From the Institute of Biochemistry and Biophysics, Polish Academy
of Sciences, Pawiskiego 5A, 02-106 Warsaw, Poland
Received for publication, February 2, 2001, and in revised form, March 14, 2001
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ABSTRACT |
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It was previously shown that
1,N6-ethenoadenine ( 1,N6-Ethenoadenine
( Ethenoadenine is eliminated from DNA by N-methylpurine-DNA
glycosylases. Eukaryotic glycosylases from yeast, rat, and human excise this lesion about 500-fold more efficiently than bacterial AlkA protein (8). Molecular dosimetry experiments suggest, however,
that it is a persistent lesion, because 2 weeks after exposure of rats
to vinyl chloride, the All known exocyclic DNA adducts are mutagenic. In bacteria, Early investigations by Tsou et al. (12) and Basu et
al. (13, 14) have shown that in alkali, but also under
physiological conditions, Materials--
1,N6-
Oligodeoxynucleotide (40-mer) containing a single
Four complementary oligodeoxynucleotides containing T, C, G, or A
opposite
E. coli Fpg and Nth proteins were purified from
overproducing strains (JM105 supE endA sbcB15
hsdR4rpsL thi Instrumentation--
HPLC was performed using a Waters dual pump
system with a tunable UV/visible light absorbance detector
managed by Millenium 2010SS (version 2.15) controller. All separations
were performed on a Waters Nova-Pak® C18 reversed-phase column (60 Å,
4 µm, 4.6 × 250 mm). NMR spectra were measured on a UNITY
500plus (Varian) spectrometer equipped with a gradient generator unit
Performa II, Ultrashims, and a high stability temperature unit
using 5 mm 1H{13C/15N}
pulsed field gradient triple probe. Electrospray ionization mass
spectrometry of nucleosides was performed on a Mariner apparatus with
time of flight detection. UV spectra were recorded on a Cary 3E spectrophotometer.
Thin Layer Chromatography--
Silica gel 60 F254 aluminum sheets (Merck, catalog no. 1.0554)
were used for analytical purposes. For preparative purposes 20 × 20 cm plates were prepared using silica gel 60 PF254
(Merck, catalog no. 1.07747). The following solvent systems were used: methanol/chloroform, 15:85 (I); isopropyl alcohol/25% aqueous NH3/water, 70:10:10 (II) and 70:5:5 (III).
Solvolytic Degradation of Isolation and Purification of NMR Measurements--
Samples for the NMR measurements were
prepared in D2O or
Me2SO-d6 at concentrations of
~5 mM. Spectra were measured at 25 °C using proton 1D,
TOCSY (19-21), ROESY (22, 23) and {1H/13C}gHSQC (23-26) experiments under
standard conditions and with standard parameters. All spectra were
analyzed using VNMR 5.1A (Varian) software. Proton spectra in aqueous
solution were calibrated against a water signal (27). In
Me2SO-d6 solution the residual solvent signal was used as a reference (28) in both proton and carbon
dimensions in {1H/13C} correlation spectra.
Ethenoadenine Decomposition in Oligodeoxynucleotides--
The
kinetics of compounds B and C formation in
oligodeoxynucleotides as well as the identification of substrates for
repair glycosylases was performed by HPLC. Excision of Kinetic Studies with Determination of Km and
Vmax for the Fpg Protein--
Kinetic constants were
established in Alkali-induced Rearrangements of
1,N6-Ethenodeoxyadenosine--
1,N6-Ethenodeoxyadenosine
at pH 12 was degraded sequentially into three products:
We were searching for other than high pH factors that could stimulate
Depurination of Degradation of Ethenoadenine in
Oligodeoxynucleotides--
Composition of Structure of Ethenodeoxyadenosine Products: NMR and MS
Analysis--
Proton chemical shifts of
The NMR signals in the spectra of
The spectra of product C differ from those of compound
B in the values of chemical shifts of the H1' and H2' protons and lack the signal at ~8.25 ppm corresponding to the proton H2. A comparison of the signals of all other non-exchangeable protons of the sugar moiety indicates that proton and carbon chemical shifts are almost identical for both the B and C products as well as the parental compound
The structures of the B and C compounds
assigned by NMR were confirmed additionally by mass spectroscopy.
The most abundant peaks in the B spectrum, the
protonated deoxynucleoside (m/z = 294.1) and
protonated base (m/z = 178.1), differ by 18 mass units from the corresponding peaks in the Enzymatic Excision of Ethenoadenine and Its Degradation Products
from DNA--
Human glycosylase-ANPG-40 protein effectively excised
Identification of
For the Fpg protein, the Km and
kcat values for excision of the Decomposition of The general scheme of 1,N6-ethenoadenine
rearrangements was originally proposed by Tsou and co-workers
(12). Tsou and co-workers (12), employing 1H NMR
and MS spectroscopy, have proven the formation of deformylated bi-imidazole nucleoside (corresponding to deoxy-cogener C, Fig. 1) from 1,N6-ethenoadenosine. Here we
present evidence, based on 1H and 13C NMR as
well as on MS spectroscopy, that the first step of reaction involves
formation of We have additionally found that We have found that Compound C is most probably not excised by the Fpg and Nth
proteins, since we did not observe significant elimination of
C in HPLC analysis (Fig. 5). Basu et al. (14)
have shown that compound C in DNA is 20-fold more mutagenic than A) in DNA rearranges
into a pyrimidine ring-opened derivative of 20-fold higher mutagenic
potency in Escherichia coli (AB1157 lac
U169) than the parental
A (Basu, A. K., Wood, M. L.,
Niedernhofer, L. J., Ramos, L. A., and Essigmann,
J. M. (1993) Biochemistry 32, 12793-12801). We have
found that at pH 7.0, the stability of the N-glycosidic
bond in
dA is 20-fold lower than in dA. In alkaline conditions, but
also at neutrality,
dA depurinates or converts into products:
dA
B
C
D. Compound B is a product of water molecule addition
to the C(2)-N(3) bond, which is in equilibrium with a product of
N(1)-C(2) bond rupture in
dA. Compound C is a deformylated
derivative of ring-opened compound B, which further depurinates
yielding compound D. Ethenoadenine degradation products are not
recognized by human N-alkylpurine-DNA glycosylase, which repairs
A. Product B is excised from oligodeoxynucleotides by
E. coli formamidopyrimidine-DNA glycosylase (Fpg) and
endonuclease III (Nth). Repair by the Fpg protein is as efficient as
that of 7,8-dihydro-8-oxoguanine when the excised base is paired with dT and dC but is less favorable when paired with dG and dA.
Ethenoadenine rearrangement products are formed in
oligodeoxynucleotides also at neutral pH at the rate of about 2-3%
per week at 37 °C, and therefore they may contribute to
A mutations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A)1 and other exocyclic
DNA adducts such as 3,N4-ethenocytosine (
C)
or N2,3-ethenoguanine (
G) are introduced to
DNA by the human carcinogen vinyl chloride and related compounds (1).
These DNA lesions are also formed during interaction with DNA of the
peroxidation products of
-6-polyunsaturated fatty acids (2).
Ethenoadenine has been found in the DNA of unexposed humans and rodents
at highly variable levels, ranging from 0.043 to 31.2
A
molecules/108 of unmodified adenine residues (3, 4). Upon
treatment of animals with vinyl chloride, the level of
A
increased in the DNA of rat liver, lung, lymphocytes, and testis (in
liver and lung severalfold) (3). The level of
-DNA adducts
correlates with increased oxidative stress, such as observed during the
accumulation of transient metal ions in Wilson disease, a human
metal storage disease, and with increased content of polyunsaturated
fatty acids in the diet (5-7).
A level in liver DNA remains very similar to
that obtained directly after treatment (9).
A is
recognized mostly as an unmodified adenine by DNA polymerases, infrequently giving rise to AT
TA substitutions (10). In mammalian cells,
-DNA adducts are classified among lesions with the highest mutagenic potency. In site-directed mutagenesis either 70 (10) or 10%
(11) of
A residues in DNA were replicated erroneously, giving rise
mainly to AT
GC (10) but also to AT
TA (preferential mutation
on the leading strand) and AT
CG substitutions (11). In the same
studies only 0.3% of 7,8-dihydro-8-oxoguanine (8-oxoG) residues
induced GC
TA transversions (11). Treatment of mammalian cells with
compounds inducing etheno-DNA adducts additionally triggers chromosomal
aberrations and sister chromatid exchanges (1).
A is rearranged into a pyrimidine
ring-opened derivative, 4-amino-5-(imidazol-2-yl)imidazole, which has
about 20-fold higher mutagenic potency in Escherichia coli
(AB1157 lac
U169) than the parental
A (14). Because the
secondary lesions arising from
A might contribute significantly to
its mutagenesis, we undertook a detailed study of the chemical
stability of
A in DNA, during which we found enzymes
repairing a derivative formed during
A chemical rearrangement and
identified excised lesion.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
dA was synthesized
using the modified procedure of Barrio et al. (15) described
for the synthesis of its ribo-cogener. The material obtained (needles,
melting point, 163-164 °C) was 99% pure (HPLC). Its
identity was confirmed by UV, NMR, and electrospray MS.
A at position 20 in the sequence 5'-d(GCT ACC TAC CTA GCG ACC T
AC GAC TGT CCC ACT GCT CGA A)-3'
was purchased from Eurogentec Herstal, (Herstal, Belgium). The
purity and identity of this oligomer was verified by HPLC and mass
spectrometry. The oligomer was digested enzymatically to
deoxynucleosides, which were separated by HPLC (for the details see
below and Fig. 5). The deoxynucleoside content calculated on the basis
of peak areas was for dC, 16.1, dG, 7.5, dT, 8.0, dA, 7.5, and
dA, 0.9 residues/molecule, which is in good agreement with the
previewed values of dC, 16, dG, 7, dT, 8, dA, 8, and
dA, 1 residue(s)/molecule. The identity of the
A-oligomer was confirmed by
mass spectrometry using an electrospray Quadrupol-time of flight
instrument (Q-TOF, Micromass). The measured molecular mass of
A-oligomer was 12,145.3 ± 0.4, which is consistent with the
expected mass of 12,144.9. No significant traces of the compound, the
molecular weight of which would correspond to the presence in the
oligomer of compound B instead of
dA
(Mr 12,163), were detected (less than 2%). No
peak corresponding to the presence of compound C in this
oligomer (Mr 12,134) was recorded. Both methods
confirmed the identity and purity of
A-oligomer.
A were either purchased from Eurogentec or synthesized according to standard procedures using a Beckman Oligo 1000 M synthesizer (Oligonucleotide Synthesis Laboratory,
Institute of Biochemistry and Biophysics, Polish Academy of Sciences).
(lac-proAB) carrying the pFPG230 plasmid and BH410 (as JM105 but fpg-1:Kn harboring the
pNTH10) as previously described (16, 17). Human
N-methylpurine-DNA glycosylase (ANPG-40) as well as
bacterial strains overproducing Fpg and Nth glycosylases were a kind
gift from Dr. Jacques Laval (Institut G. Roussy, Villejuif, France). T4
kinase was from TaKaRa, nuclease P1 from Amersham Pharmacia Biotech,
snake venom phosphodiesterase from PL Biochemicals, and E. coli alkaline phosphatase from Sigma.
dA--
The solutions of
dA
(1-2 mM) were incubated in pH 12 (0.02 N
NaOH), pH 9.2 (0.1 M
Na2B4O7), or pH 7.5 (0.1 M phosphate buffer) at 37 °C or at room temperature
(23 °C) for various periods (15 min to 30 days) and were analyzed by
HPLC. The representative separations, retention times, and
chromatographic conditions are given in Fig. 2. The neutral
depurination of 2'-deoxyadenosine and
dA was studied in 0.1 M phosphate buffer, pH 7.5, at 60 °C by HPLC. The HPLC
analysis of depurination was performed similarly to the analysis of
alkaline degradation of
dA with the exception that gradient was
present for 30 min (the relevant retention times under these conditions
(in minutes) were: dA, 11.5; A, 9.7;
dA, 13.0;
A, 11.9; and
product B, 8.4).
dA Degradation Products--
A
reaction mixture contained ~0.1 mmol of
dA in 1 ml of 0.05 N NaOH. After 3-7 days at 37 °C, TLC in solvent II
showed the presence of product B
(Rf = 0.52) and C
(Rf = 0.67), some nonreacted
dA
(Rf = 0.60), D
(Rf = 0.38), and other products. The separation
of products was done using preparative silica gel plates run 2-4 times
in the same direction in solvent III. The appropriate bands were eluted
by methanol, and the purity of products was verified by HPLC. The final
purification was done by preparative TLC in solvent I. The products
obtained were more than 95% pure, and they were used for studies by
UV, NMR, and MS. UV (
max, nm): B, 259 (H20), 265 (pH 12), 267 (pH 1); C, 274 (H2O), 265 (pH 12), 282 (pH 1) (in conformity with spectra
of ribo-cogener (18)); D, 247 (H2O), 247 (pH 12), 242 (pH 1). Electrospray-MS analytical data
(m/z assignment and relative abundance in
parentheses):
dA, 276.1 (MH+, 100), 160.1 (BH+, 35); B, 294.1 (MH+, 100),
178.1 (BH+, 35); C, 266.1 (MH+, 100), 150.1 (BH+, 15);
D, 288.3 (not assigned, 100), 316.3 (not assigned, 40). NMR
data are gathered in supplemental Tables 1S and 2S.
A-40-mer was incubated in
0.2 N NaOH for 1-4 h, neutralized by the addition of an
equivalent amount of 1 N HCl and 1/10 volume of 1 N Tris-HCl, pH 7.0, ethanol-precipitated, and washed. Then
oligomers were digested enzymatically to nucleosides. The reaction
mixture (50 µl) contained 1.5 nmol of oligomer, 1.5 units of nuclease
P1, 0.075 units of snake venom phosphodiesterase, 0.3 units of E. coli alkaline phosphatase in 20 mM Tris-HCl, pH 8.5, and 10 mM MgCl2. After digestion (1 h,
37 °C), proteins were ethanol-precipitated, and the supernatant
containing the nucleosides was evaporated to dryness, dissolved in
water, and subjected to HPLC. The representative separations, retention
times, and chromatographic conditions are given in Fig. 5.
A and Its Degradation Products from Oligomers by
Repair Glycosylases--
The 40-mer oligodeoxynucleotide containing a
single
A or its rearrangement products obtained by oligomer
incubation in 0.2 N NaOH for 1-16 h was radiolabeled with
32P at the 5'-end and annealed to the complementary strand
(double molar excess). The release of
A or its rearrangement
products by glycosylases was assessed by measuring the cleavage of
40-mer at the site of lesion. The standard reaction mixture (20 µl)
contained 5' 32P-labeled duplex (1 pmol), 100 mM KCl, 1 mM EDTA, and 5 mM
-mercaptoethanol in 70 mM Hepes-KOH, pH 7.6 for the Fpg
protein or pH 7.8 for ANPG-40 or Nth glycosylases. The mixtures were
incubated at 37 °C for a 10 min in the presence of excess of repair
glycosylases (100-150 ng of each protein/sample) and then subjected to
20% PAGE in the presence of 7 M urea. To cleave the
oligomer at the apurinic/apyrimidinic site remaining after excision of
A by ANPG-40 protein, the reaction mixture prior to PAGE was
incubated in 0.2 N NaOH at 70 °C for 30 min. Gels were
exposed to x-ray film, scanned in an LKB densitometer, and quantified
using Microcal Origin.
A-oligomer incubated in 0.2 N NaOH for
4 h. The concentration range of the oligomer was 0.6-48
nM when the modified base was paired with dT, 4.8-65 nM when paired with dA, 0.3-75 nM paired with
dC, and 2-160 nM paired with dG. The amounts of pure Fpg
protein used in the reaction were adjusted to obtain less than 50%
utilization of substrate and equaled 0.4 ng when modified base was
paired with dT and dG, 2 ng when paired with dA, and 0.2 ng with dC. In
each experiment two control samples were set: negative without enzyme,
to quantify nonspecific breakage of oligodeoxynucleotide; and positive
with excess enzyme (150 ng of Fpg), to get 100% cleavage of oligomer. The reaction was performed precisely for 10 min and stopped by adding
sequencing kit stop solution, and reaction products were separated by
PAGE. Autoradiograms were scanned (LKB scanner), and the peaks on
resulting plots, corresponding to cleavage product and nonreacted
oligomer, were quantified using multi-peak Lorentzian fitting in
Microcal Origin. In calculations the average substrate concentration
(i.e. ([S]0 + [S]t)/2) and average velocity (i.e.
([S]0
[S]t)/t) were used (29).
Vmax and Km values were
calculated by two methods: a program using the
Eisenthall-Cornish-Bowden nonparametric algorithm (30) and direct
fitting of the hyperbolic Michaelis-Menten equation to the data
points in Microcal Origin. Values obtained by both methods differed
usually by no more than 3-6%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
dA
B
C
D (Fig. 1), which could be separated by HPLC
(Fig. 2, A and B) or by TLC. The first stage,
dA
B, was the fastest
(t1/2 at 23 °C was 2.5 h). The B
C and C
D reactions at pH 12 were at least 10 times slower than the conversion
dA
B (Fig. 2). The rearrangement of
dA
B is strongly pH-dependent; a half-time of this reaction at
37 °C equals 1.5 h at pH 12, 7 days at pH 9.2, while at pH 7.5 about 1 year, as estimated on the basis of 30-day measurements (not
shown). A half-time for B
C and C
D reactions at pH 7.5, 37 °C is about 12 days, as
judged by the HPLC analysis of the isolated compounds B and
C. Thus, at physiological pH the first step of rearrangement
in nucleosides occurs very slowly, and the subsequent steps are much
faster.
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Fig. 1.
Proposed fate of
1,N6-ethenodeoxyadenosine in DNA including
structures of the parent dA and products of
its degradation. The numbering of atoms as in the purine ring was
retained in all structures for simplicity and convenience.
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Fig. 2.
Products of
1,N6-ethenoadenine rearrangement in
alkali. Panels A and B show HPLC separation of
dA and products of its degradation formed at 23 °C after 3 h
(A) or 4 days (B) in 0.02 N NaOH.
Chromatographic conditions were the following: linear gradient of 20 mM NH4HCO2, pH 9.0, 90% methanol
in water over 60 min with a flow rate of 1 ml/min and UV
absorbance detection at 260 nm. Panel C, kinetics of
dA
rearrangement in monomer (pH 12 at 23 °C) as measured from the peak
areas obtained in HPLC. Panel D, kinetics of
dA
rearrangement in polymer (pH 13 at 37 °C) as measured from peak
areas obtained in HPLC analysis and as described in Fig. 5.
dA rearrangements under physiological conditions. The degradation of
dA was tested in the presence of several amino acids (glycine,
L-proline, L-lysine, L-serine, all
at 80 mM, and 80% saturated
DL-tyrosine), 50 mM glutathione (reduced form), and 80 mM mercaptoethanol, as well as 100 mM
NaHS, NaN3, KF, and KI, all at 37 °C in 0.1 M phosphate, pH 7.5. None of these compounds accelerated
dA
B conversion (not shown).
dA at Neutrality--
HPLC analysis of neutral
dA and dA depurination at 60 °C shows that the initial rate of
A formation is 0.08%/h, whereas that of A is 0.004%/h.
Depurination of
dA is concomitant with formation of product
B with a rate of 0.2%/h (not shown); this shows that the
glycosyl bond in
dA is 20-fold less stable than that in dA and that
the pyrimidine ring opening in
dA is 2.5 times faster than the
rupture of the glycosyl bond under neutral conditions.
A-oligomer incubated for
1-16 h in NaOH at 37 °C was analyzed by HPLC. Only
dA, products
B and C, could be identified, because compound
D was masked by components of the buffer used for enzymatic
digestion of oligomer (Figs. 2B and 5D). To reach
a rate of
dA
B conversion similar to that in
nucleoside, oligomer was treated with NaOH at a concentration 10-fold
higher than that used for nucleoside. Similarly, the
fluorescence loss, because of decomposition of
A (the only
fluorescent component of the pathway) was observed at a 10-fold higher
concentration of NaOH in polymer than in monomer (not shown). Compounds
B and C were already found in
A-oligomer not
treated with NaOH (Fig. 2D), although their amount was
negligible and differed from batch to batch; usually they
constituted 2-6% of the expected
dA amount (not shown). After
4 h of oligomer incubation in 0.2 N NaOH,
A
B and C were found in comparable amounts (Fig.
2D), whereas a 4-h incubation of monomer in 0.02 N NaOH resulted in the conversion of 80% of
dA into
B (Fig. 2C). This suggests that in polymer
the reaction is shifted toward the formation of compound C
under conditions the same as those in the monomer rate of
A
B conversion.
dA compounds B
and C were assigned using TOCSY and ROESY; the spectra are
listed in supplemental Table 1S and carbon chemical shifts in
supplemental Table 2S.
dA have been assigned following
the assignment for the ribo-cogener (31). The spectra of compound
B at room temperature contain two sets of signals (Fig.
3) that are
temperature-dependent, with the coalescence in
Me2SO-d6 at 80 °C. Each of the
signal sets probably belongs to one of the isomers of compound
B (Fig. 1). We assigned the isomer B1 to be that
in which the adenine ring has the hydroxyl group at position 2 and the
hydrogen atom at position 3. The isomer B2 has the opened
pyrimidine ring with the carbonyl group at position 2 and the proton at
position 1. The ratio of B1 and B2 isomers in
Me2SOd6 solution at 25 °C is 1:1,
whereas in D2O the ratio is 13:87.
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Fig. 3.
Part of the TOCSY spectrum of the product B
in Me2SO-d6. Two traces
corresponding to the sugar moieties of both isomers B1 and
B2 are indicated. The two cross-peaks observed at 5.92/5.74
and 5.74/5.92 ppm corresponding to H1' protons are due to
B1 B2 equilibrium.
dA. This means that the
sugar conformation remains very similar in all these compounds. The
only differences observed are those indicating the shielding effects
originated from the changes occurring within the base moiety.
However, the NMR spectra of compound D are very different.
The signals of the sugar moiety are absent, and only two very broad
signals in the proton spectrum at ~3.5 and 6.9 ppm were observed. We
have not assigned these signals and do not propose any structure for
the compound.
dA spectrum (276.1 and 160.1, respectively). Then, the peaks in the C spectrum (266.1 and 150.1) differ by 28 units from the corresponding peaks in
the B spectrum. These data are consistent with the following reaction scheme:
dA + H2O (18)
B
CO (28)
C. The presence in the mass spectrum of
D peaks of m/z 316.3 and 288.3, together with the
NMR data showing lack of deoxyribose in this compound, would indicate
that D is a dimeric form of base moiety of compound
C. The conclusion can be drawn that depurination is the
final step of
dA rearrangements.
A from the
A:T pair in the 40-mer duplex. However, its capability to cleave
A-oligomer pretreated with NaOH decreased proportionally to the time of incubation in 0.2 N NaOH (Fig.
4A). The
A-oligomer incubated in NaOH was cleaved by E. coli
formamidopyrimidine-DNA glycosylase (Fpg protein), leaving behind the
-
-elimination product, and by endonuclease III (Nth protein),
which worked by the
-elimination mechanism (Fig. 4B). At
the same enzyme concentration 40-mer was cleaved to a greater extent by
the Fpg than the Nth protein. When, however, both enzymes were used,
only the Fpg cleavage product was found (Fig. 4B), which
suggests that both enzymes recognized the same lesion, but/or Nth had a
lower affinity to the lesion. Prolonged incubation of
A-oligomer in
NaOH resulted also in a partial breakage of DNA at the
A site (Fig.
4, A and B). This could be because of
alkali-triggered hydrolysis of abasic sites formed after non-enzymatic
depurination of either
A or D (Fig. 1) or to other,
unknown mechanism.
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Fig. 4.
Enzymatic recognition of A and its degradation
product(s) in
A-oligomer by human 3-methyladenine DNA glycosylase
(ANPG) (A) and E. coli Fpg and Nth proteins
(B).
A-oligomer was incubated in 0.2 N NaOH
for 1-16 h and then subjected to cleavage by repair glycosylases, and
products were separated by PAGE. Lanes: Ctrl, control
A-oligomer untreated with repair glycosylases; ANPG, Fpg,
and Nth, oligomer digested with ANPG, Fpg, or Nth
glycosylases, respectively; 19, 20, 19- or 20-mer
standards. Panel C, quantification of the efficiency of
oligomer digestion by repair glycosylases. The percent of oligomer
cleavage was measured by phosphorimaging (Molecular Dynamics
PhosphorImager) or scanning of autoradiograms. Spontaneous breaks as
measured in the control lane were subtracted from ANPG and Fpg cleavage
products. For the Nth glycosylase, only the
-elimination
product was quantified, because it reflected the amount of excised
base.
A Rearrangement Product Excised by the Fpg and
Nth Proteins--
HPLC analysis shows that the
A-oligomer incubated
for 4 h in 0.2 N NaOH contains
dA and both products
of its conversion, compounds B and C (Fig.
5A). The amount of product B decreased substantially when oligonucleotide was digested with both the Fpg protein (Fig. 5B) and the Nth glycosylase
(Fig. 5C), suggesting that both enzymes recognize and excise
the modified base present in compound B. However, we were
unable to discriminate between both isomers of compound
B.
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Fig. 5.
HPLC identification of
A derivatives excised by Fpg and Nth proteins.
A-oligomer was incubated for 4 h in 0.2 N NaOH at
37 °C, treated or not with the excess of DNA repair glycosylases,
and precipitated to remove excised base. Precipitated oligomer was
digested to the nucleosides, which were separated subsequently by HPLC
using isocratic elution with 20 mM
NH4HCO2, pH 6.0, for 10 min and then a linear
gradient of 20 mM NH4HCO2, pH 6.0, 30% methanol in water over 30 min at a flow rate 1 ml/min) and UV
absorbance detection at 260 nm. A,
A-oligomer incubated
for 4 h in 0.2 N NaOH; B, the same as in
A but digested with the Fpg protein; C, the same
as in A but digested with the Nth protein; D,
background signals derived from enzymatic solutions used for
preparation of samples for HPLC. The relative quantity of
dA (peak
areas [A260*s*104]) in
A-oligomer untreated and treated with Fpg and Nth glycosylases was
12.9 (A), 15.9 (B), and 15.6 (C),
whereas the quantity of compound B equaled 8.1 (A), 3.7 (B), and 3.4 (C). The
quantity of compound C was 19.3 (A), 18.3 (B), and 18.3 (C).
A derivative
when paired with dT and dC were very similar to the kinetic constants
of known Fpg substrates 8-oxoG and Fapy-7MeG. Interestingly, the
Km for excision of the
dA derivative paired with
dA was an order of magnitude higher (~60 nM) and when
paired with dG two orders of magnitude higher than when it was paired
with dT and dC (~6 nM, Table
I). For the Nth protein, the
Km for excision of B from the
B:T pair was 44 nM (kcat
=13 min
1).
Kinetic constants for the removal of modified bases from
oligonucleotides by the Fpg protein
A in Oligomers in Physiological pH--
The
A-oligomer stored for several months in aqueous solutions at
30 °C was susceptible to cleavage by the Fpg and Nth proteins, suggesting that chemical rearrangement of
A also occurs
spontaneously at neutrality. To determine the rate of this process,
A-40-mer was incubated up to 30 days at pH 7.4, 37 °C and
digested with the Fpg and Nth proteins. Both enzymes cleaved the 40-mer
in a time-dependent manner (Fig.
6). We have also observed formation of
DNA breaks at the
A-site but not at other sites within an oligomer.
The migration rate of the breakage product formed at neutrality was
consistent with the
-elimination pattern. When, however,
enzymatically nondigested oligomer was incubated additionally in 0.2 N NaOH for 30 min at 70 °C, the migration rate of the
breakage product increased to that observed for
-
-elimination,
suggesting that spontaneous breaks at the
A site in neutral pH
occurred by
-elimination at abasic sites, formed because of
depurination of
A or compound D (Fig. 6A). The
sum of enzymatic and non-enzymatic breaks of
A-oligomer obtained
after its incubation for different periods at neutrality, 37 °C
gives an estimate of the rate of
A rearrangement in DNA under
physiological conditions. It equals 2% of
A in DNA/week. Because,
however, in this assay we were unable to quantify product C,
this rate is higher, approximately by 1% more per week, as judged by
the rate of B and C formation in oligomer (Fig.
2D).
View larger version (49K):
[in a new window]
Fig. 6.
Panel A, enzymatic recognition of A
degradation product(s) formed spontaneously in
A-oligomer after 7 or
30 days of incubation at 37 °C, pH 7.6. Lanes:
Ctrl, control sample stored at
30 °C for several
months; Fpg, Nth oligomer digested with repair glycosylases
Fpg or Nth or both proteins; A,
-elimination
A
oligomer incubated at pH 7.6, 37 °C for 30 days; spontaneous
breaks are seen at the
A site due to
-elimination at the
apurinic/apyrimidinic site; B,
-
-elimination, the same oligomer
as in panel A (
-elimination) but
incubated additionally in 0.2 N NaOH at 70 °C for 30 min. A product of
-elimination changed the migration rate because of
subsequent
-elimination of deoxyribose. Panel B,
quantification, performed as described in Fig. 4, of spontaneous breaks
and enzymatic cleavage in
A-oligomer incubated up to 30 days at
37 °C, pH 7.6. For the Fpg and Nth proteins, spontaneous breaks
(Ctrl) and enzymatic breaks observed in control
oligomer (0 days of incubation at 37 °C) were subtracted and thus
represent only the amount of modified base, which was formed during
incubation at 37 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
dA derivative hydrated at the C(2)-N(3) bond
(B1), which is in equilibrium with the pyrimidine ring-opened bi-imidazole product still retaining the formyl group (B2). In contrast to Basu et al. (13, 14), in our
studies we did not observe any reversal of compound B to the
parental
dA during prolonged incubation of isolated B at
pH 7.5. Because our studies were performed at the deoxynucleoside
level, whereas the former authors (13, 14) presented evidence
for this reversibility at the oligodeoxynucleotide level, one cannot exclude the possibility that
A in monomer and in oligomer behaves differently.
dA depurinates with a rate 20-fold
higher than that of unmodified dA. Basu and co-workers (13) claimed
that they did not find differences in release of
A versus
A from an oligodeoxynucleotide; however, they measured depurination at
pH 2 but not at neutrality. In the rearrangement pathway, we have also
shown the second depurination of compound C. As judged by
PAGE, under physiological conditions, about 1.5% of
A residues per
week give rise to spontaneous phosphodiester bond disruption because of
A or compound D depurination (Fig. 6). This is 1-2
orders of magnitude slower than the rate of depurination of methylated
bases 7MeA, 3MeA, 7MeG (32) as well as of another exocyclic adduct,
N2,3-ethenoguanine (33). Nevertheless, we have
shown for the first time that
A in DNA might be a source of abasic
sites and spontaneous DNA strand breaks.
A rearrangement products are not eliminated from
DNA by N-methylpurine-DNA glycosylase, which participates in
the repair of parental
A (Fig. 4). This conclusion is reached from
the observation that the rate of alkali-induced
dA
B conversion in oligomer (Fig. 2D) was almost the same as the
rate of decrease in ANPG ability to cleave 40-mer at the
A site
(Fig. 4, A and C). One of these derivatives,
compound B, is excised from DNA by E. coli DNA
glycosylases participating in the repair of the oxidized bases, Fpg and
Nth proteins (34, 35). We surmise that the Fpg protein excises
pyrimidine ring-opened isomer B2, because all known
substrates of the enzyme are characterized by the presence of the
carbonyl group, e.g. 8-oxoG,
4,6-diamino-5-formamidopyrimidine (FapyA),
2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), its alkylated
derivatives, e.g. Fapy-7MeG, and 5-hydroxycytosine (36). The
A derivative is a novel substrate for the Fpg protein. The
only other pyrimidine ring-opened base excised by the enzyme is
ring-ruptured thymine (37). Interestingly, compound B is
excised by the Fpg protein with high efficiency but only when paired
with T or C but not with A or G; and thus pairs B:A or
B:G might be not repaired and potentially mutagenic. The
excision by Nth glycosylase of
A rearrangements product was not
studied as thoroughly; nevertheless, it is clear that the enzyme acts
only on product B and that Km for this
lesion is an order of magnitude higher than for the majority of
oxidized pyrimidines, e.g. thymine glycol (17). The other purine derivative excised by this enzyme, also with a low efficiency, is FapyA but not FapyG (38). The active center cleft in the Nth protein
is probably too narrow for relatively large purine residues, but when
fragmented, e.g. oxazolone, they can be fit into it quite
efficiently and excised (39).
A and induces a different spectrum of mutations in E. coli than the parental lesion. Taking into account the fact that
formation of C under physiological conditions may be as high
as 1% of
A residues per week and that it is not excised by three
major DNA-glycosylases, ANPG, Fpg, and Nth, it cannot be excluded that this derivative is persistent in DNA and that some
A-induced mutations derive from its degradation products. DNA base ring fragmentation increases its flexibility and its potential to
form different hydrogen bond faces. Examples of changing coding
properties by DNA base ring disruption are known. Both FapyA and
4,6-diamino-5N-methyl-formamidopyrimidine (Fapy-7MeA), in
contrast to parental bases, induce SOS-dependent A
G transitions in E. coli (40, 41). In fact, mutation
spectra of
A show quite a high divergence (10, 11), which might
reflect in part the proneness of
A for chemical decomposition and
the formation of persistent mutagenic lesions.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. J. Laval (URA 147 CNRS, Institut
G. Roussy, Villejuif, France) for the kind gift of ANPG protein. We
acknowledge the use of the NMR facility of the Laboratory of Biological
NMR, Institute of Biochemistry and Biophysics, Polish Academy of
Sciences (supported in part by the State Committee for Scientific
Research). We also thank Dr. A. WysA-oligomer identity by mass spectrometry.
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FOOTNOTES |
---|
* This work was supported by European Commission Grant ENV4-CT97-0505 through a collaborative research agreement between the International Agency for Research on Cancer, Lyon, France, and the Institute of Biochemistry and Biophysics, Polish Academy of Sciences (PAS) (GI/43/4), by grant C-2/VII/11 from the Polish-French Center of Plant Biotechnology, and by Grants 6 PO4A 065 18 and E-35/SPUB/P04/206/97 from the State Committee for Scientific Research (Poland). This project was realized within the framework of the activity of the Center of Excellence in Molecular Biotechnology, Institute of Biochemistry and Biophysics PAS (WP10).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. Tel.: +4822-658-47-24;
Fax: +48-39121623; E-mail: tudek@ibb.waw.pl.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M100998200
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ABBREVIATIONS |
---|
The abbreviations used are:
A, 1,N6-ethenoadenine;
dA, 1,N6-ethenodeoxyadenosine;
ANPG-40, human
N-methylpurine-DNA glycosylase, truncated form;
C, 3,N4-ethenocytosine;
8-oxoG, 7,8-dihydro-8-oxoguanine;
HPLC, high performance liquid chromatography;
Fapy, 7MeG-2,6-diamino-5N-methyl-formamidopyrimidine;
Fpg
protein, E. coli formamidopyrimidine-DNA glycosylase;
Nth
protein, E. coli endonuclease III;
PAGE, polyacrylamide gel
electrophoresis;
MS, mass spectrometry.
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