The increased lifetime under the latter conditions was attributed to a reversible reaction between Tris and the aldehydic functionality of O(6)-PAG to give a more stable oxazolidine. Under conditions similar to those that would be used for hydrolysis of DNA [0.1 M HCl-DMSO (98:2), pH 1.3, 70 degrees C, 30 min], there was an estimated 10-35% loss of O(6)-PAG. Under the same conditions, O(6)-EAG had apparent half-lives of 6.6 h (phosphate-DMSO) and 2.5 days (Tris-DMSO) and the estimated loss at pH 1.3 over 30 min (70 degrees C) was 15-20%. Ab initio quantum chemical calculations were used to understand the energy factors that underlie the occurrence of O- versus N-alkylations as well as possible, subsequent intramolecular cyclizations. Simulations of the free energies of reactions between acrolein and guanine indicated that N-alkylation was favored over O(6)()-alkylation and that cyclizations to tautomers were most favorable if they involved the N-1 or NH(2) positions.

Mechanism of formation of guanine as one of the major products in the 254 nm photolysis of guanine derivatives: concentration and pH effects.

The quantum yield of formation of guanine (Gua), one of the major products formed in the 254 nm steady-state photolysis of the following Gua derivatives (9-ethylguanine [9Et-Gua], deoxyguanosine [dGuo], guanosine [Guo], guanosine 5'-monophosphate [GMP]) was determined under different conditions. The formation yield increases in the following order: 9Et-Gua < dGuo approximately Guo < GMP. Electron scavengers or triplet quenchers were incorporated into the irradiated solutions with the purpose of reacting specifically with postulated or previously identified intermediates in the photolysis of Gua derivatives. A decrease in the yield of formation of Gua is observed with increasing electron scavenger concentration or with pH. These results suggest a major contribution from Gua-derivative electron adducts on the process of N(9)-R bond breakage. At an acidic pH a tautomer of the radical cation (E) of Guo is proposed as the precursor for Gua formation. The relative efficiency of the radical cation for initiating the release of free Gua depends on the pH of the solution, being less than 39% in neutral pH. Reactions from OH radicals may also result in base release as shown using N2O as additive. Finally, the formation of aggregates by the bases at concentrations used, plays an important role in the deactivation of the excited states and also in the probability of formation of the free base.

Hydroxylation and deglycosylation of 2′-deoxyguanosine in the presence of magnesium and nickel

Nickel (Ni), a carcinogenic and genotoxic metal, has been shown to enhance deglycosylation and hydroxylation of 2′-deoxyguanosine (dG) that has been caused by ascorbic acid and H 2O 2. There is evidence that Mg is a competitive antagonist of the toxicological effects of Ni. A factorial design was used to examine the interactive influence of Mg and Ni on the deglycosylation and hydroxylation of dG under a range of pH conditions in which ascorbate (Ascb) and H 2O 2 were added. Formation of guanine (Gu) (deglycosylation) and 8-hydroxy-2′-deoxyguanosine (8-OH-dG) (hydroxylation) appeared in large amounts in samples in which both H 2O 2 and Ascb were present. The largest amounts of Gu appeared where both Ni or magnesium (Mg) were present. When Mg alone was present, the amounts of Gu was intermediate between these two. Slightly less 8-OH-dG was formed where only Mg was present. The reaction mixtures were more sensitive to the pH than to the respective presence or absence of metals. At slightly acid or neutral pH (6.2–7.0) large amounts of both Gu and 8-OH-dG were formed. Gu formation decreased dramatically between pH 7.0 and 7.2. There was no 8-OH-dG formed at pH 7.8 and only small amounts at pH 7.6. The formation of 8-OH-dG was generally less where Mg was present. When Ni was absent, 8-OH-dG formation was greater in the pH 6.8 mixtures. The formation of Gu and 8-OH-dG from 2′-deoxyguanosine are directly a function of pH. Slight changes in pH greatly effected the formation of these biomarkers of oxidatively damaged DNA. Additional research is needed to determine if this is a cause or effect, i.e. does pH enhance toxicity conditions, thus permitting formation of 8-OH-dG, or does pH permit the reaction to proceed.

Molecular Mechanisms of Radiation Induced Dna Damage: H-Addition to Bases, Direct Ionization and Double Strand Break

Structures and properties of G.(-H) and of TH. were obtained from quantum mechanical calculations. New AMBER parameters for these radicals were obtained to fit their structures and charge distributions. Molecular mechanics simulations of the conformational changes induced in a 12-mer of DNA, d(CGCGAATTCGCG), by these radicals show that the distances between the base and the C2' of the sugar becomes shorter. Such changes suggest that the base radical can abstract the H2' and transfer the radical from the base to the sugar. Once the radical becomes centered on the sugar a strand break can follow. A simultaneous formation of guanine and thymine radicals on opposite strands may lead to a double strand break.

Physicochemical Properties of Carbovir, a Potential Anti-HIV Agent

(±)-Carbovir [(±)-9-[4α-(hydroxymethyl)-cyclopent-2-ene-1α-yl]guanine; NSC 614846] is a novel carbocyclic nucleoside analogue which has been shown to be a potent and selective inhibitor of HIV in vitro. As part of an effort to develop a parenteral formulation for subsequent clinical and toxicological evaluation of this compound, the aqueous solution stability of carbovir as a function of pH and temperature and various physicochemical properties of carbovir including its pKa, solubility versus pH and solvent composition, and octanol–water partition coefficient have been examined. Ultraviolet spectrophotometry indicated that carbovir has pKa values of 3.15 and 9.68, respectively, at 25°C and 0.01 ionic strength. The aqueous solubility of carbovir over the pH range 7–10.5 was consistent with that expected of a weak acid with a pKa of 9.65 and an intrinsic solubility of 1.24 mg/mL. Due to the limited solubility of carbovir at physiological pH, methods for solubilizing carbovir in aqueous solution were explored, including propylene glycol–water cosolvents and complexation with hydroxypropyl-β-cyclodextrin. As expected for carbovir, a semipolar compound with an octanol–water partition coefficient of 0.29, propylene glycol:water cosolvents were not highly effective in enhancing solubility. Complex formation between carbovir and 2-hydroxypropyl-β-cyclodextrin was found to be more effective, with a K1:1 of 105M−1 for the complexation. The pH profiles generated at 50, 70, and 90°C were accounted for by acid-catalyzed degradation at low pH leading to the formation of guanine and a neutral degradation pathway which dominates above pH 4. Prototype lyophilized formulations containing (after reconstitution) 10 mg/mL of carbovir at a pH of 10.6 were developed and evaluated.

Removal of O6-methylguanine from DNA by human liver fractions.

In in vitro assays using methylated DNAs as substrates, human liver fractions were shown to be able to catalyze the removal of O6-methylguanine. The amount of removal was proportional to the amount of protein added, and the loss of O6-methylguanine occurred with stoichiometric formation of guanine in the DNA and S-methylcysteine in protein. This indicates that human liver contains a protein similar to that previously found in bacteria exposed to alkylating agents. This protein acts as a transmethylase, transferring the intact methyl group from O6-methylguanine in DNA to a cysteine residue on that protein. A similar activity is present in rodent liver, but it was found that human liver was about 10 times more active in carrying out this reaction. In contrast, there was no difference between the human and rat liver extracts in catalyzing the loss of another methylation product, 7-methylguanine, from alkylated DNA. The liver is the organ most likely to be alkylated after exposure to exogenous potential alkylating agents such as dimethylnitrosamine. The present results show that human liver has a significant capacity to repair O6-methylguanine in DNA, which has been implicated as a critical product in carcinogenesis and mutagenesis.

Alterations in purine nucleotide biosynthesis induced by 2-amino-6-chloropurine

Exposure of sarcoma 180 ascites cells to 2-amino-6-chloropurine (6-C1G) in vivo resulted in several metabolic lesions in the pathways of purine nucleotide biosynthesis. Pronounced inhibition of the incorporation of glycine-2- 14C into the guanine and adenine nucleotides of the nucleic acids, coupled with blockade of the utilization of 4-amino-5-imidazolecarboxamide-2- 14C solely for the formation of guanine nucleotides, indicated the presence of a site of inhibition on the pathway of purine nucleotide formation de novo. The conversion of hypoxanthine-8- 14C to nucleic acid guanine was markedly decreased by 6-CIG, whereas the utilization of this labeled precursor for the formation of nucleic acid adenine was not lowered by this agent. Under these conditions neither the incorporation of xanthine-8- 14C nor of guanine-8- 14C into the guanine moiety of the nucleic acids was depressed, indicating that 6-CIG or a derivative thereof inhibited the conversion of inosine 5′-phosphate to xanthosine 5′-phosphate. Blockade of this site was confirmed by the finding that 2-amino-6-chloropurine ribonucleoside 5′-phosphate inhibited the activity of inosine 5′-phosphate dehydrogenase. Molar equivalent levels of 2-amino-6-chloropurine ribonucleoside and 2-amino-6-chloropurine ribonucleoside triacetate caused a degree of inhibition of the conversion of hypoxanthine-8- 14C to guanine nucleotides comparable to that produced by 6-C1G. 2-Amino-6-iodopurine ribonucleoside was at least as active an inhibitor of the formation of nucleic acid guanine as the chloro-derivatives.