Nitric Oxide-induced Deamination of Cytosine and Guanine in Deoxynucleosides and Oligonucleotides*

Jennifer L. CaulfieldDagger , John S. Wishnok§, and Steven R. TannenbaumDagger §

From the Dagger  Department of Chemistry and the § Division of Toxicology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The autoxidation of nitric oxide (NO·) forms the nitrosating agent N2O3, which can directly damage DNA by deamination of DNA bases following nitrosation of their primary amine functionalities. Within the G:C base pair, deamination results in the formation of xanthine and uracil, respectively. To determine the effect of DNA structure on the deamination of guanine and cytosine, the NO·-induced deamination rate constants for deoxynucleosides, single- and double-stranded oligonucleotides, and a G-quartet oligonucleotide were measured. Deamination rate constants were determined relative to morpholine using a Silastic membrane to deliver NO· at a rate of ~10-20 nmol/ml/min for 60 min, yielding a final concentration of ~600-1200 µM NO2-. GC/MS analysis revealed formation of nanomolar levels of deamination products from millimolar concentrations of deoxynucleosides and oligomers. Deamination rate constants for cytosine and guanine in all types of DNA were lower than the morpholine nitrosation rate constant by a factor of ~103-104. Xanthine was formed at twice the rate of uracil, and this may have important consequences for mechanisms of NO·-induced mutations. Single-stranded oligomers were 5 times more reactive than deoxynucleosides toward N2O3. Double-stranded oligomers were 10-fold less reactive than single-stranded oligomers, suggesting that Watson-Crick base pairing protects DNA from deamination. G-quartet structures were also protective, presumably because of hydrogen bonding. These results demonstrate that DNA structure is an important factor in determining the reactivity of DNA bases with NO·-derived species.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Nitric oxide (NO·)1 is an important physiological messenger that is involved in many processes in vivo, including inhibition of platelet aggregation, blood vessel relaxation, and neurotransmission (1). NO·, along with its many essential roles in vivo, can also undergo reactions that may result in cytotoxic or mutagenic events by a number of possible mechanisms. Nitric oxide reacts with oxygen, for example, to form the nitrosating agent nitrous anhydride, N2O3, which can then react with amines, thiols, and other available nucleophiles. N2O3, which is also formed from acidic nitrite, can nitrosate primary amine functionalities on DNA bases, leading to direct DNA damage via deamination. Deamination of cytosine, adenine, guanine, and 5-methylcytosine forms uracil, hypoxanthine, xanthine, and thymine, respectively.

DNA bases can also undergo "spontaneous" hydrolytic deamination (2). Both spontaneous and nitrosative deamination presumably occur via nucleophilic aromatic substitution, but the nitrosative pathway would be favored by the better leaving group, i.e. -N2OH2+ or -N2+ versus -NH3+ (3, 4). These pathways are summarized, with cytosine as the example, in Fig. 1. Pyrimidine bases in DNA are more susceptible to spontaneous deamination than are the purine bases (5, 6). This is demonstrated by the previous observation of significant cytosine deamination but no guanine deamination upon heating of the synthetic copolymer poly(dG-dC):poly(dG-dC) and the homopolymer poly(dG):poly(dC) (7, 8). Purine constituents, however, were found to be more easily deaminated by nitrous acid (9). Deamination of DNA bases can lead to mutagenesis through misincorporation by DNA polymerase, misrepair, or no repair of the resulting deamination products. The types of mutations that potentially arise from deamination of DNA bases are summarized in Table I. Given the reports of nitric oxide-induced toxicity (10) and the observation of deamination products in NO·-treated cells and DNA (10, 11), it is believed that deamination may play a key role in nitric oxide-induced mutagenesis. The predominant mutation resulting from NO· treatment is G:C right-arrow A:T (12, 13). It is potentially important because it has been observed in numerous human diseases, including hemophilia, retinoblastoma, familial Alzheimer's disease, and colon cancer (14-17). This mutation could arise from the deamination of either guanine or cytosine. Our main interest, therefore, was to determine the rate constants for deamination of these two compounds. The deaminations of adenine and 5-methylcytosine were not included in this study. The rates of nitric oxide-induced deamination of DNA bases in different environments are not known. In order to reveal the effects of base pairing and helix structure on nitrosation chemistry, we investigated the rates of guanine and cytosine deamination when these bases are components of 2'-deoxynucleosides, single- and double-stranded oligonucleotides, and a G-quartet oligonucleotide. G-quartet structures (i.e. tetraplexes of four parallel strands in which each guanine donates and accepts two hydrogen bonds) form among the tandem repeats of G-rich sequences in telomeric DNA (18). The G-quartet structure provides an additional hydrogen-bond-containing polymer for study of the reactivity of N2O3 with different DNA structures.


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Fig. 1.   Reaction pathway for deamination of cytosine.

Two different systems, including a Silastic membrane and a kinetic reactor, were used to deliver nitric oxide. The Silastic membrane delivery system, which maintains a steady state and a very low dose rate (~10-20 nmol/ml/min) of NO·, was used for most experiments. This system works ideally for compounds, such as the nucleosides and oligomers, with similar nitrosation rate constants. However, in order to determine the actual rate constant for the 2'-deoxyguanosine/N2O3 reaction, a kinetic reactor developed by Lewis et al. (19) was used to deliver nitric oxide to a deoxygenated solution of 2'-deoxyguanosine and morpholine. Subsequent introduction of oxygen results in the formation of N2O3. In this reactor, lower amounts of NO· can be delivered, resulting in a very steady pH and therefore constant concentrations of free morpholine. The Silastic system is not amenable for use with compounds such as morpholine because the higher amounts of NO· result in a slight pH drop and consequent unsteady levels of free morpholine. It is not possible to increase the buffer concentrations to maintain the pH because of the N2O3 scavenging effects that have been observed for several buffer anions (20, 21). For these reasons, the kinetic reactor was used for the morpholine/2'-dGuo reaction. The rate constant for the morpholine/N2O3 reaction relative to N2O3 hydrolysis is known (4.0 × 104 M-1). The rate constant for the 2'-deoxyguanosine/N2O3 reaction relative to N2O3 hydrolysis can therefore be found via the competitive-kinetics approach developed by Keshive et al. (22), i.e. by comparing the levels of the nitrosation product (xanthine) to the levels of N-nitrosomorpholine following exposure of a mixture of the two compounds to NO·. Using a value for k4 of 1600 s-1 (19), the rate constant for the 2'-Guo/N2O3 reaction (k7G) can then be calculated.

The rate constants for reaction of N2O3 with 2'-deoxycytidine, a G-quartet oligonucleotide, and single- and double-stranded oligonucleotides containing guanine and cytosine were based on the previously determined 2'-deoxyguanosine rate constant by using reaction mixtures containing one component for which the rate constant for reaction with N2O3 was known. Using these rate constants, it is possible to determine whether guanine or cytosine is deaminated faster within a G:C base pair. In addition, conclusions can be made regarding the effects of base pairing on deamination chemistry, which will contribute to an understanding of NO·-induced modifications to DNA.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

2'-Deoxycytidine and 2'-deoxyguanosine were purchased from Sigma, and morpholine was purchased from Aldrich. The synthetic oligonucleotides AACCCCAA, TTGGGGTT, TGTGTGTG, and CGCGCGCGCGCG were obtained from the Massachusetts Institute of Technology Biopolymers Laboratory. Phosphate buffer (0.01 M) at pH 7.4 was prepared with K2HPO4 and KH2PO4 using double-distilled water. 100% nitric oxide and 10% nitric oxide in argon (Matheson, Gloucester, MA) were passed through a column of 4-8 mesh soda lime to remove NOx impurities. Silastic tubing (0.025 in ID × 0.047 in OD) was purchased from Dow Corning Corp. (Midland, MI). Sep-Pak tC18 cartridges were obtained from Waters Associates (Bedford, MA). [1,3-15N2]Xanthine and [1,3-15N2]uracil were obtained from Cambridge Isotope Laboratories (Cambridge, MA). Silylation grade acetonitrile, pyridine, and N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MT-BSTFA) were purchased from Pierce.

Kinetic Reactor for NO· Treatment of the 2'-Deoxyguanosine and Morpholine Solution

A 200-ml kinetic reactor was used as described previously (22), with a few modifications. First, the total amount of NO· delivered was ~80 µM in these experiments versus ~30 µM in previous work. Second, the flow loop to the spectrophotometer was disconnected because UV detection cannot be used to simultaneously monitor xanthine and N-nitrosomorpholine product concentrations. Because UV detection could not be used, samples were withdrawn from the reactor immediately before O2 addition and 30 min after O2 addition (i.e. prereaction and postreaction samples). Using a gas-tight syringe that had previously been deoxygenated with argon, 600 µl of the reaction mixture was withdrawn and added to 400 µl of a 0.15 M azide solution to quench any N2O3 present. Due to the fact that there is dissolved nitric oxide in the prereaction sample, high concentrations of azide are needed to prevent reaction of N2O3 with morpholine or 2'-deoxyguanosine. Complete N2O3 scavenging was confirmed by omitting morpholine from the reaction mixture but including it in the azide solution and determining that there was no N-nitrosomorpholine formation. Approximately 80 µM NO· was delivered to a solution of 3 mM 2'-deoxyguanosine and 0.5-1.5 mM morpholine in 10 mM potassium phosphate buffer at pH 7.4. The resulting levels of xanthine and N-nitrosomorpholine were quantitated by GC/MS as described below.

Sample Preparation and GC/MS

N-Nitrosomorpholine-- A portion (200 µl) of the prereaction and postreaction samples was withdrawn, and 50 µl of a 48.4 µM nitrobenzene solution was added for use as an internal standard. The aqueous sample was extracted with methylene chloride (300 µl), and the organic layer was analyzed for N-nitrosomorpholine and nitrobenzene using a Supelcowax column obtained from Supelco (Bellefonte, PA). Analysis was performed on an HP 5989 GC/MS apparatus in the electron ionization mode. Quantitation was based on a standard curve.

Xanthine and Uracil-- A portion (100 µl) of the prereaction and postreaction samples was withdrawn, and 50 µl of a 100 pg/µl [1,3-15N2]xanthine solution and 50 µl of a 100 pg/µl [1,3-15N2]uracil solution were added as internal standards. Acid hydrolysis was performed in Reacti-Vials using 500 µl of 60% formic acid at 100 °C for 1 h. Samples were then dried in a Speed Vac. Sep-Pak tC18 cartridges were used, and the methanol eluant was dried completely in a Speed Vac. Samples were derivatized in a Reacti-Vial with 15 µl of acetonitrile, 10 µl of pyridine, and 25 µl of N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide at 130 °C for 30 min and analyzed on a Hewlett-Packard HP-5 column.

Silastic Membrane Delivery System for NO· Treatment of the 2'-Deoxynucleoside and Oligonucleotide Solutions

The oligonucleotides studied are shown in Table II. The experimental mixtures were as follows: 1) 1 mM 2'-dGuo and 1 mM 2'-dCyd, 2) 0.25 mM AACCCCAA and 1 mM 2'-dGuo, 3) 0.25 mM TGTGTGTG and 1 mM 2'-dCyd, 4) 0.25 mM TTGGGGTT and 1 mM 2'-dCyd, and 5) 0.18 mM CGCGCGCGCGCG and 1 mM 2'-dCyd. All NO· treatments were carried out in 10 mM potassium phosphate buffer at pH 7.4. After passing through a column of 4-8 mesh soda lime, NO· was introduced as a mixture of 10% NO· in argon using a Silastic membrane system shown in Fig. 2 and as described by Tamir et al. (23) with minor modifications. For sample 1, a 7-ml volume was treated via 10 cm of Silastic tubing for 1 h. For samples 2-5, a volume of 1.5 ml was treated via 2 cm of Silastic tubing for 1 h. All solutions were stirred to minimize the boundary layer at the polymer-liquid interface. The total amount of NO· actually delivered was measured at the end of each experiment as total nitrite (24). In all experiments, the nitric oxide delivery rate was ~10-20 nmol/ml/min, resulting in a final NO2- concentration of ~600-1200 µM. Samples were analyzed for xanthine and uracil by GC/MS as described above. An additional step, involving separation of the oligomer from the deoxynucleoside using Millipore Ultrafree-MC 2,000 NMWL Filter Units, was necessary for the double-stranded oligomer. The oligomer is retained by the filter and recovered in the retentate, whereas the deoxynucleoside is found in the filtrate. Separation was confirmed in individual trial experiments with each component.


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Fig. 2.   Nitric oxide membrane delivery system. Measured lengths of Silastic tubing were threaded onto hypodermic needles that pass through a rubber septum. Solutions were stirred to minimize the boundary layer at the polymer-liquid interface. 10% NO· in argon was then delivered for 1 h, resulting in a final NO2- concentration of ~600-1200 µM delivered at ~10-20 nmol/ml/min.

Kinetic Model and Reaction Scheme

In previous experiments, Lewis et al. (19) showed that the principal nitrosating agent in the NO· oxidation pathway at physiological pH is N2O3, which leads primarily to NO2- as summarized in Equations 1-3.
2<UP>NO</UP>+<UP>O</UP><SUB>2</SUB> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>1</SUB>=2.1×10<SUP>6</SUP> <UP><SC>m</SC></UP><SUP><UP>−2</UP></SUP><UP> s<SUP>−1</SUP></UP></UL></LIM> 2<UP>NO</UP><SUB>2</SUB> (Eq. 1)
<UP>NO</UP>+<UP>NO</UP><SUB>2</SUB> <LIM><OP><ARROW>⇄</ARROW></OP><LL>k<SUB>3</SUB>=4.3×10<SUP>6</SUP> <UP>s<SUP>−1</SUP></UP></LL><UL>k<SUB>2</SUB>=1.1×10<SUP>9</SUP> <UP><SC>m</SC></UP><SUP><UP>−1</UP></SUP><UP> s<SUP>−1</SUP></UP></UL></LIM> <UP>N</UP><SUB>2</SUB><UP>O</UP><SUB>3</SUB> (Eq. 2)
<UP>N</UP><SUB>2</SUB><UP>O</UP><SUB>3</SUB>+<UP>H</UP><SUB>2</SUB><UP>O</UP> <LIM><OP><ARROW>⇄</ARROW></OP><LL>k<SUB>5</SUB>=5.6 <UP><SC>m</SC></UP><SUP><UP>−1</UP></SUP><UP> s<SUP>−1</SUP></UP></LL><UL>k<SUB>4</SUB>=1.6×10<SUP>3</SUP> <UP>s<SUP>−1</SUP></UP></UL></LIM> 2<UP>HNO</UP><SUB>2</SUB> ⇄ 2<UP>NO</UP><SUP><UP>−</UP></SUP><SUB><UP>2</UP></SUB>+<UP>2H<SUP>+</SUP></UP> (Eq. 3)
N2O3 can also react with phosphate, chloride, and bicarbonate (19, 20) to enhance the hydrolysis of N2O3 represented otherwise by Equation 3. In a competitive kinetics approach, however, these effects do not need to be included in the kinetic analysis (22). Morpholine nitrosation by N2O3 is shown in Equation 4.
<UP>N</UP><SUB>2</SUB><UP>O</UP><SUB>3</SUB>+<UP>Mor</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>6</SUB>=6.4×10<SUP>7</SUP> <UP><SC>m</SC></UP><SUP><UP>−1</UP></SUP><UP> s<SUP>−1</SUP></UP></UL></LIM> <UP>NMor</UP>+<UP>NO</UP><SUP><UP>−</UP></SUP><SUB><UP>2</UP></SUB>+<UP>H<SUP>+</SUP></UP> (Eq. 4)

Determination of the 2'-dGuo/N2O3 Rate Constant Relative to N2O3 Hydrolysis (k7G/k4)-- In the above reaction scheme, all reaction rate constants are known. The first unknown rate constant to be determined was for the 2'-dGuo/N2O3 reaction shown below.
<UP>N</UP><SUB>2</SUB><UP>O</UP><SUB>3</SUB>+<UP>G</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>7</SUB><SUP><UP>G</UP></SUP></UL></LIM> <UP>X</UP>+<UP>NO</UP><SUP><UP>−</UP></SUP><SUB><UP>2</UP></SUB>+<UP>H<SUP>+</SUP></UP> (Eq. 5)

The complete kinetic analysis for estimating the unknown rate constant k7 can be found in Ref. 22. Using that analysis, the rates of formation of NMor and X are as follows.
<FR><NU>d[<UP>NMor</UP>]</NU><DE>dt</DE></FR>=k<SUB>6</SUB>[<UP>Mor</UP><SUP>0</SUP>][<UP>N</UP><SUB>2</SUB><UP>O</UP><SUB>3</SUB>] (Eq. 6)
<FR><NU>d[<UP>X</UP>]</NU><DE>dt</DE></FR>=k<SUB>7</SUB><SUP><UP>G</UP></SUP>[<UP>G</UP>][<UP>N</UP><SUB>2</SUB><UP>O</UP><SUB>3</SUB>] (Eq. 7)

Combining Equations 6 and 7, we obtain,
<FR><NU>d[<UP>X</UP>]</NU><DE>d[<UP>NMor</UP>]</DE></FR>=<FR><NU>k<SUB>7</SUB><SUP><UP>G</UP></SUP>[<UP>G</UP>]</NU><DE>k<SUB>6</SUB>[<UP>Mor</UP><SUP>0</SUP>]</DE></FR> (Eq. 8)
where [Mor0] is the concentration of unprotonated morpholine that is available for nitrosation. Integration of Equation 8 with the assumption that [G] and [Mor0] are essentially constant during the reaction, gives the equation,
k<SUB>7</SUB><SUP><UP>G</UP></SUP>=k<SUB>6</SUB><FR><NU>&Dgr;[<UP>X</UP>][<UP>Mor</UP><SUP>0</SUP>]</NU><DE>&Dgr;[<UP>NMor</UP>][<UP>G</UP>]</DE></FR> (Eq. 9)
where Delta [X] and Delta [NMor] represent the changes in the respective concentrations during the reaction (i.e. postreaction amounts minus prereaction levels). Rearrangement of Equation 9 gives the following equation.
&Dgr;[<UP>X</UP>][<UP>Mor</UP><SUP>0</SUP>]=<FR><NU>k<SUB>7</SUB><SUP><UP>G</UP></SUP>/k<SUB>4</SUB></NU><DE>k<SUB>6</SUB>/k<SUB>4</SUB></DE></FR> &Dgr;[<UP>NMor</UP>][<UP>G</UP>] (Eq. 10)

The rate constant for 2'-dGuo/N2O3 (k7G/k4) was determined from the slope of the plot of Delta [X][Mor0versus Delta [NMor][G] and the previously reported value for k6/k4 (4 × 104 M-1) (22).

Determination of the 2'-dCyd/N2O3 Rate Constant Relative to N2O3 Hydrolysis (k7C/k4)

The rate constant for the reaction of N2O3 with 2'-dCyd was determined using the rate constant for the 2'-dGuo/N2O3 reaction relative to N2O3 hydrolysis found above (k7G/k4), the amounts of xanthine and uracil formed during the 2'-dCyd/2'-dGuo treatment, and the following equation, which arises from the same theory as Equation 9 above.
<FR><NU>k<SUB>7</SUB><SUP><UP>C</UP></SUP></NU><DE>k<SUB>4</SUB></DE></FR>=<FR><NU>k<SUB>7</SUB><SUP><UP>G</UP></SUP></NU><DE>k<SUB>4</SUB></DE></FR> <FR><NU>&Dgr;[<UP>U</UP>][<UP>G</UP>]</NU><DE>&Dgr;[<UP>X</UP>][<UP>C</UP>]</DE></FR> (Eq. 11)

Determination of the Single-stranded Oligonucleotide and G-Quartet Oligonucleotide/N2O3 Rate Constants Relative to N2O3 Hydrolysis (k7C(oligo)/k4 and k7G(oligo)/k4)

The rate constants for the reaction of N2O3 with single-stranded oligomers and the G-quartet oligomer were determined using the known rate constants for the 2'-dGuo/N2O3 reaction and 2'-dCyd/N2O3 reaction relative to N2O3 hydrolysis, the amounts of xanthine and uracil formed during the oligomer/deoxynucleoside reactions, and the following equations.
<FR><NU>k<SUB>7</SUB><SUP><UP>C</UP>(<UP>oligo</UP>)</SUP></NU><DE>k<SUB>4</SUB></DE></FR>=<FR><NU>k<SUB>7</SUB><SUP><UP>G</UP></SUP></NU><DE>k<SUB>4</SUB></DE></FR> <FR><NU>&Dgr;[<UP>U</UP>][<UP>G</UP>]</NU><DE>&Dgr;[<UP>X</UP>][<UP>C</UP>(<UP>oligo</UP>)]</DE></FR> (Eq. 12)
<FR><NU>k<SUB>7</SUB><SUP><UP>G</UP>(<UP>oligo</UP>)</SUP></NU><DE>k<SUB>4</SUB></DE></FR>=<FR><NU>k<SUB>7</SUB><SUP><UP>C</UP></SUP></NU><DE>k<SUB>4</SUB></DE></FR> <FR><NU>&Dgr;[<UP>X</UP>][<UP>C</UP>]</NU><DE>&Dgr;[<UP>U</UP>][<UP>G</UP>(<UP>oligo</UP>)]</DE></FR> (Eq. 13)

Determination of the Double-stranded/N2O3 Rate Constant Relative to N2O3 Hydrolysis (k7C(oligo)/k4 and k7G(oligo)/k4)

The rate constants for the reaction of N2O3 with cytosine and guanine in the double-stranded oligomer were determined using the amounts of xanthine and uracil formed from the oligomer, the amount of uracil arising from 2'-dCyd upon treatment of the double-stranded oligomer/2'-dCyd mixture, and the following equations.
<FR><NU>k<SUB>7</SUB><SUP><UP>C</UP>(<UP>oligo</UP>)</SUP></NU><DE>k<SUB>4</SUB></DE></FR>=<FR><NU>k<SUB>7</SUB><SUP><UP>C</UP></SUP></NU><DE>k<SUB>4</SUB></DE></FR> <FR><NU>&Dgr;[<UP>U</UP>(<UP>oligo</UP>)][<UP>C</UP>]</NU><DE>&Dgr;[<UP>U</UP>][<UP>C</UP>(<UP>oligo</UP>)]</DE></FR> (Eq. 14)
<FR><NU>k<SUB>7</SUB><SUP><UP>G</UP>(<UP>oligo</UP>)</SUP></NU><DE>k<SUB>4</SUB></DE></FR>=<FR><NU>k<SUB>7</SUB><SUP><UP>C</UP></SUP></NU><DE>k<SUB>4</SUB></DE></FR> <FR><NU>&Dgr;[<UP>X</UP>(<UP>oligo</UP>)][<UP>C</UP>]</NU><DE>&Dgr;[<UP>U</UP>][<UP>G</UP>(<UP>oligo</UP>)]</DE></FR> (Eq. 15)

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Morpholine Concentration and pH-- The unprotonated form of morpholine is the substrate for nitrosation and is thus the most important form of morpholine for these experiments. Denoting total morpholine as Mor and the unprotonated form as Mor0, the respective concentrations are related by the equation,
[<UP>Mor</UP><SUP>0</SUP>]=<FR><NU>[<UP>Mor</UP>]</NU><DE>1+10<SUP><UP>pK−pH</UP></SUP></DE></FR> (Eq. 16)
where the pK for morpholine at 25 °C is 8.5. The amount of morpholine available for nitrosation at pH 7.4 is 7.4% of the total morpholine concentration. The pH during a given reaction was essentially constant, i.e. the concentration of unprotonated morpholine did not change significantly. In all experiments, some nitrosation products were present in the solution prior to introduction of O2 due to a small air leak that could not be eliminated.

Determination of the 2'-dGuo/N2O3 Rate Constant (k7G)-- From the plot of Equation 10, the 2'-dGuo/N2O3 rate constant is calculated to be k7G = 2.2 ± 0.2 M-1s-1. Results are summarized in Table III. In one experiment, morpholine was not present in the reaction mixture but was included in the azide mixture when samples were withdrawn from the reactor to ensure that there was no nitrosation occurring from the nitric oxide present in the prereaction sample. No detectable N-nitrosomorpholine was observed, indicating that the N2O3 was efficiently scavenged by the azide and that the levels of xanthine and N-nitrosomorpholine reported in the prereaction samples are accurate.

Determination of the 2'-dCyd/N2O3 Rate Constant (k7C)-- As shown in Table III, the rate constant for the 2'-dCyd/N2O3 reaction was determined to be k7C = 1.1 ± 0.2 × 104 M-1s-1 based on the rate constant for the 2'-dGuo/N2O3 reaction above and the amounts of xanthine and uracil formed upon treatment of the 2'-dCyd/2'-dGuo mixture using Equation 11.

Determination of the Rate Constants for Reaction of N2O3 with Cytosine and Guanine in Single-stranded Oligonucleotide and a G-Quartet Oligonucleotide (k7C(oligo) and k7G(oligo))-- The rate constant for N2O3 with cytosine in the single-stranded oligonucleotide AACCCCAA was found to be k7C(oligo) = 5.6 ± 1.1 × 104 M-1s-1 using Equation 12, the 2'-dGuo/N2O3 rate constant, and the amounts of xanthine and uracil found upon 2'-dGuo/AACCCCAA oligo treatment. The rate constant for guanine within the single-stranded oligonucleotide TGTGTGTG was found to be 9.8 ± 1.3 × 104 M-1s-1 using Equation 13, the 2'-dCyd/N2O3 rate constant, and the xanthine and uracil amounts found upon 2'-dCyd/TGTGTGTG oligo treatment. Similarly, the rate constant for the reaction of N2O3 with guanine in the G-quartet oligonucleotide TTGGGGTT was found to be k7G(oligo) = 7.4 ± 1.1 × 104 M-1s-1.

Determination of the Rate Constants for Reaction of N2O3 with Cytosine and Guanine in a Double-stranded Oligonucleotide (k7C(oligo) and k7G(oligo))-- As seen in Table III, the rate constant for the reaction of cytosine in the double-stranded oligonucleotide CGCGCGCGCGCG was found to be k7C(oligo) = 5.6 ± 1.0 × 103 M-1s-1 using Equation 14, the amounts of uracil arising from the oligo, amounts of uracil from 2'-dCyd, and the previously determined 2'-dCyd/N2O3 rate. Similarly, the rate constant for the reaction of guanine in the double-stranded oligonucleotide was calculated to be k7C(oligo) = 1.0 ± 0.1 × 104 M-1s-1 using Equation 15.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Mutations may ultimately result from deamination of any of the bases shown in Table I. The G:C right-arrow A:T transition, which could result from the deamination of either guanine, cytosine, or 5-methylcytosine, has frequently been observed as the primary type of mutation upon NO· treatment (26-28). Focusing on the G:C base pair, this study was intended to determine whether guanine or cytosine is deaminated faster to give rise to the G:C right-arrow A:T mutation. The possible mutagenic effects of cytosine and guanine deamination forming uracil and xanthine, respectively, are discussed below.

                              
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Table I
Mutations that potentially arise from deamination of DNA bases

Deamination of cytosine will lead to a G:C right-arrow A:T transition mutation upon replication due to pairing of uracil with adenine (27, 28). Alternatively, the uracil may be removed by the enzyme DNA uracil glycosylase prior to replication (29). Uracil glycosylase excises uracil from both single- and double-stranded DNA with preference for single-stranded DNA (30). The importance of cytosine deamination is emphasized by the fact that organisms that lack this enzyme have an increased spontaneous mutation rate and more G:C right-arrow A:T transitions (31).

Deamination of guanine to xanthine will lead to a G:C right-arrow A:T transition mutation upon replication because xanthine is believed to base pair with thymine (26, 32). There is no known repair mechanism for xanthine. The N-glycoside bond of deoxyxanthosine is acid-labile (33, 34); therefore, one possible fate of xanthine is depurination to form an abasic site. Many organisms will replicate past such a lesion by incorporating the "A rule," i.e. by inserting an adenine opposite the abasic site (35-37). The ultimate result of such an event would be a G:C right-arrow T:A transversion. The abasic site may also be cleaved by an endonuclease or by base catalysis to yield a DNA single strand break that may be toxic to the cell (38).

Indeed, the formation of deamination products and single strand breaks have been observed in Salmonella typhimurium and TK6 cells following treatment with NO· (10, 12). Also, upon treatment of calf thymus DNA, significant deamination of cytosine was observed with high doses, i.e. accumulated amounts of 0.1-1 mol of NO· per liter, delivered by syringe (12). This method (bubbling NO· into solution) is inefficient due to loss of NO· into the gas phase. The Silastic membrane system used in this study delivers nitric oxide at low, steady rates that approach the delivery rates of stimulated cells, such as macrophages. Rates of delivery in these experiments are ~10-20 nmol/ml/min and result in a final concentration of ~600-1200 µM NO2-. Using this system, Tamir et al. (23) observed increased cytotoxicity to TK6 and S. typhimurium cells as compared with bubbling NO· into the solution due to the fact that the effective nitric oxide concentration in solution is higher because NO· is not released into the gas phase, as is the case with delivery by syringe.

The rates and products of the reactions of purine and pyrimidine bases, nucleosides, and nucleic acids with nitrous acid have been previously established by the elegant study of Shapiro and Pohl (33). It is clear from this early study that relative rates of nitrous acid-induced deamination depend on both the individual base and the nucleic acid structure. Due to the many reports of nitric oxide-induced toxicity and the observation of deamination products in the DNA of NO·-treated cells (10, 12), it is likely that deamination also plays a key role in mutagenesis induced by nitric oxide (13). This is the motivation behind the current study to determine the rates of reaction of N2O3 with guanine and cytosine in different environments. In their work on the rates of reaction of DNA with N2O3, Shapiro and Pohl (33) suggested that in future studies on the chemical modification of nucleic acids, the reaction rates should be discussed with reference to the rates of suitable model compounds and that rate constants be determined. They also suggested that variables such as pH and the concentration of reactive species be as closely controlled as possible. All of these conditions have been taken into consideration here, where the pH and the concentration of reactive species is strictly controlled. In addition, the rates are expressed in relative terms and were determined by comparison with the model compound morpholine. The rate constant for the 2'-deoxyguanosine/N2O3 reaction relative to N2O3 hydrolysis was determined to be 1.4 ± 0.1 × 101 M-1s-1. This rate constant and all rate constants for cytosine and guanine deamination in oligonucleotides are several orders of magnitude lower than that for morpholine nitrosation relative to N2O3 hydrolysis (4 × 104 M-1), demonstrating that deoxynucleosides and oligomers react with N2O3 much slower than does a typical amine (19).

The single- and double-stranded oligonucleotides and the G-quartet oligonucleotide shown in Table II were treated with NO· to reveal the effects of base pairing and helix structure on nitrosation chemistry. The rate constants for the reaction of guanine and cytosine when present in each type of DNA are shown in Table III. Single-stranded oligomers are seen to be approximately 5 times more reactive than deoxynucleosides. Nguyen et al. (10) observed a similar effect: the yields of deamination products from single-stranded nucleic acids (RNA and heat-denatured calf thymus DNA) were higher than those from free bases upon nitric oxide treatment (10). Single-stranded oligomers may react differently than monomers or double-stranded nucleotides due to folding or stacking interactions. The increased rate for single-stranded oligonucleotides may also occur as a result of nearest-neighbor effects, whereby the nitrosyl group could be passed to neighboring groups within the single-stranded oligo.

                              
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Table II
Oligonucleotides used in this study

                              
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Table III
Rate constants for reaction of N2O3 with 2'-deoxynucleosides, single- and double-stranded oligomers, and G-quartet oligomers relative to N2O3 hydrolysis

Guanine is more reactive than cytosine in DNA (see Table IV). This is in contrast to the thermal hydrolytic deamination reactions, in which cytosine > adenine >>  guanine (5, 39). In several types of RNA and in calf thymus DNA, the relative reactivity of guanine to cytosine with nitrous acid has been reported to be approximately 2:1, which is in agreement with the results of this study (33). Therefore, within a G:C base pair, guanine is deaminated faster than cytosine, and their relative reactivity remains the same regardless of their environment (Table IV). The only exception is in the comparison between the single-stranded AACCCCAA oligo and the G-quartet TTGGGGTT oligo.

                              
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Table IV
Relative deamination rates of C and G in different contexts

The reduced reactivity of the G-quartet oligonucleotide TTGGGGTT as compared with the single-stranded oligonucleotide TGTGTGTG suggests that the hydrogen bonding associated with quadruplex formation decreases the reactivity of the guanine N2 amino group toward N2O3. The reactivity of the G-quartet oligonucleotide is still significantly greater than that of the double-stranded oligonucleotide, perhaps due to the accessibility of the guanine N2 amino group in the quadruplex as opposed to its internal position within the double helix of the double-stranded oligonucleotide (see Fig. 3). The G-quartet structure has been shown to form in telomeric DNA and is perhaps involved in chromosomal stabilization. The four strands of the G-quartet are centered around a cation, and tetraplex formation is therefore strongly dependent on the nature of the cation. The potassium ion is the preferred ion for tetraplex formation and is the buffer cation used in experiments here. Some cations, e.g. sodium, promote tetraplex formation to a lesser extent, whereas others, e.g. lithium, do not allow for tetraplex formation (for a detailed review of G-quartets, see Ref. 18). The deamination of guanine in G-quartet structures in vivo may not be of major importance, but the study of deamination of this short oligomer does provide information regarding the reactivity of N2O3 with different DNA structures. The important conclusion from our data is that the G-quartet oligonucleotide reactivity is lower than that of the single-stranded oligonucleotide of the same composition but different sequence, indicating the importance of hydrogen bonding and secondary structure formation in determining the reactivity with N2O3.


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Fig. 3.   G-quartet structure. Four strands of DNA containing a series of four guanines can form a stable tetraplex structure around a cation as shown here. The potassium ion is most efficient for forming a tetraplex, whereas sodium promotes tetraplex formation to a lesser extent. G-quartet structures do not form in the presence of lithium.

Watson-Crick base pairing also protects DNA from deamination as demonstrated here by the observation that double-stranded oligomers are 10-fold less reactive with N2O3 than are single-stranded oligomers. This decreased reactivity is in agreement with previous reports on the study of spontaneous and heat-induced cytosine deamination (6, 40-42), suggesting that genome integrity may be best preserved in double-stranded DNA.

Spontaneous cytosine deamination, however, was reported to occur over 100 times faster in single- than in double-stranded DNA (40, 42) whereas, in this work, NO·-induced deamination was ~10 times faster in single- than in double-stranded oligonucleotides. In the absence of other relevant data, it is therefore unknown whether single-stranded DNA is 10-fold or 100-fold more reactive than double-stranded DNA. A possible explanation for this inconsistency may be that there is a significant amount of single-stranded oligomer in equilibrium with the double-stranded oligomer. Due to the fact that the single-stranded oligomer is so much more reactive, it could be contributing to the observed reactivity of the double-stranded oligomer.

If single-stranded DNA is actually 100 times more susceptible than double-stranded DNA to deamination, then ~10% single strand character would significantly alter the observed reactivity of the double-stranded oligomer. The result would be an observed 10-fold difference between single- and double-stranded DNA, when it actually is a 100-fold difference. Melting curve data, however, indicate that the fraction of single-stranded oligomer in this buffer is <1%; i.e. little contribution from the single-stranded oligomer to the observed rate of reactivity of the double-stranded oligomer would be expected. In fact, 1% single-stranded character could affect the observed double-stranded reactivity by, at most, a factor of 2.

Steric factors may be a key reason for the reduced reactivity of DNA bases within double-stranded oligonucleotides. In addition, protonation of the cytosine N3 position has been reported to be important in deamination of cytosine in solution; however, this nitrogen cannot be protonated in double-stranded DNA due to Watson-Crick base pairing (5, 43). Therefore, steric factors and lack of protonation of the cytosine N3 position may both play a role in the lower rate of deamination of cytosine in double-stranded DNA.

It has been reported that there is a correlation between DNA duplex melting and cytosine deamination, indicating that deamination in double-stranded DNA is inversely related to duplex stability (40). Another study demonstrated that spontaneous deamination occurred selectively near AT-rich regions, possibly due to the increased single-stranded character of this stretch of DNA (43). Based on this evidence, it has been proposed that double-stranded DNA may deaminate through a single-stranded intermediate (42). DNA is single-stranded during replication, transcription, and breathing (44) in vivo.

Our results demonstrate that the rate constants for the reaction of guanine and cytosine in any environment are several orders of magnitude lower than that for morpholine nitrosation, indicating that the bases react with N2O3 much slower than a typical amine. In addition, the relative reactivity of guanine and cytosine is the same regardless of the environment (xanthine formation is always approximately double the amount of uracil formation), which may have important consequences for mechanisms of NO·-induced mutations. Due to the faster deamination rate of guanine and the high levels of uracil glycosylase in cells, guanine deamination is more likely to be responsible for the observed G:C right-arrow A:T mutations. In agreement with previous reports (10, 42, 45), single-stranded oligomers are more reactive than double-stranded oligomers and deoxynucleosides. The decreased reactivity of a double-stranded oligomer and a G-quartet oligomer toward N2O3, relative to single-stranded oligomers, suggests that hydrogen bonding and Watson-Crick base pairing may protect DNA from deamination. It is therefore important to consider the DNA structure when determining its reactivity with nitric oxide-derived species.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA09112 and CA26731.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Massachusetts Institute of Technology, Room 56-731A, Cambridge, MA 02139. Tel.: 617-253-3729; Fax: 617-252-1787; E-mail: srt{at}mit.edu.

1 The abbreviations used are: NO·, nitric oxide; GC/MS, gas chromatography-mass spectrometry; Mor, morpholine; dGuo, deoxyguanosine; dCyd, deoxycytidine.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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