Mechanistic clues to the mutagenicity of alkylated DNA bases: a theoretical study

Experiment indicates that the N7-guanine site in DNA is not "promutagenic" (mutation-inducing) on alkylation, while the O6-guanine and O4-thymine sites are so. These differences in nucleic acid template activity are attributed to corresponding differences in acidity of the Watson-Crick hydrogen bonding protons. Mechanistic indicators for ease of Watson-Crick proton loss are calculated using molecular orbital theory for DNA bases alkylated at the N7-guanine, O6-guanine and O4-thymine sites. Their values point to a definite favouring of the proton loss for the O-alkylated bases compared to the N7-alkylguanines. This may suggest the possibility that, at biological pH, the O-alkylated bases deprotonate readily while the N7-alkylguanines do not, thus accounting for observed differences in promutagenicity and nucleic acid template activity.     

Repair of O6-alkylguanine by alkyltransferases

The predominant pathway for the repair of O⁶-methylguanine in DNA is via the activity of an alkyltransferase protein that transfers the methyl group to a cysteine acceptor site on the protein itself. This review article describes recent studies on this alkyltransferase. The protein repairs not only methyl groups but also 2-chloroethyl-, benzyl- and pyridyloxobutyl-adducts. It acts on double-stranded DNA by flipping the O⁶-guanine adduct out of the DNA helix and into a binding pocket. The free base, O⁶-benzylguanine, is able to bind in this pocket and react with the cysteine, rendering it an effective inactivator of mammalian alkyltransferases. The alkylated form of the protein is rapidly degraded by the ubiquitin/proteasomal system. Some tumor cells do not express alkyltransferase despite having an intact gene. Methylation of key sites in CpG-rich islands in the promoter region are involved in this silencing and a change in the nuclear localization of an enhancer binding protein may also contribute. The alkyltransferase promoter contains Sp1, GRE and AP-1 sites and is slightly inducible by glucocorticoids and protein kinase C activators. There is a complex relationship between p53 and alkyltransferase expression with p53 mediating a rise in alkyltransferase in response to ionizing radiation but having no clear effect on basal levels. DNA adducts at the O⁶-position of guanine are a major factor in the carcinogenic, mutagenic, apoptopic and clastogenic actions of methylating agents and chloroethylating agents. Studies with transgenic mice in which alkyltransferase levels are increased or decreased confirm the importance of this repair pathway in protecting against carcinogenesis. Alkyltransferase activity in tumors protects them from therapeutic agents such as temozolomide and BCNU. This resistance is abolished by O⁶-benzylguanine and this drug is currently in clinical trials to enhance cancer chemotherapy by these agents. Studies are in progress to reduce the toxicity of such therapy towards the bone marrow by gene therapy to express alkyltransferases with mutations imparting resistance to O⁶-benzylguanine at high levels in marrow stem cells. Several polymorphisms in the human alkyltransferase gene have been identified but the significance of these in terms of alkyltransferase action is currently unknown.     

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.     

In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites

We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo[a]pyrene. DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent. The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV. We conclude that the alkali-labile sites formed in vivo by benzo[a]pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N²-guanine adducts. This finding questions the involvement of apurinic sites in the mutagenic activity of benzo[a]pyrene.     

Correlation of the guanosine exchangeable and nonexchangeable base protons in 13C-/15N-labeled RNA with an HNC-TOCSY-CH experiment

Summary A triple resonance HNC-TOCSY-CH experiment is described for correlating the guanosine imino proton and H8 resonances in ¹³C-/¹⁵N-labeled RNAs. Sequential assignment of the exchangeable imino protons in Watson-Crick base pairs is generally made independently of the assignment of the nonexchangeable base protons. This H(NC)-TOCSY-(C)H experiment makes it possible to unambiguously link the assignment of the guanosine H8 resonances with sequential assignment of the guanosine imino proton resonances. 2D H(NC)-TOCSY-(C)H spectra are presented for two isotopically labeled RNAs, a 30-nucleotide lead-dependent ribozyme known as the leadzyme, and a 48-nucleotide hammerhead ribozyme-RNA substrate complex. The results obtained on these two RNAs demonstrate that this HNC-TOCSY-CH experiment is an important tool for resonance assignment of isotopically labeled RNAs.     

Effect O6‐guanine alkylation on DNA flexibility studied by comparative molecular dynamics simulations

Alkylation of guanine at the O6 atom is a highly mutagenic DNA lesion because it alters the coding specificity of the base causing G:C to A:T transversion mutations. Specific DNA repair enzymes, e.g. O⁶‐alkylguanin‐DNA‐Transferases (AGT), recognize and repair such damage after looping out the damaged base to transfer it into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The O⁶‐alkylation of guanine may change the deformability of DNA which may facilitate the initial binding of a repair enzyme at the damaged site. In order to characterize the effect of O⁶‐methyl‐guanine (O⁶‐MeG) containing base pairs on the DNA deformability extensive comparative molecular dynamics (MD) simulations on duplex DNA with central G:C, O⁶‐MeG:C or O⁶‐MeG:T base pairs were performed. The simulations indicate significant differences in the helical deformability due to the presence of O⁶‐MeG compared to regular undamaged DNA. This includes enhanced base pair opening, shear and stagger motions and alterations in the backbone fine structure caused in part by transient rupture of the base pairing at the damaged site and transient insertion of water molecules. It is likely that the increased opening motions of O⁶‐MeG:C or O⁶‐MeG:T base pairs play a decisive role for the induced fit recognition or for the looping out of the damaged base by repair enzymes. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 23–32, 2015.     

Congenic mapping and candidate gene analysis for streptozotocin-induced diabetes susceptibility locus on mouse chromosome 11

Streptozotocin (STZ) has been widely used to induce diabetes in rodents. Strain-dependent variation in susceptibility to STZ has been reported; however, the gene(s) responsible for STZ susceptibility has not been identified. Here, we utilized the A/J-11^SM consomic strain and a set of chromosome 11 (Chr. 11) congenic strains developed from A/J-11^SM to identify a candidate STZ-induced diabetes susceptibility gene. The A/J strain exhibited significantly higher susceptibility to STZ-induced diabetes than the A/J-11^SM strain, confirming the existence of a susceptibility locus on Chr. 11. We named this locus Stzds1 (STZ-induced diabetes susceptibility 1). Congenic mapping using the Chr. 11 congenic strains indicated that the Stzds1 locus was located between D11Mit163 (27.72 Mb) and D11Mit51 (36.39 Mb). The Mpg gene, which encodes N-methylpurine DNA glycosylase (MPG), a ubiquitous DNA repair enzyme responsible for the removal of alkylated base lesions in DNA, is located within the Stzds1 region. There is a close relationship between DNA alkylation at an early stage of STZ action and the function of MPG. A Sanger sequence analysis of the Mpg gene revealed five polymorphic sites in the A/J genome. One variant, p.Ala132Ser, was located in a highly conserved region among rodent species and in the minimal region for retained enzyme activity of MPG. It is likely that structural alteration of MPG caused by the p.Ala132Ser mutation elicits increased recognition and excision of alkylated base lesions in DNA by STZ.

     

Oligonucleotide probes containing pyrimidine analogs reveal diminished hydrogen bonding capacity of the DNA adduct O6-methyl-G in DNA duplexes

Oligonucleotide hybridization probes containing nucleoside analogs offer a potential strategy for binding specific DNA sequences that bear pro-mutagenic O⁶-G alkylation adducts. To optimize O⁶-Me-G-targeting probes, an understanding of how base pairs with O⁶-Me-G are stabilized is needed. In this study, we compared the ability of O⁶-Me-G and G to hydrogen bond with three pyrimidine-like nucleobases (Z, 4-thio-U, and 3-deaza-C) bearing varied hydrogen bond donor and acceptor groups. We found that duplexes containing the pyrimidine analog nucleoside:G pairs were more thermodynamically stable than those containing pyrimidine analog nucleoside:O⁶-alkyl-G pairs. Thus, hydrogen bonding alone was not sufficient to impart selectivity to probes that target O⁶-G alkylation adducts in DNA.     

Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage

Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O⁶-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-N′-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced γH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.     

Biological effects of incorporation of O-methyldeoxyguanosine into Chinese hamster V79 cells

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O⁶-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O⁶-methyl-[8-³H]deoxyguanosine (2.4 μM, sp. act. 14 500 Ci/mole) showed an incorporation level of 4 × 10⁻⁸ nucleotides. When 1000-fold higher concentrations were employed (3–15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O⁶-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O⁶-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O⁶-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.     

Formation and subsequent removal of O6-methylguanine from deoxyribonucleic acid in rat liver and kidney after small doses of dimethylnitrosamine

1. The amounts of 7-methylguanine and O⁶-methylguanine present in the DNA of liver and kidney of rats 4h and 24h after administration of low doses of dimethylnitrosamine were measured. 2. O⁶-Methylguanine was rapidly removed from liver DNA so that less than 15% of the expected amount (on the basis of 7-methylguanine found) was present within 4h after doses of 0.25mg/kg body wt. or less. Within 24h of administration of dimethylnitrosamine at doses of 1mg/kg or below, more than 85% of the expected amount of O⁶-methylguanine was removed. Removal was most efficient (defined in terms of the percentage of the O⁶-methylguanine formed that was subsequently lost within 24h) after doses of 0.25–0.5mg/kg body wt. At doses greater or less than this the removal was less efficient, even though the absolute amount of O⁶-methylguanine lost during 24h increased with the dose of dimethylnitrosamine over the entire range of doses from 0.001 to 20mg/kg body wt. 3. Alkylation of kidney DNA after intraperitoneal injections of 1–50μg of dimethylnitrosamine/kg body wt. occurred at about one-tenth the extent of alkylation of liver DNA. Removal of O⁶-methylguanine from the DNA also took place in the kidney, but was slower than in the liver. 4. After oral administration of these doses of dimethylnitrosamine, the alkylation of kidney DNA was much less than after intraperitoneal administration and represented only 1–2% of that found in the liver. 5. Alkylation of liver and kidney DNA was readily detectable when measured 24h after the final injection in rats that received daily injections of 1μg of [³H]dimethylnitrosamine/kg for 2 or 3 weeks. After 3 weeks, O⁶-methylguanine contents in the liver DNA were about 1% of the 7-methylguanine contents. The amount of 7-methylguanine in the liver DNA was 10 times that in the kidney DNA, but liver O⁶-methylguanine contents were only twice those in the kidney. 6. Extracts able to catalyse the removal of O⁶-methylguanine from alkylated DNA in vitro were isolated from liver and kidney. These extracts did not lead to the loss of 7-methylguanine from DNA. 7. The possible relevance of the formation and removal of O⁶-methylguanine in DNA to the risk of tumour induction by exposure to low concentrations of dimethylnitrosamine is discussed.     

6‐phenylpyrrolocytosine as a fluorescent probe to examine nucleotide flipping catalyzed by a DNA repair protein

Cellular exposure to tobacco‐specific nitrosamines causes formation of promutagenic O⁶‐[4‐oxo‐4‐(3‐pyridyl)but‐1‐yl]guanine (O⁶‐POB‐G) and O⁶‐methylguanine (O⁶‐Me‐G) adducts in DNA. These adducts can be directly repaired by O⁶‐alkylguanine‐DNA alkyltransferase (AGT). Repair begins by flipping the damaged base out of the DNA helix. AGT binding and base‐flipping have been previously studied using pyrrolocytosine as a fluorescent probe paired to the O⁶‐alkylguanine lesion, but low fluorescence yield limited the resolution of steps in the repair process. Here, we utilize the highly fluorescent 6‐phenylpyrrolo‐2′‐deoxycytidine (6‐phenylpyrrolo‐C) to investigate AGT‐DNA interactions. Synthetic oligodeoxynucleotide duplexes containing O⁶‐POB‐G and O⁶‐Me‐G adducts were placed within the CpG sites of codons 158, 245, and 248 of the p53 tumor suppressor gene and base‐paired to 6‐phenylpyrrolo‐C in the opposite strand. Neighboring cytosine was either unmethylated or methylated. Stopped‐flow fluorescence measurements were performed by mixing the DNA duplexes with C145A or R128G AGT variants. We observe a rapid, two‐step, nearly irreversible binding of AGT to DNA followed by two slower steps, one of which is base‐flipping. Placing 5‐methylcytosine immediately 5′ to the alkylated guanosine causes a reduction in rate constant of nucleotide flipping. O⁶‐POB‐G at codon 158 decreased the base flipping rate constant by 3.5‐fold compared with O⁶‐Me‐G at the same position. A similar effect was not observed at other codons.     

The Escherichia coli 3-methyladenine DNA glycosylase AlkA has a remarkably versatile active site

3-Methyladenine DNA glycosylase II (AlkA) from Escherichia coli is induced in response to DNA alkylation, and it protects cells from alkylated nucleobases by catalyzing their excision. In contrast to the highly specific 3-methyladenine DNA glycosylase I (E. coli TAG) that catalyzes the excision of 3-methyl adducts of adenosine and guanosine from DNA, AlkA catalyzes the excision of a wide variety of alkylated bases including N-3 and N-7 adducts of adenosine and guanosine and O(2) adducts of thymidine and cytidine. We have investigated how AlkA can recognize a diverse set of damaged bases by characterizing its discrimination between oligonucleotide substrates in vitro. Similar rate enhancements are observed for the excision of a structurally diverse set of substituted purine bases and of the normal purines adenine and guanine. These results are consistent with a remarkably indiscriminate active site and suggest that the rate of AlkA-catalyzed excision is dictated not by the catalytic recognition of a specific substrate but instead by the reactivity of the N-glycosidic bond of each substrate. Damaged bases with altered base pairing have a modest advantage, as mismatches are processed up to 400-fold faster than stable Watson-Crick base pairs. Nevertheless, AlkA does not effectively exclude undamaged DNA from its active site. The resulting deleterious excision of normal bases is expected to have a substantial cost associated with the expression of AlkA.     

Site-resolved energetics in DNA triple helices containing G*TA and T*CG triads

Recognition of specific sites in double-helical DNA by triplex-forming oligonucleotides has been limited until recently to sites containing homopurine-homopyrimidine sequences. G*TA and T*CG triads, in which TA and CG base pairs are specifically recognized by guanine or by thymine, have now extended this recognition code to DNA target sites of mixed base sequences. In the present work, we have obtained a characterization of the stabilities of G*TA and T*CG triads, and of the effects of these triads upon canonical triads, in triple-helical DNA. The three DNA triplexes investigated are formed by the folding of the 31-mers d(GAAXAGGT(5)CCTYTTCT(5)CTTZTCC) with X = G, T, or C, Y = C, A, or G, and Z = C, G, or T. We have measured the exchange rates of imino protons in each triad of the three triplexes using nuclear magnetic resonance spectroscopy. The exchange rates are used to map the local free energy of structural stabilization in each triplex. The results indicate that the stability of Watson-Crick base pairs in the G*TA and T*CG triads is comparable to that of Watson-Crick base pairs in canonical triads. The presence of G*TA and T*CG triads, however, destabilizes neighboring canonical triads, two or three positions removed from the G*TA/T*CG site. Moreover, the long-range destabilizing effects induced by the T*CG triad are larger than those induced by the G*TA triad. These findings reveal the molecular basis for the lower overall stability of G*TA- and T*CG-containing triplexes.     

Proton exchange and base pair opening in a DNA triple helix

Nuclear magnetic resonance spectroscopy has been used to characterize opening reactions and stabilities of individual base pairs in two related DNA structures. The first is the triplex structure formed by the DNA 31-mer 5'-AGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3'. The structure belongs to the YRY (or parallel) family of triple helices. The second structure is the hairpin double helix formed by the DNA 20-mer 5'-AGAGAGAACCCCTTCTCTCT-3' and corresponds to the duplex part of the YRY triplex. The rates of exchange of imino protons with solvent in the two structures have been measured by magnetization transfer from water and by real-time exchange at 10 degrees C in 100 mM NaCl and 5 mM MgCl2 at pH 5.5 and in the presence of two exchange catalysts. The results indicate that the exchange of imino protons in protonated cytosines is most likely limited by the opening of Hoogsteen C+G base pairs. The base pair opening parameters estimated from imino proton exchange rates suggest that the stability of individual Hoogsteen base pairs in the DNA triplex is comparable to that of Watson-Crick base pairs in double-helical DNA. In the triplex structure, the exchange rates of imino protons in Watson-Crick base pairs are up to 5000-fold lower than those in double-helical DNA. This result suggests that formation of the triplex structure enhances the stability of Watson-Crick base pairs by up to 5 kcal/mol. This stabilization depends on the specific location of each triad in the triplex structure.     

Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIα and β were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N⁶-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage ~10-fold. 1,N⁶-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.     

Molecular Dynamics Simulations of Chlorambucil/DNA Adducts. A Structural Basis for the 5′ -GNC Interstrand DNA Crosslink Formed by Nitrogen Mustards

Abstract

The alkylation of DNA by chlorambucil has been studied using a computational approach. Molecular dynamics simulations were performed on the fully solvated non-covalent complex, two monoadducts and a crosslinked diadduct of chlorambucil with the d(CGG₃G₂CGC).- d(GCG₁CCCG) duplex, in which the N7 atoms of G₁, G₂ and G₃ are potential alkylation sites. The results provide a structural basis for the preference of nitrogen mustards to crosslink DNA duplexes at a 5′-GNC site (a 1,3 crosslink, G₁ -G₃) rather than at a 5′-GC sites (a 1,2 crosslink, G₁ -G₂).

In the non-covalent complex simulation the drug reoriented from a non-interstrand crosslinking location to a position favorable for G₁ -G₃ diadduct formation. It proved possible to construct a G₁ -G₃ diadduct from a structure from the non-covalent simulation, and continue the molecular dynamics calculation without further disruption of the DNA structure. A crosslinked diadduct developed with four B_II conformations on the 3′ side of each alkylated guanine and of their respective complementary cytosine. In the first monoadduct simulation the starting point was the same DNA conformation used in the crosslinked diadduct simulation with alkylation at G₁. In this simulation the DNA deformation was reduced, with the helix returning to a more canonical form. A second monoadduct simulation was started from a canonical DNA conformation alkylated at G₃. Here, no significant motion towards a potential crosslinking conformation occurred. Collectively, the results suggest that crosslink formation is dependent upon the drug orientation prior to alkylation and the required deformation of the DNA to permit 1,3 crosslinking can largely be achieved in the non-covalent complex.     

Carcinogen-Nucleic Acid Interactions: Equilibrium Binding Studies of Aflatoxins B1 and B2 with DNA and the Oligodeoxynucleotide d(ATGCAT)2

Abstract

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B₁ and B₂ with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B₂ with the oligodeoxynucleotide d(ATGCAT)₂. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B₁ binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin ? 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels ? 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a “two-site” binding model [L.S. Rosenberg, M J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002–1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B₂ binding to d(ATGCAT)₂ indicates that the aflatoxin B₂/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1–0.5 ppm are observed for the aflatoxin B₂ 4-OCH₃, H5, and H6a protons. Much smaller chemical shift changes ? 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T₁ experiments demonstrate a 15–25 % decrease in the relaxation time for the adenine H2 protons when aflatoxin B₂ is added to the solution. This result suggests that aflatoxin B₂ protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B₁ and B₂ complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B₁,-guanine N7 DNA adducts.     

O6-Methylguanine-DNA methyltransferase in human cells

O⁶-Methylguanine-DNA methyltransferase activity was measured in extracts of human tumor cells and was partially purified from human placenta. Repair of O⁶-methylguanine in DNA inactivated the methyltransferase, and treatment of cells with MNNG, which produces this alkylated base in DNA, depleted the cells of active methyltransferase. RNA and protein synthesis were required for restoration of methyltransferase activity, which transiently exceeded the original levels by 50% 48 h after treatment. One species of methyltransferase of M_r = 22 kd was present in human tumor cells and human placenta.     

A role for mismatch repair in production of chromosome aberrations by methylating agents in human cells

We have shown previously that certain alkylation products, or alkylation derived lesions, which induce chromosome aberrations (abs) persist for at least two cell cycles in Chinese hamster ovary cells. The increase in abs in the second cycle after treatment contrasts with the classical observation of reduction in ab yield with successive mitoses following ionizing radiation. Here we present evidence that processing of lesions by mismatch repair is a mechanism for ab induction by methylating agents.Our previous studies implicated O⁶-methylguanine (O⁶MeG) as an important lesion in induction of abs, particularly in the second cell cycle after treatment. In the absence of repair of O⁶MeG by alkylguanine DNA alkyltransferase (AGT), new abs were induced in the second cycle after treatment with e.g. methylnitronitrosoguanidine (MNNG) and methylnitrosourea (MNU). Thus, we hypothesized that abs were produced not by O⁶MeG or its repair in the first S phase, but by subsequent processing of the lesions. We suggested that after replication proceeded past the O⁶MeG lesion in the first S phase, inserting an incorrect base on the newly synthesized strand, recognition and repair by mismatch repair in the second S phase led to a chromosome ab. Here we used MT1 cells, a human lymphoblastoid cell line that has a defect in strand-specific mismatch repair. MT1 cells are alkylation tolerant and have a mutator phenotype, compared with their parent line, TK6; both MT1 and TK6 cells lack AGT so do not remove the methyl group from O⁶MeG. While the initial levels of abs at the first metaphase were similar in MT1 and TK6 cells, ab levels in MT1 cells were greatly reduced in the second and third cell cycles following treatment with MNNG, dimethylnitrosamine and MNU, in contrast with the parent TK6 cells, which had more abs in the second cell cycle than in the first. This supports the hypothesis that repair of mismatched base pairs involving O⁶MeG is one mechanism for induction of chromosome abs. In contrast to the difference in response to methylating agents between TK6 cells and mismatch repair-deficient MT1 cells, the profile of ab induction by an ethylating agent, ethylnitronitrosourea, was similar in MT1 cells to those for TK6 cells and CHO cells.