3954-3961
Nucleic Acids Research, 1995, Vol. 23, No. 19
© 1995 Oxford University Press
Photooxidation of d(TpG) by ribofiavin and methylene
blue. Isolation and characterization of thymidylyl-(3,5>
2-amino-5-[(2-deoxy-(3-D-eryt/7ro-pentofuranosyl)amino]4Af imidazol-4-one and its primary decomposition
product thymidylyl-(3r,5)-2,2-diamino-4-[(2-deoxy-|3-DefyfAiro-pentofuranosyl)amino]-5(2H)-oxazolone
Garry W. Buchko+, Jean Cadet1, Benedicte Morin1 and Michael Weinfeld*
Radiobiology Department, Cross Cancer Institute, Edmonton, Alberta, Canada T6G 1Z2 and 1CEA/Department
de Recherche Fundamentale sur la Matiere Condensee, 38054 Grenoble Cedex 9, France
Received May 30,1995; Revised and Accepted August 25,1995
ABSTRACT
The major initial product of ribofiavin- and methylene
blue-mediated photosensitization of 2'-deoxyguanosine
(dG) in oxygen-saturated aqueous solution has previously been identified as 2-amino-5-[(2-deoxy-(3-D-e/yf/7ro-pentofuranosyl)amino]-4H-imidazol-4-one (dlz). At
room temperature in aqueous solution dlz decomposes
quantitatively to 2,2-diamino~4-[(2-deoxy-p-D-eryf/7/%>pentofuranosyl)amino]-5(2W)-oxazolone (dZ). The data
presented here show that the same guanine photooxidation products are generated following ribofiavin- and
methylene blue-mediated photosensitization of thymidylyl-(3',5>2'-deoxyguanosine [d(TpG)]. As observed for
the monomers, the initial product, thymidylyl(3',5>2-amino-5-[(2-deoxy-p-D-eryf/jro-pentofuranosyl)amino]-4W-imidazol-4-one [d(Tplz>], decomposes in
aqueous solution at room temperature to thymidylyl(3',5>2,2-diamino-4-[(2-deoxy-p-D-eo^/i«>-pentofuranosyl)amino]-5(2H)-oxazolone [d(TpZ)]. Both modified
dinucleoside monophosphates have been isolated by
HPLC and characterized by proton NMR spectrometry,
fast atom bombardment mass spectrometry, chemical
analyses and enzymatic digestions. Among the chemical
and enzymatic properties of these modified dinucleoside
monophosphates are: (i) d(Tplz) and d(TpZ) are alkalilabile; (ii) d(Tplz) reacts with methoxyamine, while d(TpZ)
is unreactive; (iii) d(Tplz) is digested by snake venom
phosphodiesterase, while d(TpZ) is unaffected; (iv)
relative to d(TpG), d(TpZ) and d(Tplz) are slowly digested
by spleen phosphodiesterase; (v) d(Tplz) and d(TpZ) can
be 5'-phosphorylated by T4 polynucleotide kinase. The
first observation suggests that dlz and dZ may be
responsible for some of the strand breaks detected
following hot piperidine treatment of DNA exposed to
photosensitizers.
INTRODUCTION
While the cytotoxic effect of light-activated compounds on cells
wasfirstreported almost 100 years ago (1), the biological basis for
cell death is still not fully understood (2). Many of these
light-activated compounds, called photosensitizers, are endogenous and may play a role in the natural aging process and the
deleterious effects of solar radiation (3). Other photosensitizers,
such as some polycyclic aromatic hydrocarbons, are exogenous
and may contribute to the biological hazards associated with
environmental pollutants. However, the cytotoxic effects of
photosensitizers may be used beneficially, as illustrated by
photodynamic therapy currently used to treat various skin diseases
and malignant tumors (4-6). While DNA may not be the primary
target biomolecule for these photoagents, oxidation of DNA has
mutagenic and potentially carcinogenic consequences (7-10). The
characterization of the genotoxic photolesions arising from DNA
oxidation may therefore shed light on how photosensitizers kill
cells and on the initial events of carcinogenesis.
Photosensitizers, light-activated into excited states, generate
DNA modifications on their return to the ground state via two
competitive mechanisms, termed type I and type II (11). Type I
mechanisms involve direct proton or electron transfer by the
excited photosensitizer to or from the substrate (DNA) to
generate a free radical. Type II mechanisms involve the initial
generation of singlet oxygen ('02) by the excited photosensitizer.
Each mechanism generates a different set of DNA photoproducts
specifically at guanine residues (12-16). Photoexcited methylene
blue generates oxidation decomposition products predominantly,
but not exclusively, through a type II reaction mechanism, while
* To whom correspondence should be addressed
+
Present address: Macromolecular Structure and Dynamics, Pacific Northwest Laboratories, PO Box 999, Richland, WA 99352, USA
Nucleic Acids Research, 1995, Vol. 23, No. 19 3955
dlz
dZ
Figure 1. Structure and numbering scheme for 2-amino-5-[(2-deoxy-p-D-ery''"i'-pentofuranosyl)amino]-4//-imidazol-4-one
[(2-deoxy-P-D-eryf/iro-pentofuranosyl)amino]-5(2H)-oxazolone (dZ), the modified components of d(Tp!z) and d(TpZ), respectively.
photoexcited riboflavin generates decomposition products primarily through a type I mechanism (16-18).
The major type II oxidation products generated using 3',5'-di0-acetyl-2'-deoxyguanosine and 2'-deoxyguanosine (dG) as
substrates for photosensitization are N-(2-deoxy-$-D-erythropentofuranosyl)cyanuric acid, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and the 4R* and 45* diastereoisomers of
4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine (dO) (17,19).
Of these lesions, only cyanuric acid has not been identified at the
oligomer level (20-22). On the other hand, using dG as the
substrate for photosensitization the major type I oxidation product
generated is 2-amino-5-[(2-deoxy-P-D-ery?/iro-pentofuranosyl)amino]-4//-imidazol-4-one (dlz), which decomposes in
aqueous solution to 2,2-diamino-4-[(2-deoxy-P-D-eryr/iro-pentofuranosyl)amino]-5(2//)-oxazolone (dZ) (Fig. 1) (23). Both of
these latter compounds can also be produced by type II
photooxidation of 8-oxodG (24). While dlz and dZ have been
observed at the nucleoside level, it remained to be determined
whether these modifications are generated within oligonucleotides.
Using thymidylyl-(3',5')-2'-deoxyguanosine [d(TpG)] as a substrate for riboflavin- and methylene blue-mediated photosensitization, we report here that the major photoproduct generated is
thymidyly l-(3 ',5 ')-2-amino-5- [(2-deoxy- |}-D-eryrftrc>-pentofuranosyl)amino]-4//-imidazol-4-one [d(TpIz)], which slowly converts
in aqueous solution to thymidylyl-(3',5')-2,2-diamino-4[(2-deoxy-P-D-e/7fW-r^ntofuranosyl)amino]-5(2/y)-oxazolone
[d(TpZ)]. Various physical and biochemical properties of these
latter two products are described which should assist in establishing protocols to identify the presence of these lesions in larger
DNA substrates.
(dlz) and 2,2-diamino-4-
(Baie d'Urfe, Quebec, Canada); calf alkaline phosphatase
(1 U/|il) was from Boehringer Mannheim Canada (Dorval,
Quebec, Canada); spleen phosphodiesterase (0.02 U/(il) was
from Worthington Biochemicals (Freehold, NJ); SI nuclease
(100 U/(J.l) was from BRL Canada (Burlington, Ontario, Canada).
Definitions of the units of these enzymes were those given by the
supplier.
Methylene blue-mediated photosensitization of d(TpG)
In a 14.5 mm diameter glass vial a 300 (xl solution of 0.2 mM
d(TpG) and 0.2 mM methylene blue was irradiated with a 100 W
spotlight (Canadian General Electric 45-T8) at a distance of
16 cm (21) for 5-120 rnin using a Kodak 23A filter to eliminate
radiation shorter than 590 nra. During the irradiation the solution
was maintained near room temperature with circulating water and
constant stirring. Aliquots (30-50 (J.1) were periodically removed
to monitor photoproduct formation by high performance liquid
chromatography (HPLC). Experiments were repeated replacing
H2O with 98% D2O.
Riboflavin-mediated photosensitization of d(TpG)
A 3.0 ml saturated solution of riboflavin plus 5 mg d(TpG) was
irradiated in a 20 ml beaker at 365 nm (two Syvania F8T5/BL
light tubes) in a Brinkmann Chromato-vue model CC-20
ultraviolet box at a distance of 5 cm. After a significant build up
of photoproducts most of the solvent was removed by rotary
evaporation (<30°C) and the undissolved riboflavin removed by
passage through a Millipore Millex-GV4 0.2 mm syringe filter
prior to HPLC analysis.
MATERIALS AND METHODS
Chromatographic separation of photoproducts
Chemicals and enzymes
Samples were injected onto either a 5 \im Hypersil (10 x 250 mm;
Phenomenex) semi-preparative or nBondapak (4 x 300 mm;
Waters) analytical octadecylsilyl reverse-phase column. The
gradient programs used to monitor photochemical or enzymatic
reactions were as follows: 100% solution A (0.1 M ammonium
acetate, pH 6.0) and 0% solution B (methanol) for 1 rnin, followed
by a linear gradient to 75% solution A and 25% solution B over
50 min at a flow rate of 1.2 ml/min (semi-preparative column) or
1.0 ml/min (analytical column) (Table 1). The product profile was
monitored by UV absorbance at 260 nm with a Tracor 970A
The dinucleoside monophosphate d(TpG), prostatic acid phosphatase (0.1 U/u.1) and snake venom phosphodiesterase (Crotalus
adamanteus, 1 U/ml) were purchased from Sigma Chemical
Company (St Louis, MO); [y-*2P] ATP (4500 Ci/mmol) was from
ICN Canada (Montreal, Quebec, Canada); piperidine was from
Aldrich Chemical Co. (Milwaukee, WI); methylene blue was
from Fluka Chemika-BioChemika (Ronkonkoma, NY); T4
polynucleotide kinase (10 U/(il) was from Pharmacia Canada
3956 Nucleic Acids Research, 1995, Vol. 23, No. 19
Variable Wavelength Detector. Quantities of d(TpIz) sufficient
for mass spectral and NMR studies were isolated with the
semi-preparative column using the elution conditions described
above, except for the substitution of unbuffered H2O for 0.1 M
ammonium acetate. Riboflavin was used in the photosensitization
reactions to obtain such quantities of d(TpIz) because of the
difficulty of removing methylene blue from the octadecylsilyl
column. Following photosensitization the major peaks were
collected, pooled, concentrated by rotary evaporation (<30°C)
and lyophilized at least once. The substitution of H2O for
ammonium acetate in the HPLC gradient program is critical
because d(TpIz) will otherwise decompose during rotary evaporation and lyophilization.
recorded at a proton frequency of 600.13 MHz on a Bruker
AI^X600 spectrometer. Conventional homonuclear decoupling
and two-dimensional coherence (25) and relay coherence (26)
experiments were conducted for proton spectral assignments. The
chemical shifts of the base and anomeric protons are provided in
Table 3.
Table 3. Proton chemical shifts (a, p.p.m. from DSS) of the base and
anomeric protons of d(TpG), d(TpIz) and d(TpZ)a
Molecule
H6
Me5
HI'
H8
HI'
7.41
1.84
6.06
8.04
6.18
d(TpIz)
7.56
1.86
6.24
5.83
d(TpZ)
7.64
1.89
6.31
5.78
d(TpG)
Table 1. HPLC retention times (min) for selected dinucleoside
monophosphates and nucleosides on semi-preparative and analytical columns
pX
Tp
b
a
Molecule
Retention time
Semi-preparative
Analytical
Spectra obtained at 23°C for d(TpIz) and d(TpZ) and 10°C for d(TpG) in 0.1
M NaCl.
b
Data for d(TpG) obtained from Buchko and Cadet (32).
d(TpG)
53
35
d(TpIz)
45
26
Fast atom bombardment (FAB) mass spectrometry
d(TpZ)
39
20
d(TpAP)
41
22
Negative ion mode FAB mass spectra {glycerol [d(TpZ) and
d(TpG)] or 6:1 (w/w) dithiothreitol:dithioerythritol [d(TpIz)]
matrix, 8-keV xenon atoms} were obtained on a modified
AEI/Kratos MS-9 spectrometer. Some of the more diagnostic
peaks are reported here as follows: mlz (relative intensity, [ionic
species]) with the relative intensity normalized to the molecular
ion [M - H]~. d(TpIz): 553(138, [M + Na - H]~), 531(100, [M H]-), 329(67, [5'-dIzMP + Na - H]-), 307(57, [5'-dIzMP - H]"),
343(28, [3'-TMP + Na - H]-), 321(63, [3'-TMP - H]-). d(TpZ):
571(100, [M + Na - H]-), 549(100, [M - H]-), 347(163,
[5'-dZMP + Na - H]-), 325(250, [5'-dZMP - H]-), 343(200,
[3'-TMP + Na - H]-), 321(787, [3'-TMP - H]-). d(TpG):
570(100, [M - H]~), 346(20, [5'-TMP - H]-), 321(42, [3'-TMP
-H]-).
d(TpM)
50
36
d(TpO)-A
37
18
d(TpO)-B
40
20
dlz
26
6
dZ
12
6
dG
43
20
dT
46
21
To detect 5'-32P-labeled nucleotides a Berthold HPLC-Radioactivity Monitor (LB504) was connected to the HPLC system.
Products were separated with the semi-preparative column using
the following elution program: 100% solution A for 10 min
followed by a linear gradient to 75% solution A and 25% solution
B over 50 min at a flow rate of 1.2 iruVmin (Table 2).
Table 2. HPLC retention times (min) for selected 5'-phosphorylated
dinucleoside monophosphates on a semi-preparative column
Molecule
Retention time
pTpIz
45
pTpZ
38
pTpAP
38
pTpM
53
Absorbance spectroscopy
The following UV absorption data were obtained in 10 mM
NaH2PC<4 buffer, pH 7.4, on a Beckman DU-640 spectrophotometer: d(TpIz): ^ a x = 259 nm, X^n = 229 nm; d(TpZ): A^ax =
268 nm, Xmjn = 229 nm; d(TpG): Xmax = 256 nm (major), 264
(shoulder), X^n = 228 nm; dlz: A^^ = 259 nm, k^n = 229 nm;
dZ: ^max = 207 nm (major), X^n = 232 nm (shoulder); 3'-TMP:
=
268 nm, A-rnin = 235 run.
Hot alkali treatment
Dinucleoside monophosphate samples (-1.0 OD260) were suspended in 100 nl 1.0 M piperidine and incubated at 90°C for
30 min. Following incubation the piperidine was removed by
three freeze drying cycles. The samples were resuspended in
water and the reactions monitored by HPLC.
Proton nuclear magnetic resonance ( ] H NMR) spectroscopy
Samples were lyophilized twice from 99.8% D2O prior to
resuspension in 400 \i\ NMR buffer (pD 7.0) consisting of 0.1 M
sodium chloride, 0.01 M sodium phosphate and 0.001 M
ethylenediaminetetraacetic acid (EDTA). A trace of t-butanol was
added to serve as an internal NMR reference (assigned a chemical
shift of 1.231 p.p.m. on the DSS scale). The proton spectra were
Methoxyamine treatment
Dinucleoside monophosphate samples (-2.0 OD260) were incubated for 30 min at 37° C in a total volume of 100 u.1 containing
50 mM methoxyamine and 40 mM NaH2PO4, pH 7.6. Reactions
were monitored by direct injection onto the semi-preparative
HPLC column.
Nucleic Acids Research, 1995, Vol. 23, No. 19 3957
pH stability profile of d(TpIz)
d(Tplz)
A 300 ul solution containing 5.0 OD260 of d(TpIz) was incubated
in 10 mM NaH2PO4 buffers at pH 4.8, 7.4 and 10.4. A 30 |il
aliquot was removed at various time intervals and analyzed
directly by HPLC.
dfTpG)
Spleen phosphodiesterase digestions
Dinucleoside monophosphate samples (-2.0 OD260) were incubated at 37°C in 100 u.1 5 mM Tris-HCl, pH 7.4, with 0.02 U
spleen phosphodiesterase. After 30 min the reaction was stopped
with 1.0 ul 0.5 M EDTA and immediately analyzed by HPLC
using the semi-preparative column.
d(TPZ)
u
\
n
o
w
B
Snake venom phosphodiesterase digestions
Dinucleoside monophosphate samples (-1.0 OD260) were incubated overnight at 37 °C in 40 ul digestion buffer (10 mM
Tris-HCl, pH 7.5, 4 mM MgCl2) containing 0.002 U snake
venom phosphodiesterase and 0.4 U calf alkaline phosphatase.
Reactions products were analyzed by direct injection onto the
semi-preparative HPLC column.
0
SI nuclease and acid phosphatase digestions
Dinucleoside monophosphate samples (-2.0 OD260) were incubated for 30 min at 37° C in a total volume of 50 ul containing
50 mM NaCl, 10 mM ammonium acetate, 1 mM ZnC^, 100 U
51 nuclease and 0.25 U acid phosphatase buffered at pH 5.3.
Reaction products were analyzed by direct injection onto the
semi-preparative HPLC column.
20
40
Retention time (min)
Figure 2. Reverse phase HPLC profiles of the photodamage to dfTpG)
immediately following exposure to visible light in oxygenated aqueous
solutions (H2O) containing riboflavin (A) or methylene blue (D2O) (C). (B)
Reverse phase HPLC profile of the products obtained after isolating the HPLC
peak in chromatograms (A) and (C) that elutes at 26 min and incubating it at
37°C for 24 h. The chromatograms were obtained on an analytical column
using the elution conditions described in the text. The peak marked with an
asterisk contains d(Tp8-oxoG).
5-Phosphorylation with T4 polynucleotide kinase
Approximately 0.01 OD260 units of d(TpIz) and d(TpZ) were
incubated for 30 min at 37°C with 1.1 pmol (5 uCi) [Y- 3 2 P]ATP
and 5 U T4 polynucleotide kinase in 10 ul lx One-Phor-AllPLt/5
buffer (Pharmacia). Reactions were monitored by direct injection
onto the semi-preparative HPLC column on line with a radioactivity detector.
RESULTS AND DISCUSSION
Physicochemical characterization of d(TpIz) and d(TpZ)
Figure 2 A is an HPLC chromatogram (analytical column) of the
products of riboflavin-mediated photosensitization of d(TpG) in
aerated aqueous solution. The initial major HPLC peak generated, with a retention time of 26 min, has been identified as
d(TpIz). Isolation of this major product followed by its incubation
in aqueous solution at 37 °C leads to its conversion to a peak with
a retention time of 20 min (Fig. 2B). The content of the latter peak
has been identified as d(TpZ). The HPLC peak which elutes at
45 min (under asterisk, Fig. 2A) contains a dinucleoside monophosphate with a 7,8-dihydro-8-oxo-2'-deoxyguanosine lesion
[d(Tp8-oxoG)].
The HPLC profile of the products of methylene blue-mediated
photosensitization of d(TpG) in D2O immediately following a
5 min exposure to visible light also contains a significant peak
which elutes at 26 min (Fig. 2C). This peak co-elutes with the
major peak observed in Figure 2A when the photoproducts are
co-injected on the HPLC column and this isolated product
similarly decomposes to a peak which elutes at 20 min. However,
in comparison with Figure 2A there is the significant presence of
other photoproducts. The peaks with retention times of 18 and
20 min have previously been observed following phthalocyanine-mediated photosensitization of d(TpG) and shown to
contain dinucleoside monophosphates containing the 4R* and
45* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine [d(TpO)-A and d(TpO)-B] (22). The peak with a
retention time of 18 min also contains a minor product identified
as thymidylyl-(3',5')- l-(2-deoxy-P-D-«ry/Aro-pentofuranosyl)oxaluric acid (G.W.Buchko and J.Cadet, in preparation). The
peak which elutes at 22 min co-migrates and shares chemical
properties with the authentic apurinic dinucleoside monophosphate d(TpAP). The peaks which elute at 16 and 24 min disappear
following overnight incubation at room temperature and efforts
are currently in progress to identify and characterize these latter
compounds and their decomposition products. Note the absence
of an HPLC peak corresponding to d(Tp8-oxoG), which eluted at
45 min in Figure 2A.
The exposure of dG to photoexcited phthalocyanines and
methylene blue generates oxidation products primarily through a
type II reaction mechanism (17,27). On the other hand, the
exposure of dG to photoexcited riboflavin generates oxidation
decomposition products primarily through a type I reaction
mechanism (16). It is possible to recognize photoproducts
generated through a type II reaction mechanism by conducting
the experiment in deuterated solvent, which increases the lifetime
of '02 (28) and, consequently, may increase the yield of type II
products (16). Such an increase was observed not only for
3958 Nucleic Acids Research, 1995, Vol. 23, No. 19
endoperoxide intermediate (?)
dG
neutral radical intermediate
Figure 3. Photosensitization pathways proposed for the generation of dlz from dG. R = 2-deoxy-P-D-eryr/iro-pentofuranosyl. The endoperoxide intermediate has
recently been observed at-80°C following photosensitized oxygenation of 2',3',5'-tri(t-butyldimethylsilyl)oxy-7,8-dihydro-8-oxoguanosine with tetraphenylporphine
and Rose Bengal in organic solvents (64).
As mentioned, the nucleoside dlz has previously been characterized as a major product of type I-mediated photosensitization of dG and one of at least four products of type II-mediated
photosensitization of 8-oxodG. In aqueous solution dlz is
hydrolyzed to dZ, a process enhanced by heating or the presence
of base (23,30). Because the mononucleosides have been
characterized, HPLC markers exist to identify these lesions in
oligomers following enzymatic digestion. Such a digestion of
d(TpIz) with S1 nuclease and acid phosphatase for 30 min results
in products with HPLC retention times identical to thymidine
(dT), dlz and a minor peak corresponding to dZ. Increasing the
period of d(TpIz) digestion to 24 h results in only two HPLC
peaks, corresponding to dT and dZ. On the other hand, a 30 min
S1 nuclease and acid phosphatase digestion of d(TpZ), obtained
from the prior heating of d(TpIz), only generates HPLC peaks
with retention times corresponding to dT and dZ, as illustrated in
Figure 4.
The negative ion mode FAB mass spectra of d(TpIz) and
d(TpZ) indicate that the modified components of d(TpIz) and
d(TpZ) are 2,5-diamino-4//-imidazol-4-one and 2,2,4-triamino-
dZ
\
Q)
U
re
.Q
d(TpZ)
\
dT
/
*
bsoi
d(TpO)-A and -B, but also for d(TpIz) when the methylene blue
reaction was conducted in D2O [the HPLC chromatogram pattern
after 5 min of methylene blue-mediated photosensitization of
d(TpG) in H2O is similar to that of Fig. 2C except for a drastic
decrease in intensities of all the peaks]. An increase in the yield
of d(TpIz) was also observed, to a lesser extent, when the
riboflavin-mediated photosensitization experiment was performed in D2O. These latter observations may be interpreted in
the light of similar photosensitization experiments performed on
dG and 8-oxodG, which show that dlz may be generated through
type II photooxidation of 8-oxodG, as well as through type I
photooxidation of dG, as illustrated in Figure 3 (23,24). Indeed,
8-oxodG is a better substrate for type II photooxidation than dG
itself, being converted to dlz, the AR* and 45* diastereoisomers
of dO and another as yet unidentified product with a molecular
weight of 287 (24,29). Therefore, the observed increase in yield
of d(TpIz) in D2O is likely due to type II photooxidation of the
photoproduct d(Tp8-oxoG), which is generated through both type
I and type II mediated photosensitization of d(TpG) (18,20). Such
an event explains the absence of an HPLC peak containing
d(Tp8-oxoG) following methylene blue-mediated photosensitization of d(TpG) (Fig. 2C), a product which is detectable following
riboflavin-mediated photosensitization of d(TpG) (Fig. 2A).
<
0
25
50
Retention time (min]
Figure 4. Reverse phase HPLC profile of the products generated by incubation
of d(TpZ) with SI nuclease and acid phosphatase for 30 min. The chromatogram was obtained on a semi-preparative column using the conditions
described in the text. The peak marked with an asterisk is from the digestion
cocktail.
5(2/f)-oxazolone, respectively. For d(TpIz) a pseudomolecular
ion at mlz 531 is observed, indicating a molecular mass of 532,
which is 39 mass units, or two carbons, one nitrogen and one
hydrogen, smaller than d(TpG). Fragmentation ions at mlz
307([5'-dIzMP - H]-) and 321([3'-TMP - H]") further support
the structure of this photoproduct. On the other hand, a
pseudomolecular ion at mlz 549 is observed for d(TpZ),
indicating that it has a molecular mass of 550. In comparison with
d(TpIz), d(TpZ) is larger by 18 mass units, or one molecule of
H2O, which is expected after hydrolysis. Fragmentation ions at
mlz 325([5'-dZMP - H]") and 321([3'-TMP - H]~) further
support the structure of this decomposition product. Additional
evidence for our assignments of these ions are the observation of
a sodium ion 22 mlz units larger for each species containing a
phosphate group.
Further support for the structures proposed for d(TpIz) and
d(TpZ) are the >H NMR spectra for both molecules. Unlike the
proton spectra of d(TpG), which contains two downfield
resonances assigned to thymine H6 and guanine H8 (31,32), only
the downfield thymine H6 resonance is observed in the spectra of
d(TpIz) and d(TpZ). Furthermore, the H6 chemical shifts of
d(TpIz) and d(TpZ) are >0.2 p.p.m. downfield relative to the H6
resonance of d(TpG), suggesting that the modified dimers are
experiencing weaker ring current effects from the neighboring
Nucleic Acids Research, 1995, Vol. 23, No. 19 3959
d(TpIz) and d(TpZ) are sensitive to hot piperidine
treatment
Hot piperidine treatment of oxidized DNA generates strand
breaks at sites of base modification or base loss (33). Such
piperidine-labile sites have been found at the sugar-phosphate
bond 5' of guanine residues following treatment of doublestranded DNA with chemically generated singlet oxygen (34,35)
and with photosensitizers such as methylene blue (36,37) and
riboflavin (38). Because apurinic sites only account for a small
fraction of the DNA damage produced by both methods, as
determined by repair studies using AP endonucleases (39,40), it
has been postulated that chemically modified guanine residues,
including 8-oxodG (41,42), account for the remaining piperidinelabile sites (23). Both d(TpIz) and d(TpZ) are decomposed by hot
piperidine treatment to a product with a HPLC retention time
identical to 3'-TMP and, therefore, these compounds may be
responsible for a proportion of the alkali-labile sites observed
following photosensitizer-mediated oxidation of DNA.
pH stability of d(TpIz)
Figure 5 is a plot of the time-dependent decomposition of d(TpIz)
at 37°C in sodium phosphate buffers of pH 4.8,7.4 and 10.4. The
dinucleoside monophosphate is most stable at pH 7.4, more
unstable under alkaline conditions, but most unstable under acidic
conditions. This is in contrast to the behavior of the monomer,
where dlz was shown to be hydrolyzed at equal rates at pH 4.8 and
7.4, but decomposed most rapidly at pH 10.4 (30). The major
initial decomposition product of d(TpIz) is d(TpZ). However,
with increasing length of time, especially under alkaline conditions, d(TpZ) also decomposes, with the release of guanidine
(23).
Methoxyamine treatment of d(TpIz) and d(TpZ)
The apurinic dinucleoside monophosphate d(TpAP) has an
HPLC retention time near that of d(TpZ) (Table 1). A common
method of detecting apurinic sites is by chemical modification
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base. Such observations dictate that the guanine base has been
modified and may have a less aromatic character. Because the
remaining protons of the modified guanines are exchangeable
with D2O and are not detectable, the *H NMR spectra provide no
further direct information regarding the chemical modification.
However, the latter spectra each show the presence of two
2-deoxyribose moieties in a 1:1 ratio, indicating that dinucleoside
monophosphates have been isolated and that these samples are
>95% pure.
The UV absorption spectra for d(TpIz) and d(TpZ) also
indicate that a chemical reaction altered the electronic structure
of d(TpG). This is because in comparison with d(TpG) the UV
absorption spectra of d(TpIz) and d(TpZ) are different, both
lacking a A.max shoulder at 264 nm. Examination of the UV
spectra of the modified nucleosides indicates that dlz still has
some aromatic character, showing an absorption maximum at
259 nm, while dZ has lost all its aromatic character, as reflected
in the continuous decrease in UV absorbance at wavelengths
longer than 232 nm (23). The latter loss of UV absorption for dZ
is apparent in the UV absorption spectrum of d(TpZ), which is
now similar to that of 3'-TMP, with A^^ of 268 and 267 nm,
respectively.
n -i
U
0
2
4
7.4
^10.4
>« 4.8
6
8
1(
Time (h)
Figure 5. Plot of the kinetics of decomposition of d(TpIz) at 37°C in 10 mM
sodium phosphate at pH 4.8, 7.4 and 10.4.
with methoxyamine (43—45), which alters their HPLC retention
times. Such a treatment of d(TpAP) leads to its conversion to the
methoxyamine derivative d(TpM), which has an HPLC retention
time of 50 min on the semi-preparative column, which is distinct
from the 41 min retention time of d(TpAP). A similar treatment
of d(TpZ) saw no change in its HPLC retention time of 39 min,
indicating that this lesion was not an apurinic site. On the other
hand, methoxyamine treatment of d(TpIz) does produce a
derivative with an HPLC retention time identical to d(TpM). In
the latter case d(TpIz) is converted to d(TpAP) and then reacts
with methoxyamine, because methoxyamine treatment of dlz
generates a methoxyamine-modified 2-deoxyribose (B.Morin
and J.Cadet, in preparation).
Snake venom and spleen phosphodiesterase treatment
ofd(TpIz)andd(TpZ)
Incubation of d(TpIz) with snake venom phosphodiesterase for
30 min produced two major HPLC peaks which co-eluted with dT
and dlz and two minor HPLC peaks which co-eluted with dZ and
d(TpZ). Increasing the period of incubation to 24 h saw the total
conversion of the dlz peak to dZ, while the intensity of the d(TpZ)
peak remained unchanged. The latter observation suggested that
once d(TpIz) was hydrolyzed to d(TpZ) it was resistant to snake
venom phosphodiesterase and this was confirmed using pure
d(TpZ) as starting material. Impediment of snake venom
phosphodiesterase's ability to hydrolyze the phosphodiester bond
attached to other altered 3'-deoxynucleosides has previously been
observed. These modified nucleosides include apurinic sites
(46,47), thymine glycols (48), 6-methyluracil (49), 4,8-dihydro-4-hydroxy-8-oxoguanine (22) and C^-isopropylthymine
(50). Interestingly, snake venom phosphodiesterase can act on the
base modification of dlz, a planar 5-member ring separated from
the sugar by a nitrogen. However, when the same base loses its
aromaticity and becomes bulkier as a result of geminal amino
groups and ring puckering, snake venom phosphodiesterase can
no longer function.
Digestion of d(TpZ) with spleen phosphodiesterase generates
3'-TMP and dZ, although relative to d(TpG) the digestion rate is
dramatically reduced. In less than 1 h spleen phosphodiesterase
3960 Nucleic Acids Research, 1995, Vol. 23, No. 19
completely hydrolyzed d(TpG), while the same quantity of
enzyme produced marginally detectable quantities of d(TpZ)
digestion products. Indeed, using 10 times the quantity of
enzyme, 15% of d(TpZ) remained undigested after 24 h. Similar
results were obtained for the spleen phosphodiesterase digestion
of d(TpIz), although quantitation was hampered by the significant
conversion of d(TpIz) and dlz to d(TpZ) and dZ at 37°C. The
reduced rate of hydrolysis of both d(TpZ) and d(TpIz) relative to
d(TpG) is consistent with other observations that suggest that
spleen phosphodiesterase interacts with the 3' base (as well as the
5' base) flanking an internucleotide phosphodiester bond
(47,51-54).
CHCM program (contract no. CHRX 93.0275 to JC) and an
Alberta Cancer Board postdoctoral fellowship (GWB).
Phosphorylation of d(TpIz) and d(TpZ) by T4
polynucleotide kinase
8
9
10
11
It is possible to 5'-end-label both d(TpIz) and d(TpZ) with
[y-32P]ATP using T4 polynucleotide kinase and detect distinct
HPLC peaks with retention times of 45 and 38 min, respectively
(Table 2). Overnight incubation of 32pTpIz in aqueous solution at
37 °C results in its complete conversion to 32pTpZ, as determined
by HPLC analyses. Treatment of the 5'-labeled molecules with
methoxyamine converts 32pTpIz to 32pTpM (retention time
53 min), while 32pTpZ is unaffected. Because d(TpZ) resists
hydrolysis by snake venom phosphodiesterase and is a substrate
for T4 polynucleotide kinase, it should be possible to detect this
lesion in DNA using an appropriate 32P-postlabeling assay
(48,55).
REFERENCES
1
2
3
4
5
6
7
12
13
14
15
16
17
18
19
20
CONCLUSIONS
21
The data reported here show that the major products of methylene
blue- and riboflavin-mediated photosensitization of d(TpG) are
dinucleoside monophosphates containing dlz and dZ. To date the
only guanine lesions unambiguously identified in DNA exposed
to methylene blue plus light are 8-oxodG (20) and 2,6-diamino4-hydroxy-5-formamidopyrimidine (FapydG) (56), with the
level of FapydG ~20-fold lower than the level of 8-oxodG. On the
other hand, the only guanine modification unambiguously
identified in DNA exposed to riboflavin plus light is 8-oxodG
(18,38). The latter observation also applies to DNA exposed to a
chemical source of singlet oxygen (20,57). In all three instances
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progress to verify the generation of dlz and dZ in DNA exposed
to excited photosensitizers or to singlet oxygen. Others who have
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other than 8-oxodG are formed (58-60). One reason for such a
conclusion is that while unrepaired 8-oxodG most frequently
leads to G—>T transversions (61-63), G—*C trans versions are the
dominant mutation in single-stranded M13mp2 bacteriophage
DNA exposed to methylene blue plus light when transfected into
SOS-induced Escherichia coli (58).
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
ACKNOWLEDGEMENTS
We thank Drs A.Hogg and A.Morales (University of Alberta,
Edmonton) for recording the mass spectra. This research was
funded by NCI Canada (MW), the European Commissions
40
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