From the Department of Chemistry and the
§ Division of Toxicology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139
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ABSTRACT |
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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.
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INTRODUCTION |
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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 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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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 M1). 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.
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EXPERIMENTAL PROCEDURES |
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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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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.
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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(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.
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(Eq. 5) |
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(Eq. 6) |
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(Eq. 7) |
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(Eq. 8) |
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(Eq. 9) |
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(Eq. 10) |
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.
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(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.
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(Eq. 12) |
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(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.
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(Eq. 14) |
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(Eq. 15) |
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RESULTS |
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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,
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(Eq. 16) |
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 M1s
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
M1s
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 M1s
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 M1s
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.
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DISCUSSION |
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Mutations may ultimately result from deamination of any of the
bases shown in Table I. The G:C 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
A:T mutation. The possible mutagenic effects of cytosine and guanine
deamination forming uracil and xanthine, respectively, are discussed
below.
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Deamination of cytosine will lead to a G:C 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
A:T transitions
(31).
Deamination of guanine to xanthine will lead to a G:C 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
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
M1s
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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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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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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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 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.
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FOOTNOTES |
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* 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.
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