[CANCER RESEARCH 49. 3463-3467, July I, 1989]
Structure and Mechanism of Hydroxyl Radical-induced Formation of a DNAProtein Cross-Link Involving Thymine and Lysine in Nucleohistone
Mirai Dizdaroglu1 and Ewa Gajewski
Center for Chemical Technology, National Institute of Standards and Technology, Gaithersburg, Maryland 20899
ABSTRACT
Hydroxyl radical-induced formation of a DNA-protein cross-link in
volving thymine and lysine in calf thymus nucleohistone in vitro is
reported. Basic amino acids such as lysine constitute a very high propor
tion of the amino acids of histones, and help histones to bind to DNA in
chromatin. For this reason, basic amino acids are likely to participate in
DNA-protein cross-linking. For identification of the thymine-Iysine cross
link in nucleohistone, hydroxyl radical-induced cross-linking of thymine
to lysine was investigated first using a model system, i.e., an aqueous
mixture of thymine and lysine. Hydroxyl radicals were generated by
exposure of this mixture to ionizing radiation after VO saturation. The
technique of gas chromatography-mass spectrometry was used to analyze
the samples for possible cross-links. One thymine-Iysine cross-link was
found and its structure was elucidated. Using gas chromatography-mass
spectrometry with selected-ion monitoring, this thymine-Iysine cross-link
was identified in acidic hydrolysates of calf thymus nucleohistone yirradiated in N2O-saturated aqueous solution. The yield of this DNAprotein cross-link was also measured and found to be a linear function of
radiation dose between 15 and 200 Gy. This yield amounted to 0.0085
ftmol/J. Possible mechanisms for the formation of this DNA-protein
cross-link in nucleohistone were proposed.
INTRODUCTION
DNA-protein cross-links are one of various types of damage
in cellular DNA induced by ionizing and UV radiations, and
by a number of carcinogenic and chemotherapeutic chemicals
(1-4). There is evidence that the chemical bonds involved in
DNA-protein cross-links are of a covalent nature (4-6). Hy
droxyl radicals appear to play an important role in the forma
tion of ionizing radiation-induced DNA-protein cross-links in
chromatin in vitro and in intact cells (4, 5). The involvement of
core histones and nonhistone proteins in DNA-protein crosslinking in 7-irradiated chromatin has been demonstrated (5, 7).
However, little is known about the chemical nature of DNAprotein cross-links and their mechanisms of formation. An
understanding of the mechanisms of this DNA damage in cells
will depend on the chemical characterization of the constituents
of DNA and proteins, and the bonds involved in cross-linking.
The knowledge of the chemical nature of DNA-protein cross
links will also be necessary for assessment of their role in free
radical-induced biological endpoints such as cell lethality, mu
tation, and carcinogenesis.
Recently, we described the chemical nature of a number of
OH radical-induced DNA-protein cross-links involving Thy2
and various amino acids in calf thymus nucleohistone in
aqueous solution, and we also proposed mechanisms for their
formation (8, 9). In the present work, \ve investigated DNAReceived 2/1/89; revised 3/30/89; accepted 4/5/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed.
2The abbreviations used are: Thy, thymine; Lys. lysine; BSTFA,
bis(trimethylsilyl)trifluoroacetamide;
Phe-Phe. phenylalanyl-phenylalanine; GCMS, gas chromatography-mass
spectrometry; Me3Si. trimethylsilyl; a.m.u.,
atomic mass unit; SIM. selected-ion monitoring; RMRF, relative molar response
factor.
protein cross-links involving the basic amino acid Lys in calf
thymus nucleohistone in aqueous solution. Basic amino acids
are present in a very high proportion in histones, and the
positive charge of these amino acids helps the histones to bind
tightly to DNA regardless of the DNA sequence (10, 11). For
these reasons, basic amino acids of histones are likely to play
an important part in formation of DNA-protein cross-links in
chromatin. Here we present the evidence for OH radical-in
duced cross-linking of Lys to Thy, and the chemical character
ization of the resulting DNA-protein cross-link in calf thymus
nucleohistone.
MATERIALS
AND METHODS
Materials'. Thymine, lysine, calf thymus nucleohistone, and PhePhe were purchased from Sigma Chemical Company. BSTFA, acetonitrile, and 6 M HC1 were from Pierce Chemical Company. Dialysis
membranes with a molecular weight cutoff of 3,500 were purchased
from Fisher Scientific Company.
Irradiations. Aqueous mixtures of thymine (0.25 mivi) and lysine
(0.75 HIM)were saturated with N2O for 30 min and irradiated in a 60Co7 source (Gammacell-220; Atomic Energy of Canada Ltd.) at a dose of
400 Gy (dose rate 150 Gy/min). Samples were then lyophilized. Solu
tions of calf thymus nucleohistone (0.35 mg/ml) in 10 mM phosphate
buffer (pH 7.0) were saturated with N2O and irradiated at doses ranging
from 15 to 200 Gy (dose rate, 150 Gy/min). After irradiation, nucleo
histone solutions were dialyzed extensively against water and then
lyophilized.
Treatment with Hydrochloric Acid. Aliquots (2 mg) of lyophilized
samples were treated with 1 ml of 6 M HC1 in evacuated and sealed
tubes for 6 h at 120°C.Samples were then lyophilized.
Derivatization. HCl-treated and lyophilized samples were trimethylsilylated in polytetrafluoroethylene-capped hypovials (Pierce) with 0.15
ml of a BSTFA/acetonitrile (2:1, v/v) mixture by heating for 30 min at
130°C.After cooling, samples were injected directly onto the injection
port of the gas Chromatograph without further treatment.
GC-MS. Analysis of derivatized samples was performed using a Mass
Selective Detector interfaced to a gas Chromatograph (both from Hew
lett-Packard) equipped with an automatic sampler. The split mode was
used for injections. The injection port, the ion source and the interface
were maintained at 250°C.Separations were carried out using a fused
silica capillary column ( 12.5 m x 0.20 mm i.d.) coated with cross-linked
5% phenyl methylsilicone gum phase (film thickness, 0.11 ¿¿m)
(Hew
lett-Packard). Helium was used as the carrier gas at an inlet pressure
of 40 kPa. Mass spectra were obtained at 70 eV.
RESULTS
The purpose of the present work was to determine whether
OH radicals cause formation of DNA-protein cross-links be
tween Thy and Lys in a DNA-histone complex in aqueous
solution, and also to elucidate the chemical nature of such
cross-links and their mechanism of formation. Hydroxyl radi
cals were generated by ionizing radiation in N2O-saturated
aqueous solution. In dilute aqueous solutions, ionizing radia
1Certain commercial equipment or materials are identified in this paper in
order to specify adequately the experimental procedure. Such identification does
not imply recommendation or endorsement by the National Institute of Standards
and Technology, nor does it imply that the materials or equipment identified are
necessarily the best available for the purpose.
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FORMATION OF A DNA-PROTEIN CROSS-LINK
tion interacts with water to produce three radical species, i.e.,
OH radicals, hydrogen atoms and hydrated electrons (for a
review see Ref. 12). When N2O is present in solution, hydrated
electrons are converted into additional OH radicals in a diffu
sion-controlled reaction with N2O (12). In terms of radical
species, the system contains mainly OH radicals (~9Q%) and a
small amount of hydrogen atoms (~10%). Thus the radiolysis
of water in the presence of N2O is a most suitable tool to
generate almost exclusively OH radicals and to study their
reactions with various substrates.
In the present work, calf thymus nucleohistone was chosen
as a DNA-histone complex. The GC-MS technique was used
to elucidate the chemical nature of OH radical-induced DNAprotein cross-links in nucleohistone. As was determined in a
previous work, this commercial preparation consisted of 39%
DNA, 46% protein, and 15% unidentified material (by weight),
and contained histones H2A, H2B, H3, and H4 (9). In order
to use the GC-MS technique for chemical characterization of
the cross-linked components of DNA and proteins, nucleo
histone must be hydrolyzed first. The standard protein hydrol
ysis, i.e., hydrolysis with 6 M HC1, appears to be the simplest
way for this purpose, and this was used here. Furthermore, it
is necessary to know the gas Chromatographie and mass spectral
properties of cross-links between a DNA base and an amino
acid of interest. For this reason, we first investigated the OH
radical-induced cross-linking of Thy to Lys in a model system
consisting of a mixture of Thy and Lys in aqueous solution.
N2O-saturated aqueous mixtures of Thy and Lys were 7-irradiated, treated with 6 M HC1, and then analyzed by GC-MS
after trimethylsilylation. The reason for the HC1 treatment was
to have the same experimental conditions as those in subsequent
experiments with nucleohistone because HC1 was used to hydrolyze nucleohistone prior to analysis by GC-MS.
Figure 1 illustrates a typical total-ion chromatogram obtained
from a 7-irradiated mixture of Thy and Lys after HCl-treatment
and trimethylsilylation. Peaks 1 and 4 represent the Me3Si
derivatives of Thy and Lys, respectively. Peaks 2 and 3 corre
spond to the Me.,Si derivatives of 5,6-dihydrothymine and 5hydroxy-5,6-dihydrothymine,
respectively, which are the monomeric products of Thy. Peaks 5 and 6 represent some dimeric
products of Thy, which have been described previously (13).
Mass spectra taken from the other peaks in Fig. 1 were analyzed
for possible Thy-Lys cross-links. Peak 7 was found to represent
the Me3Si derivative of a Thy-Lys cross-link on the basis of the
typical fragmentation patterns of the Me.,Si derivatives of DNA
1 .4E7-
bases, amino acids, and DNA base-amino acid cross-links (8,
13-15). No other compound, which would correspond to a ThyLys cross-link, was observed. It should be stated here that the
Thy-Lys cross-link represented by peak 7 in Fig. 1 was not a
result of the HC1 treatment of the mixture of Thy and Lys.
This statement is based on the evidence obtained from the GCMS analysis of an unirradiated mixture of Thy and Lys. The
mass spectrum taken from peak 7 is illustrated in Fig. 2. In the
mass spectra of the Me.iSi derivatives of DNA base-amino acid
cross-links, the molecular ion (M+ ) is either not present or is
observed with a very low intensity (8, 13). In Fig. 2, M* was
not observed. The ion at m/z 687 was attributed to the typical
(M—Me)+ ion, which results from M+ by loss of a Me radical
(13-15). The ion at m/z 659 is formed from the (M—Me)+ ion
(m/z 687) by typical loss of CO (15). The following two struc
tures are proposed for this Thy-Lys cross-link along with frag
mentation patterns leading to characteristic ions in the mass
spectrum:
- 219
-.216
Me>^
Me3SO
,CH3 «o
N^X-CH2-CH-j-CH2-CHNHS.Me3
^,AH
,'„N(&Me3)2
CH2
,;4 N(&Me3)2
i
n
Cleavage of the bond between 5- and e-carbons of Lys with
charge retention on the t-carbon gives rise to the second most
intense ion at m/z 174, which is a characteristic fragment of
the Lys moiety (15). The presence of the m/z 174, 188, 514,
and 528 ions clearly excludes the 5- and «-carbonsas the site of
cross-linking on the Lys moiety of this molecule. Loss of
HOSiMe, (90 a.m.u.) from m/z 514 presumably accounts for
the intense ion at m/z 424. Loss of CO2SiMe, (117 a.m.u.)
from M+ leading to the intense ion at m/z 585 is a characteristic
fragmentation of Me,Si derivatives of amino acids and DNA
base-amino acid cross-links (8, 13, 15). Another characteristic
fragmentation involving the cleavage of the bond between aand 0-carbons with charge retention on the a-carbon produces
the ions at m/z 218 and 219 (with an H atom transfer). The
presence of the m/z 218, 219, and 484 ions excludes the acarbon as the site of cross-linking on the Lys moiety. Likewise,
the presence of m/z 470 excludes the /3-carbon as the site of
cross-linking. The abundant ion at m/z 297 presumably arises
from m/z 470 by loss of CH2N(Me3Si)2 accompanied with an
H atom transfer. The origin of the intense ion at m/z 498 is
not known. It might be the result of a rearrangement reaction
of the m/z 585 ion [e.g., m/z 585 - HNSiCH2(CH,)2 + H].
I .2E7! . 0E78.0E66 . 0E61.0ES
2.0ESH
e
Tie
(min.
B
)
Fig. 1. A total-ion chromatogram obtained from a -y-irradiated mixture of
thyminc and lysine after HCI-trcatment and trimethylsilylation (radiation dose
400 Gy). The column was programmed from 150 to 270°Cat 10'C/min after 1
min at 150"C. For other details see "Materials and Methods." Peaks: /, thymine:
2, 5,6-dihydrothymine; 3. 5-hydroxy-5,6-dihydrothymine;
4, lysine; 5 and 6,
dimeric products of thymine; 7, thymine-lysine cross-link (all compounds as their
Me3Si derivatives). Other peaks were not defined.
100
200
300
400
500
600
Fig. 2, Mass spectrum taken from peak 7 ¡nFig. I.
3464
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FORMATION OF A DNA-PROTEIN
The ions that result from cleavages of the bonds of the Lys
moiety as discussed above clearly indicate the 7-carbon of Lys
as the site of cross-linking. The prominent ion at m/z 269 and
the ion of low intensity at m/z 433 apparently represent the
Thy and Lys moieties of the molecule, respectively. The m/z
73 and 147 ions are common for MejSi derivatives and are not
used for diagnostic purposes (14, 15). The site of cross-linking
on the Thy moiety cannot be defined with certainty from the
mass spectrum. There are two possible sites of cross-linking as
illustrated in Structures I and II. The presence of the unsaturated heteroatom (nitrogen-1) next to the carbon-6 in Structure
I should facilitate the cleavage of the bond between Thy and
Lys moieties [a cleavage (16)] yielding an abundant m/z 269
ion. On the other hand, the high abundance of the m/z 269 ion
might be an indication for the methyl group of Thy as the site
of cross-linking because the m/z 269 ion being an allyl cation
is expected to be abundant in the mass spectrum of Structure
II as a result of resonance stabilization (16).
Having determined the gas Chromatographie and mass spectrometric characteristics of the Thy-Lys cross-link, the GC-MS
technique with SIM was used to search for this cross-link in
trimethylsilylated HCl-hydrolysates of calf thymus nucleohistone 7-irradiated in N2O-saturated aqueous solution. A num
ber of characteristic ions from the mass spectrum in Fig. 2 were
monitored during the GC-MS/SIM analysis in the time period
between 11 and 12 min where the Me3Si derivative of the ThyLys cross-link was expected to elute under the Chromatographie
conditions used in Fig. 1. Unirradiated samples were prepared
and analyzed in the same manner. Fig. 3, A and B. illustrates
the ¡on-currentprofiles of the m/z 174, 269, 470, 498, 585, and
687 ions obtained during the GC-MS/SIM analysis of unirra.Ion
0E5-5.0E41.
174
g(\JV_Fon
.0E5-5.0E4-10000-
174
0-10000269"
269JuJ^Ion
5000-10000-5000-010000-5000-10000-5000"
5000-10000-5000-0-"10000-5000-10000-5000-
"VIon
470Ion
470_^
,Ion
498»Ion
498JU_Ion
585Ion
5851JIon
0-1000-500-Ion
0-1000-500:Ion
687\V^W*/Ã-A\jMvkv~JlW1
687\/Y^f*
VKYMvyr^MÃ-l'l
t
1
f
T i me
12
Cm 1 n . )1
T i me
12
Cm i n . )
Fig. 3. Ion-current profiles of the ions m/z 174, 269, 470, 498, 585, and 687
obtained during the GC-MS/SIM analysis of trimethylsilylated hydrolysates of
nucleohistone. I, unirradiated; lì.
7-irradiated in NjO-saturated aqueous solution
at a dose of 100 Gy. Chromatographie conditions were as in Fig. 1.
CROSS-LINK
dialed and irradiated samples, respectively. Twelve ions were
monitored in the same time period; however, for practical
reasons, only profiles of six ions are shown in Fig. 3. The
signals of the monitored ions are seen in Fig. 3B at the expected
retention times (indicated with an arrow) of the Me3Si deriva
tive of the Thy-Lys cross-link. No signal was observed for the
same ions when unirradiated samples were analyzed (Fig. 3,4),
meaning that the Thy-Lys cross-link identified here was not a
result of any treatment of nucleohistone including HCl-hydrolysis other than irradiation. Subsequently, a partial mass spec
trum was obtained on the basis of the monitored ions and their
relative intensities (Fig. 3B), because relative intensities of
monitored ions must also match those of the same ions in the
mass spectrum of the authentic compound for an unequivocal
identification. This partial spectrum (not shown here) was
identical in terms of the monitored ions and their relative
abundances to the mass spectrum in Fig. 2, meaning the une
quivocal identification of the Thy-Lys cross-link in 7-irradiated
nucleohistone. The presence of the Thy-Lys cross-link in nu
cleohistone could be detected in the present study at radiation
doses as low as 5 Gy by the use of the GC-MS/SIM technique.
The yield of the Thy-Lys cross-link in 7-irradiated nucleo
histone was measured by GC-MS/SIM. For quantitative analy
sis using this technique, the mass spectrometer should be cali
brated with known quantities of both the analyte and an internal
standard which is added to the sample, by obtaining the relative
molar response factor4 for a typical ion of the analyte relative
to a typical ion of the internal standard (17). In the present
work, a dipeptide (Phe-Phe) was used as an internal standard.
Due to lack of a sufficient amount of the authentic Thy-Lys
cross-link, the RMRF could not be determined experimentally.
The RMRF was calculated by comparing the relative abundance
of the m/z 174 ion in terms of the percentage of the total ion
current in the mass spectrum of the Me3Si derivative of the
Thy-Lys cross-link (Fig. 2) to that of the m/z 192 ion in the
mass spectrum of the Me^Si derivative of Phe-Phe (18). For
this purpose, both mass spectra were recorded under the same
tuning conditions of the mass spectrometer. After irradiation,
dialysis and hydrolysis of aliquots of nucleohistone samples, an
aliquot of Phe-Phe was added. Samples were then frozen im
mediately in liquid nitrogen and then lyophilized and tri
methylsilylated. Ion currents of the m/z 174 and 192 ions were
measured during the GC-MS/SIM analysis. Using the calcu
lated RMRF, which amounted to 3.2, the yield of the Thy-Lys
cross-link in nucleohistone 7-irradiated at doses ranging from
15 to 200 Gy was determined, and was found to be a linear
function of radiation dose. At least three independent measure
ments of the yield were done for each of five radiation doses
used between 15 and 200 Gy. The G value (yield per 1 J of
radiation energy), which was calculated from the linear doseyield plot, amounted to 0.0085 ±0.0008 /¿mol/J.This value
represents approximately 1.5% of the total yield of OH radicals
generated by ionizing radiation in an aqueous system saturated
with N2O [i.e., 0.56 ¿¿mol/J
(12)] and corresponds to formation
of one Thy-Lys cross-link per 1 Gy in every approximately 3 x
IO4 nucleotides in nucleohistone. The yield of the Thy-Lys
cross-link is the highest among those of the DNA-protein cross
links identified so far in nucleohistone in vitro (8, 9).
4 RMRF is obtained as follows:
Amount of the analyte
Amount of the standard
Peak area of the ion of the standard
Peak area of the ion of the analyte
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FORMATION OF A DNA-PROTEIN CROSS-LINK
DISCUSSION
The formation of DNA-protein cross-links found in this work
can be explained as a result of reactions of OH radicals with
Thy moiety of DNA and Lys moiety of proteins in nucleohistone. Hydroxyl radicals react with Thy mainly by addition
to the carbon-5=carbon-6
double bond, and to a lesser extent
by abstraction of an H atom from the methyl group (19). The
predominant reaction of OH radicals with aliphatic amino acids
is the H atom abstraction from the side chain (20, 21). In the
case of Lys, electron spin resonance spectroscopic evidence has
been presented for the formation of a 7-carbon-centered radical
as one of the three possible side chain radicals (20). The ThyLys cross-link with two possible structures (see above Structures
I and II), which was found in the model system consisting of
Thy and Lys, should be a result of radical-radical reactions
under the experimental conditions used:
Ajo.
o
11
CO~2
CHi
*
^N-^-H
c°2
CH-CH2-CH
CH2
(1)
*NH3
1
*NH3
CH2
H3N-CH2
H3N-CH2
CO~2
JLJL
O^N^H
*
2 I
i
2 I
CH2
2i
*NH3
H3N-CH2
B
The dehydration step in Reaction 1 might be spontaneous or
induced by acidic treatment. Reaction 1 should be more likely
than Reaction 2 because, when aqueous solutions of Thy are
irradiated, Structure III predominates among the radicals
formed and Structure IV amounts to only 10% of radicals (19).
On the other hand, a Thy-Tyr cross-link, which was found in a
mixture of Thy and Tyr -/-irradiated under similar conditions,
has been shown to have Structure IV as one of its precursors
(22). Moreover, formation of A as shown in Reaction 1 might
be sterically hindered because of the presence of the methyl and
OH groups at the carbon-5 of Thy. As was mentioned above,
the Me.,Si derivatives of A and B (i.e., Structures I and II) could
not be distinguished from each other by their mass spectra (see
"Results").
In the case of nucleohistone, the following radical-radical or
radical addition reactions are proposed as possible mechanisms
for formation of DNA-protein cross-links involving Structure
formation of Structure A. Since the final product of Reaction
3 is the same as that of Reaction 4, the two mechanisms shown
in Reactions 3 and 4 cannot be distinguished from each other
by their final product.
Mechanism for formation of DNA-protein cross-links in
volving Structure B should involve a radical-radical reaction:
(5)
I
DNA
Mechanisms for DNA-protein cross-linking in nucleohistone
via radical-radical reactions as illustrated in Reactions 3 and 5
require formation of two radicals in close proximity because of
the impaired mobility of macromolecules and because of DNAhistone associations. The track model of the energy deposition
of ionizing radiation in a medium provides the concept of
formation of two or more radicals in track entities such as spurs
and blobs (24). Two OH radicals escaped from track entities
without undergoing radical-radical combinations might mediate
formation of two radicals in close proximity, one on DNA and
one on a protein in nucleohistone. According to this concept,
radical-radical Reactions 3 and 5 might represent possible
mechanisms for DNA-protein cross-linking involving Thy and
Lys. At low radiation doses used in this work, the likelihood of
such processes or their contribution to the formation of cross
links is not known. If Structure B is the structure of the ThyLys cross-link found in nucleohistone, radical-radical Reaction
5 would be the only possible mechanism. All mechanisms
proposed above (Reactions 3, 4, and 5) require the close prox
imity of a Lys moiety to a Thy moiety in the DNA-histone
complex. This close proximity should be possible because Lys
uniquely forms a hydrogen bond between its i-NH,+ group and
the oxygen at the carbon-4 of an adjacent Thy molecule (25,
26).
In summary, the OH radical-mediated formation of a DNAprotein cross-link involving Thy and Lys moieties in nucleo
histone in aqueous solution was described, and possible mech
anisms for its formation were proposed. The analytical ap
proach used might be useful in elucidation of DNA-protein
cross-links induced by ionizing radiation or other free radicalgenerating processes in living cells.
REFERENCES
A (R depicts a protein with Lys radical V):
I
•fR'
O**N
DNA
3
n
I
DNA
H*
A
I
DNA
I
DNA
4
I
DNA
Highly selective addition of alkyl radicals to the carbon-6 of
pyrimidines in model systems as shown in Reaction 4 has been
reported recently (23). It should be pointed out that addition of
the protein radical to the carbon-5 of Thy would not lead to
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FORMATION
OF A DNA-PROTEIN CROSS-LINK
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Structure and Mechanism of Hydroxyl Radical-induced
Formation of a DNA-Protein Cross-Link Involving Thymine and
Lysine in Nucleohistone
Miral Dizdaroglu and Ewa Gajewski
Cancer Res 1989;49:3463-3467.
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