Carcinogenesls vol.17 no.3 pp.525-532, 1996
Effect of sulphur mustard on the initiation and elongation of
transcription
Andrew Masta1, Peter J.Gray1*2 and Don R.Phillips1-3
'School of Biochemistry, La Trobe University, Melbourne, Australia 3083
and 2 Aeronautical and Maritime Research Laboratory, Defence Science and
Technology Organisation, Melbourne, Australia 3032
3
Tb whom correspondence should be addressed
Sulphur mustard is a potent alkylating agent that causes
severe vesication as well as systemic and genotoxic effects.
Despite its long history as a chemical warfare agent,
the mechanism of its toxicity remains unknown and no
successful pharmacological intervention has yet been
found. In this study we have examined the effects of
mustard alkylation of DNA on transcriptional processes.
Gel mobility shift analysis shows that mustard alkylation
of the lac UV5 promoter increases the stability of the
promoter-RNA polymerase binary complex. Following
formation of the initiation complex and addition of elongation nucleotides, -45% of the RNA polymerase in the
initiated complex remained associated with the alkylated
promoter, compared to only 7% remaining associated with
the unalkylated promoter. For the RNA polymerase able
to escape the initiation complex, mustard alkylation of the
DNA template resulted in the production of truncated
transcripts. Analysis of these truncated transcripts revealed
that sulphur mustard alkylates DNA preferentially at 5'AA, 5-GG and 5-GNC sequences on the DNA template
strand and this is significantly different from the alkylation
sites observed with nitrogen mustard. This study represents
the first report at the molecular level of sulphur mustardinduced effects on transcriptional processes.
Introduction
Sulphur and nitrogen mustards are potent bifunctional alkylating agents (1). While these compounds share a similar chemical
structure, nitrogen mustard and its derivatives have received
more attention due to their widespread use in cancer chemotherapy. Sulphur mustard (mustard gas, bis(2-chloroethyl)suphide) has been used several times this century as a chemical
warfare agent (2,3) with devastating acute effects such as
erythema and blistering on human victims, as well as systemic
and genotoxic effects (4). Cell death is thought to be the causal
event in the pathogenesis of sulphur mustard lesions, and is
attributed to alkylation of critical molecules within cells (5).
In aqueous solution sulphur mustard spontaneously forms
reactive sulphonium ions (6) that can react readily with many
biological nucleophiles. Several mechanisms of the toxic
effects of sulphur mustard have been proposed and these
include inhibition of glycolysis, activation of poly(ADPribose)polymerase, thiol-Ca2+mediated oxidative-electrophilic
stress-induced cell death and glutathione depletion-induced
lipid peroxidation (5). At this stage, none of these theories can
be regarded as proven, although it is generally believed that
•Abbreviations: GpA, guanylyl(3'-5')adenosine; TEMED, tetraethylmethylethyldiamine; DTT, dithiothreitol; TBE, Tris-borate-EDTA; TE, Tris-EDTA.
© Oxford University Press
alkylation of specific biopolymers is the initial event leading
to the observed toxicities. Because the molecular mechanism(s)
of sulphur mustard's toxicity remains unresolved there are no
known antidotes or prophylactic treatments for this alkylating agent.
There is a substantial body of evidence which suggests the
involvement of DNA in the mode of action of sulphur mustard.
Ultrastructural studies show that the first visible indication
of damage by sulphur mustard appears in the nucleus (7).
Furthermore, sulphur mustard has been shown to possess
mutagenic and carcinogenic activity and also has the ability
to produce chromosomal aberrations and a variety of other
types of DNA damage (8). The most compelling evidence that
DNA represents a sensitive cellular target for sulphur mustard
is the observation that cells incapable of repairing sulphur
mustard-induced DNA lesions were considerably more sensitive than those capable of carrying out this repair (9).
Mustard-induced DNA lesions would be expected to affect
the DNA directed processes of replication and transcription.
Transcription is a multi-step process which results in the
synthesis of mRNA molecules that are subsequently translated
into the proteins required in virtually all cellular processes.
The steps in the transcription process are: binding of RNA
polymerase to the promoter on the DNA; formation of an
initiated transcription complex; elongation of the initiated
complex; and termination of transcription at specific sites. In
eukaryotes each of these steps is a complex process involving
transcription factors and regulatory proteins in addition to
RNA polymerase. In prokaryotic systems, this process is much
simpler, with no requirements for such co-factors, and is
therefore the system of choice to examine the basic aspects of
effects of sulphur mustard-induced DNA lesions on transcriptional processes.
Although sulphur and nitrogen mustards have similar
chemical structures and reactivity (6), sulphur mustard induces
more toxic effects. In order to develop effective treatments for
these agents it is important first to determine the molecular
basis of their differences in toxicity. In this report the effects
of alkylation by sulphur mustard of a DNA fragment containing the lac UV5 promoter were investigated in terms of (i)
the binding of Escherichia coli RNA polymerase to the
promoter; (ii) formation of the transcription initiation complex; and (iii) elongation of the transcriptional complex. We
demonstrate that sulphur mustard alkylation of the DNA
impedes the processivity of the RNA polymerase from the
initiated transcription complex and also inhibits transcription
elongation, resulting in the production of truncated transcripts.
Materials and methods
Materials
Sulphur mustard (bis(2-chloroethyl)sulphide) was synthesized at the Aeronautical and Maritime Research Laboratory, DSTO, and was >98% pure as
assessed by 'H NMR. Nuclease-free E.coli RNA polymerase, ribonuclease
inhibitor (human placenta), ultrapure ribonucleotides and deoxynbonucleotides, 3-O-methyl nucleoside triphosphates and BSA (RNase/DNase free)
525
A.Masta, PJ.Gray and D.R.Phillips
were obtained from Pharmacia. [a-32P]ATP and [a-32P]dATP were from
Amersham. Nitrogen mustard (mechloroethamine hydrochloride), heparin and
guanylyl(3'-5')adeonsine (GpA*) were purchased from Sigma and tetraethylmethylethyldiamine (TEMED) was obtained from Promega. Bisacrylamlde, acrylamide, ammonium persulphate and dithiothreitol (DTT) were obtained
from BioRad as electrophoresis grade reagents; urea was obtained from
Amresco. Restriction enzymes and KJenow fragment were obtained from
either New England Biolabs or Boehringer-Mannheim. DNA fragments were
recovered from electrophoresis gels using a Schleicher and Schuell Bio-Trap
Eluter. Nensorb 20 chromatographic columns were from Duponl. All other
chemicals were of analytical grade and all solutions were prepared using
distilled, deionized and filtered water from a Milli-Q four-stage water
purification system (Millipore).
DNA templates for transcription
For the gel mobility shift studies, a 188 bp PvulVEcoRl restriction fragment
of pCCl (derived from the vector pBR322) containing the lac UV5 promoter
was used as the template for E.coli RNA polymerase (10). The 3-overhang
generated by £coRI was end-filled by Klenow fragment. Briefly, the labelling
reaction mixture contained -10 ug of the 188 bp fragment, 0.125 |ig/ml
BSA, 1.25 mM DTT, 200 nCi [a-32P]dATP and 10 units of Klenow fragment
in 50 mM Tris-HCl and 10 mM MgCl2, pH 7.5, was incubated at room
temperature for 20 min. A mixture of all four deoxynbonucleotides was then
added (2 mM final concentration of each) and incubated for a further 20 min
at room temperature. The labelled DNA fragment was purified using a Nensorb
20 chromatographic column according to the Dupont protocol, dried and
then resuspended in an appropriate volume of TE buffer (10 mM Tris—HC1
and 1 mM EDTA, pH 8.0). For transcription elongation studies, a 497 bp
PvulVSall restriction fragment from pRWl containing the lac UV5 promoter,
was used for in vitro transcription with E.coli RNA polymerase (11). The
isolation and purification of these DNA fragments was according to standard
procedures (12).
Alkylation of the DNA template
Sulphur mustard was initially dissolved in ethanol and appropriate concentrations were prepared prior to use in TE buffer. For studies of the RNA
polymerase-promoter binding and formation of the transcription initiation
complex, the alkylation reaction contained -0.2 ng of the 3'-end labelled 188
bp DNA fragment and an appropriate concentration of sulphur mustard. For
the transcription elongation studies, the alkylation reaction contained -0.5 \ig
of the 497 bp DNA fragment and an appropriate concentration of sulphur
mustard. The alkylation reactions were normally incubated at 37°C for 60 min.
For reaction time studies the alkylation reactions were carried out at 37°C for
the appropriate times and the reactions terminated by freezing with liquid
nitrogen. The alkylated DNA samples were precipitated with ethanol to
remove unreacted mustard, dried and resuspended in TE buffer and were
either used immediately or stored at -20°C until required.
Gel mobility shift assay of the promoter—RNA polymerase complex
The RNA polymerase-promoter complex was formed by adding -0.2 ng of
the labelled DNA fragment to 600 nM RNA polymerase in transcription
buffer and this mixture (5.0 |il) was incubated at 37°C for 15 min. The
transcription buffer contained 40 mM Tris-HCl, pH 8.0, 100 mM KC1, 3 mM
MgCl2, 0.1 mM EDTA, 0.125 ug/ml BSA, 10 mM DTT and 1.5 units RNase
inhibitor. Heparin (2.5 |il) was added to a final concentration of 400 (ig/ml to
remove any non-specifically bound RNA polymerase and the mixture was
incubated for a further 5 min at 37°C. For the transcription initiation studies,
the ternary complex was then formed by adding 2.5 |il of an initiation
nucleotide mixture consisting of GpA (final concentration of 200 nM), ATP,
GTP and UTP (5 uM final concentration of each). The transcription elongation
studies were performed by adding 5.0 \i\ of the elongation nucleotide mixture
to the initiated transcription complexes. The elongation nucleotide mixture
consisted of UTP, CTP, GTP and ATP (2 mM final concentration of each)
and KC1 at a final concentration 400 mM. A 1/10 volume of a loading buffer
comprising 60% sucrose, 0.01% xylene cyanol and 0.01% bromophenol blue
was added to the RNA polymerase-promoter complex and the samples were
analysed on a 5% (29:1) native polyacrylamide gel containing 100 |iM MgCI2
in TBE buffer (90 mM Tris-HCl, pH 8.3, 90 mM boric acid and 2.5 mM
EDTA). Electrophoresis was carried out at 100 V for 3 h and the gel was
then fixed in 10% glacial acetic acid for 10 min. The gel was dried using a
BioRad gel drier prior to exposure to a phosphor image screen for 15 h.
In vitro transcription
The transcription elongation experiments were performed essentially as
described previously (11,13). Briefly, -0.5 ng of the 479 bp DNA fragment
and 100 nM E.coli RNA polymerase were incubated in transcription buffer
to form a binary complex as described above. Heparin was added to a final
concentration of 400 u.g/ml to remove any non-specifically bound RNA
polymerase, and the mixture was incubated at 37°C for 5 min. A stable ternary
526
complex was formed by addition of the initiation nucleotides and incubated
at 37°C for a further 5 min. The initiation nucleotide mixture was essentially
as described above except that [a-^PJATP (50 uCi) was used instead of ATP.
The resulting initiated complexes were elongated by the addition of the
elongation nucleotides. For studies on the effect of mustard concentration and
reaction times, transcription elongation was carried out at 37°C for 60 min.
For studies on the effect of elongation time, aliquots from the elongation
reaction were removed at various times between 5 and 180 min. The elongation
reaction was stopped with the addition of an equal volume of loading/
termination buffer comprising 10 mM urea, 10% sucrose, 0 1% xylene cyanol
and 0.1% bromophenol blue in 2X TBE. Electrophoresis was carried out as
previously described (II).
Gel analysis and quanlitation
Gel analyses were performed using a Molecular Dynamics Model 400B
Phosphorlmager and Molecular Dynamics ImageQuant software.
Results
RNA polymerase binding and transcription initiation
The effect of mustard alkylation of the lac UV5 promoter on
the binding of E.coli RNA polymerase, and formation of the
transcription initiation complex was investigated using a gel
mobility shift assay. Figure I (A) shows the effect of reaction
time of mustard (100 U.M) with the labelled DNA fragment
(188 bp) on the binding of RNA polymerase to the lac UV5
promoter. The amount of binary complex increased with
reaction time from 0 to 60 min. No significant increase of this
complex was observed thereafter (Figure IB). The effect of
varying concentrations of mustard on RNA polymerase binding
was also investigated (Figure 2A). The amount of the binary
complex increased with increasing mustard concentration up
to 200 (iM (60 min reaction time) and this is shown in Figure
2(B). The free probe in lane F of Figures 1(A) and 2(A)
contains an additional band. This extra band is most likely
due to the association of residual Klenow DNA polymerase
(as used in the DNA labelling procedure) and the labelled
DNA fragment. This band is not present in other lanes because
they have been subjected to heparin treatment to displace any
non-specifically bound RNA polymerase and this has also
served to remove the residual Klenow DNA polymerase.
Figure 3(A) shows a gel mobility shift analysis of the
formation of the transcription initiation complexes and subsequent elongation of these complexes. Quantitation of the
bands corresponding to the DNA-RNA polymerase complexes is shown in Figure 3(B). In the absence of mustard
treatment, the transcription initiation complex comprised -30%
of the total DNA. Following elongation of this complex, the
DNA-RNA polymerase band decreased to - 2 % of the total
DNA. With the alkylated template (100 |xM mustard), the
transcription initiation complex represented -35% of the total
DNA, whereas following elongation, -16% of the RNA
polymerase remained associated with the promoter region on
the DNA template (i.e. -45% of the RNA polymerase in
the initiated transcription complexes remained bound at the
alkylated promoter).
Transcriptional elongation
The 497 bp DNA fragment was alkylated with 100 |iM
mustard for various times prior to analysis by transcriptional
footprinting. Figure 4 shows the effect of reaction time on
transcription elongation. The control elongation lanes show
no significant pausing of transcription and only full-length
transcripts were synthesized. Exposure of DNA to sulphur
mustard for increasing periods of time resulted in decreasing
amounts of the full-length transcript and increasing amounts
of shorter (truncated) transcripts. The amount of full-length
Inhibition of transcription by sulphur mustard
UNA POLYMERASE-DNA
BINARY COMPLEX
RNA POLYMERASE-DNA
BINARY COMPLEX
B
40
^
—
-•
160
200
X
pie
B
30
E
o
/
o
z 20
Q
/
Q.
Z
rr
# 10
7
1r
n
40
80
120
Mustard Concentration/uM
30
60
90
120
Alkylation Time/min
Fig. 1. Alkylation lime dependence of the RNA polymerase-promoter
binary complex. (A) Autoradiogram of a gel mobility shift analysis of RNA
polymerase-promoter binary complex. Labelled DNA (188 bp) was
alkylated with 100 nM sulphur mustard for various times (0-180 min) at
37°C prior to the formation of the binary complex. F denotes labelled DNA
with no RNA polymerase. (B) Quantitation of the binary complex. The
percentage of the DNA in the RNA polymerase-promoter complex is shown
as a function of reaction time.
transcript also decreased with increasing alkylation time up to
-30 min, after which no further decrease in the full-length
product was observed. This is evident by the appearance of
bands corresponding to shorter length RNA after 1 min, and
their increase in intensity up to a reaction time of approximately
60 min. Some of the truncated transcripts remained constant
with increasing reaction time, while others slowly decreased
(e.g. 43mer and 102mer). The total transcriptional activity as
measured by the intensity of all bands in each lane also
decreases with increasing alkylation time.
The effect of concentration of sulphur mustard on transcription elongation was also investigated. The DNA fragment
was alkylated prior to transcription with concentrations of
Fig. 2. Concentration dependence of the RNA polymerase-promoter binary
complex. (A) Autoradiogram of a gel mobility shift analysis of the RNA
polymerase-promoter binary complex. Labelled DNA (188 bp) was reacted
with varying concentrations of sulphur mustard (0-200 LLM) at 37°C for 60
min prior to the formation of the binary complex. F denotes labelled DNA
with no RNA polymerase. (B) Quantitation of the DNA in the RNA
polymerase-promoter complex is shown as a function of sulphur mustard
concentration.
mustard ranging from 0 to 400 H.M. Figure 5 shows the
concentration dependence of elongation of these alkylated
DNA templates. As the mustard concentration was increased
from 0 to 100 \iM (60 min alkylation time), the amount of
full-length transcript decreased and there was a corresponding
increase in shorter length transcripts. At 400 ^M mustard,
no full-length transcript was observed, and transcriptional
elongation was almost completely inhibited, with 43mer and
75mer truncated transcripts being the only major products
observed.
The transcription system employed yields a synchronized
population of the initiated transcripts that are mainly 10
nucleotides in length (14). The extent of RNA polymerase
processivity can therefore be measured from elongation of the
initiated transcripts. Figure 6 shows the effect of mustardinduced (200 |iM) transcriptional blockages on the processivity
of E.coli RNA polymerase as a function of elongation time.
At some sites the polymerase is able to continue past the
initial blockages, as if the blockage was removed or bypassed
527
A.Masta, PJ.Gray and D.K.Phillips
CONTROL C
I
5
15
30
60
120
180
FIT
RNA POLYMERASE-DNA
COMPLEXES
B
40
43
30
o
20
rr 10
U
CONTROL
I
E
ALKYLATED
Fig. 3. Effect of sulphur mustard on the formation of transcription initiation
and elongation complex. (A) Autoradiogram of a gel mobility shift analysis
of the RNA polymerase-promoter complex. Labelled DNA (188 bp) was
reacted with 100 |iM sulphur mustard at 37°C for 60 min prior to the
formation of the these complexes. Control expenments were performed in
the absence of sulphur mustard. U denotes the complex formed before the
addition of initiation nucleotides. I denotes the initiated complexes formed
by the addition of initiation nucleotides and E denotes those initiated
complexes elongated with the addition of elongation nucleotide mixture.
(B) Quantitation of the RNA polymerase-DNA complexes.
(see, for example, 61 and 98 nucleotide length transcripts
where blockages were evident in the 5 min lane and absent
thereafter). At other sites of transcription termination, RNA
polymerase is able to transcribe past the initial blockage sites
by several nucleotides with increasing elongation time (43mer
and 44mer). At most sites, however, the transcriptional
blockages are maintained with increasing elongation time. In
the 100 bp probed in this assay, the major transcriptional
blockages observed were at 5'-GG (sites 1, 5 and 6), 5'-A A
(sites 2 and 4) and 5'-GNC (sites 1, 3 and 6) sequences on
the DNA template strand (Figure 7B). Because sulphur and
nitrogen mustard have a similar chemical structure, it was
necessary to establish whether or not they induce the same
transcriptional blockages. Figure 7(C) represents a transcrip528
Fig. 4. Effect of alkylation time on transcription elongation. The
autoradiogram shows the effect of alkylation time on the formation of
truncated transcnpts. Sulphur mustard (100 u.M) was reacted with -0.8 U-M
DNA (497 bp) at 37°C for 0-3 h. Aliquots containing -80 nM DNA were
removed from 1-180 min. Transcription was earned out as described in
Materials and methods. Control lanes represent transcription carried out in
the absence of sulphur mustard. Lane C is the sequencing lane resulting
from termination by 3-0-methyl CTP and FLT corresponds to the fulllength transcript. The length of several major truncated transcripts are
shown on the right hand side of the autoradiogram.
tional analysis of nitrogen mustard alkylated DNA (497 bp
fragment) subjected to the same conditions as shown for
sulphur mustard (Figure 7B). Transcriptional blockages were
observed mainly at 5'-G, 5'-GG and 5'-GNC sequences on
the DNA template for nitrogen mustard. However, there
were two major differences: (i) while both mustards alkylate
DNA preferentially at 5'-GG and 5'-GNC sequences, sulphur
mustard has a higher tendency to alkylate adenine residues;
(ii) nitrogen mustard is able to alkylate isolated guanine
residues, whereas there is no evidence that sulphur mustard
can act in this manner.
Discussion
Inhibition of transcription
The first step in transcription is the correct binding of the
RNA polymerase to the promoter. Although this process
is fundamental to gene expression, the effect of covalent
Inhibition of transcription by sulphur mustard
400 100 50
30
15 I
CONTROL
180 120 60
30
15
5
C
G
FIT
102
43
Fig. 5. Effect of concentration on transcription elongation. The
autoradiogram shows the effect of sulphur concentration on the production
of truncated transcripts. Sulphur mustard was reacted with -80 nM DNA
(497 bp) at 37°C for 1 h prior to transcription. Mustard concentration
ranges from 1 to 400 |iM. Control lanes represent unalkylated DNA. FLT
denotes full-length transcript and I denotes the initiated complex. Lane G is
the sequencing lane resulting from termination by 3-O-methyl GTP. The
length of several major truncated transcripts are shown on the left hand side
of the autoradiogram.
modification of the promoter by sulphur mustard on RNA
polymerase binding and initiation of transcription has received
little attention. Since the lac UV5 promoter region contains a
number of the sulphur mustard preferred alkylation sequences
(five of 5'-AA, three of 5'-GG and three of 5'-GNC sequences),
alkylation at these sites may therefore interfere with the RNA
polymerase binding and initiation of transcription. The gel
mobility shift assay has demonstrated that alkylation of the
promoter by sulphur mustard increases the formation of a
complex between DNA and RNA polymerase. This binary
complex is normally unstable (15) and mustard alkylation
increases the stability of this complex significantly. This
increased stability was dependent on both mustard concentration and alkylation time. There are several possible explanations
for this increased stability of the binary complex. Firstly, the
RNA polymerase may be covalently bound to the DNA by
sulphur mustard, resulting in a DNA-protein crosslink. This
may not necessarily occur at the promoter region as RNA
Fig. 6. Effect of elongation time. The autoradiogram shows the dependence
of the production of truncated transcripts on elongation time. Sulphur
mustard (200 jlM) was reacted with 80 nM DNA (497 bp) at 37°C for
60 min prior to transcription. Transcription was carried out essentially as
described in Materials and methods except that elongation times varied from
5 to 180 min. At appropriate times, aliquots of the elongation reaction
mixture were removed and added to an equal volume of the loading/
termination buffer. Lanes C and G are sequencing lanes resulting from
termination by 3-O-methyl CTP and 3-O-methyl GTP respectively. The
length of several truncated transcripts are shown on the left hand side of the
autoradiogram.
polymerase is known to undergo a one-dimensional diffusion
along the DNA 'in search' of the promoter (16) and may
therefore make contact and become covalently attached to the
DNA at sites other than in the promoter region. The occurrence
of DNA-protein crosslinks has been reported with nitrogen
mustard and suggested to play a significant role in its mode
of action (17). An alternative explanation for the increased
stability of the binary complex is that DNA alkylation may
distort the DNA structure and create an altered conformation
which 'traps' the RNA polymerase. RNA polymerase binding
to the promoter induces changes in the DNA structure, resulting
in an equilibrium between the closed and open transcriptional
complexes (18). Mustard-induced DNA lesions and the
distortion they induce, particularly the DNA interstrand
crosslink, may interfere with this equilibrium and result in
the increased stability observed between DNA and RNA
polymerase. There was no further stabilization of the binary
529
A.Masta, PJ.Gray and D.R.Philllps
A
1
ik.li
B
u1 till
I
I III i
£
c
1
OJ
•—
* <u
«2
h
A
c
HI
11
II Mi
2
J ii., iIi
nI
MI
i
Fig. 7. Quantitation of the truncated transcripts. The relative intensity of
RNA transcript is shown for the major truncated transcripts. The relative
intensities of the major bands at 5 min elongation time and 120 min
elongation time (Figure 6) are shown in (A) and (B) respectively.
(C) represents the relative intensity of nitrogen mustard-induced truncated
transcripts. The experimentaJ conditions are the same as those used for
sulphur mustard (B). For clarity, both the DNA non-template (upper) and
template (lower) sequences are shown. The numbering corresponds to the
+1 site of the transcribed message.
complex after 60 min of reaction, and this indicates that
sulphur mustard reaction is complete by this time, due to either
the alkylation of all available sites on the DNA or the competing
mustard hydrolysis (19). The rate of adduct formation was
calculated from Figure 4 (total amount of adducts as a
percentage of the total transcriptional activity at each alkylation
time) and showed that adducts were formed rapidly within the
first 20 min of alkylation, with completion at ~60 min. This
is consistent with the rate of formation of RNA polymerasepromoter binary complexes shown in Figure 1(B).
Addition of initiation nucleotides to the RNA polymerasepromoter binary complex results in the formation of a number
of stable ternary complexes (20). In the presence of high
concentrations of ribonucleotides almost all the initiated complexes formed with the unalkylated DNA have elongated. With
the alkylated template a more stable complex resulted, and in
the presence of a high concentration of all ribonucleotides a
significant amount of the initiated complex remained associated with the DNA. This suggests that the processivity of
the RNA polymerase is retarded and the complex is no
longer transcriptionally functional. A similar effect has also
been reported for DNA alkylated with nitrogen mustard (21).
A number of studies have shown that covalent adducts produced
by alkylation of DNA with alkylating agents are capable of
terminating the transcription elongation process and result in
the formation of truncated transcripts (22). This study is
the first to demonstrate that sulphur mustard adducts result in
the production of similar truncated transcripts. Furthermore,
the total transcriptional activity as measured by the total
intensity of all bands in each lane decreases with increasing
530
alkylation time (Figure 4), and this appears to be due to the
stabilizing effect of sulphur mustard on the RNA polymerase
in the initiated transcription complexes.
Transcription termination
Reaction of sulphur mustard with DNA resulted in accumulation of truncated transcripts. The transcriptional blockages of
the processivity of RNA polymerase were located at (or 1 bp
prior to) the alkylation sites on the DNA template strand
(13,22). Five of the six major sites observed were located at
5'-AA or 5'-GG sequences on the template strand, suggesting
the likelihood of adenine-adenine or guanine-guanine intrastrand crosslinks between adjacent purine residues. Since no
blockages were associated with 5'-AA and 5'-GG sequences
on the non-template strand (positions 30, 32, 63 and 65), this
provides good evidence that it is only the intrastrand crosslink
on the template strand that is sufficiently disruptive to impede
the transcriptional process, and this has been noted previously
with other alkylating agents (22). Three possible interstrand
crosslinks were evident (sites 1, 3 and 6; Figure 7B) and
appear to involve an intervening base pair between the two
crosslinked residues of 5'-GNC sequences—the same
interstrand linkage reported with nitrogen mustard (23).
The use of the transcription assay to elucidate the sequence
specificity of mustard alkylation gives a more biologically
relevant insight into the mechanism of action of mustards since
transcription termination is detected at individual blockage sites
under conditions of active transcription of the DNA. The
similarity of the chemical structures of nitrogen and sulphur
mustards suggests that their alkylation patterns might be
essentially the same. Others have used in vitro transcription
systems to probe for the alkylation sites of nitrogen mustard
and have shown that alkylation occurred specifically at
guanine residues on the DNA template strand (13,24,25)
with no evidence of alkylation of adenine residues. The
sequence specificities of DNA alkylation by sulphur and
nitrogen mustards are therefore significantly different, with
sulphur mustard producing a clear propensity to alkylate
adenine residues, and this difference is important for a number
of reasons. Several of the current theories of the mechanism
of sulphur mustard toxicity involve DNA alkylation. Modified
nucleotides, particularly alkylated adenine, are sensitive to
endonuclease cleavage, resulting in DNA strand breaks (9)
and this initiates a cascade of molecular events leading to the
release of tissue proteases and ultimately to cell necrosis (4).
Furthermore, since di(adeninyl-ethyl)sulphide products have
been reported with DNA alkylated with sulphur mustard (26),
and if the indication of adenine-adenine intrastrand crosslink
formation is correct, then this is the first report of the potentially
lethal genotoxic effects of this form of DNA crosslink.
The preferential alkylation of adenine residues by sulphur
mustard may also impede the binding of adenine-specific
DNA-binding proteins such as transcription factors. Since
adenine N-l and N-3 alkylation sites are located at the DNA
minor groove, enzymes that bind to the DNA minor groove
may also be hindered. Because nitrogen mustard preferentially
alkylates DNA at the N-7 position of guanine (located in the
major groove), this suggests that alkylation of the minor groove
may be the more damaging lesion and may account for the
greater toxicity observed with sulphur mustard.
The cause of the broad elongation time dependent transcriptional blockages beginning at 43mer and extending to a
47mer is not clear. The 43-45mers are probably due to
alkylation of guanine at position 44 and 45, resulting in
Inhibition of transcription by sulphur mustard
dominantly 43mer and 44mer blockages at early elongation
times. A similar behaviour has been reported with nitrogen
mustard using the same transcription system (13) and was
attributed to the polymerase being able to read-through past
the initial alkylation sites to some degree, to yield blockages
at 46mer and 47mer. Transcriptional blockages are observed
at a number of sites for the first 5 and/or 15 min of elongation,
after which the termination seems to have been overcome and
RNA polymerase is able to continue past these sites. These
sites are located at the 5'-GA (69mer and 71mer), 5'-GT
(26mer, 28mer, 35mer and 61mer) and 5'-GC (38mer, 83mer
and 98mer) sequences on the DNA template strand, all of which
are sites of possible monoadduct formation. The mechanism of
such read-through is unclear. A possible explanation for this
observation is that depurination of the alkylated base has
occurred, thereby allowing RNA polymerase to transcribe
efficiently past such depurinated sites. While this mechanism
has previously been reported for E.coli RNA polymerase at
alkylation-induced apurinic and abasic sites on the DNA
template (27,28), significant depurination events were not
observed until -20 h post-alkylation with nitrogen mustard
(27). Alternatively, this phenomenon could also be due to the
inherent ability of RNA polymerase to bypass this type of
monofunctional adduct. This is important because even if the
RNA polymerase is able to bypass the adduct or alkylationinduced apurinic site, it is not known whether the correct base
is subsequently inserted into the RNA product. If the lesion is
not repaired, an incorrect ribonucleotide is likely to be inserted
into the RNA chain (28) and this may represent a potential
mechanism of sulphur mustard-induced mutagenesis and
carcinogenesis.
Conclusions
Sulphur mustard is a powerful alkylating agent which has been
used as a chemical warfare agent. The potential for exposure
to this agent currently appears greater than at any time since
World War I. As concern over the reappearance of this threat
grows, understanding the key mechanisms of this toxicity
has become increasingly important. However, despite intense
research into the molecular and cellular mechanisms of sulphur
mustard toxicity, none of the current theories of mustard
toxicity have been fully proven, and this is the major reason
why no pharmacological intervention has yet been successful.
The results reported here have several implications for the
current understanding of the mechanism of toxicity of sulphur
mustard. Alkylation of the DNA results in retardation of the
processivity of RNA polymerase. For the RNA polymerases
that are able to escape the initiated complex, mustard alkylation
causes termination of transcription, resulting in the production
of truncated transcripts. If the same process occurs in eukaryotic
systems, then it appears that as well as inhibiting DNA
replication, sulphur mustard alkylation may also inhibit transcription of mRNA with a consequential effect on translation.
Furthermore, the observed read-through at some sites of the
mustard-induced transcriptional blockages may represent a
potentially important mechanism for the known mutagenic and
carcinogenic effect of sulphur mustard. Since the effects of
sulphur mustard on transcriptional processes reported in this
study occur at biologically relevant concentrations, it is likely
that such effects can occur in vivo. A knowledge of the effect
of sulphur mustard on similar aspects of eukaryotic gene
expression is essential and is now the subject of current
investigations. This information will ultimately improve our
capacity to minimize and provide effective therapy for the
casualties of sulphur mustard-induced toxicities.
Acknowledgements
The authors thank Dr Ian Tilley of the Aeronautical and Maritime Research
Laboratory (DSTO) for synthesizing the sulphur mustard. This work was
supported by the Australian Research Council (DRP) and a scholarship from
the Australian Agency for International Development (AM).
References
l.Brookes.P. (1990) The early history of the biological aJkylating agents,
1918-1968. Mutat. Res , 233, 3-14.
2.RobinsonJ.P. (1971) The Problem of Chemical and Biological Warfare,
Vol.1: The Use of CB Weapons. Almquist and Wiksell Humanities
Press, Sweden.
3. WillemsJ.L. (1989) Clinical management of mustard gas casualties. Ann.
Med. Milt. Belg., 3 (Suppl.).
4.BorakJ. and Sidell,R.F. (1992) Agents of chemical warfare: sulphur
mustard. Ann. Emerg. Med., 21, 303-308.
5.Papirmeister,B., FosterJ.A., Robinson.I.S. and FoixLD.R. (1991) Medical
Defense Against Mustard Gas. CRC Press, Boca Raton, FL.
6.Ross,W.C.S. (1962) Biological Alkylating Agents. Butterworths, London.
7.PetraliJ.P, Ogeslsby.S.B. and Meir.H.C. (1990) Ultrastructural correlates
of the proteins afforded by niacinamide against sulphur mustard-induced
cytotoxicity of human lymphocytes in vitro. Ultrasruct. Pathol., 14,
253-262
8,Fox,M. and Scott,D. (1980) The genetic toxicity of nitrogen and sulphur
mustard. Mutat. Res., 75, 131-168.
9. Papirmeister.B., Gross.C.L., Meier.H.L., PetraliJ.P. and Johnson J.B.
(1985) Molecular basis of mustard vesication. Fundam. Appl. Toxicol., S,
S314-S147.
lO.Cullinane.C. and Phillips.D.R. (1993) Thermal stability of DNA adducts
induced by cyanomorpholinoadriamycin in vitro. Nucleic Acids Res., 21,
1857-1862.
ll.White.R.J. and Phillips.D.R. (1988) Transcription analysis of multi-site
drug dissociation kinetics: drug induced termination of transcription.
Biochemistry, 27, 9122-9132.
12 SambrookJ., Fntsch.E.F. and Mamatis.T. (1989) Molecular Cloning: A
Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
13.Gray,P.J., Cullinane.C. and Phillips.D.R. (1991) In vitro transcription
analysis of DNA alkylation by nitrogen mustard. Biochemistry, 30,
8036-8040.
14. Phillips,D.R. and Crothers.D. (1986) Kinetics and sequence specificity of
drug-DNA interactions: an in vitro transcription assay. Biochemistry, 25,
7355-7366.
15.Gill,S.C, Yager.T.D. and von Hippel.PH. (1990) Thermodynamic analysis
of transcription cycle in E.coli. Biophys. Chem., 37, 239-250.
16. von Hippel.P.H., Bear.D.C, Morgan.W.D. and McSwiggerJ.A. (1984)
Protein-nucleic acid interactions in transcription. Annu. Rev. Biochem.,
53, 389-446.
17.Thomas,C.B., Kohn,K.W. and Bonner.W.M (1978) Characterization of
DNA-protein cross-link formed by treatment of LI210 cells and nuclei
with bis(2-chloroethyl)methylamine (nitrogen mustard). Biochemistry, 17,
3954-3958.
l8.Daube,S.S. and von Hippel.P.H. (1986) Functional transcription elongation
complexes from synthetic RNA-DNA bubble duplexes. Science, 258,
1320-1324.
19.Yang,Y.C, Szafransec.L.L., Beaudry.W.T. and WardJ.R (1983) Kinetics
and mechanisms of the hydrolysis of 2-chloroethyl sulphides. /. Org.
Chem., 53, 3293-3297.
20. Straney.D.C. and Crothers.D.M. (1985) Intermediates in transcription from
E.coli lac UV5 promoter. Cell, 43, 449^*59.
21.Gray.PJ. and Phillips.D.R. (1993) Effects of alkylating agents on the
initiation and elongation of the lac UV5 promoter. Biochemistry, 32,
12471-12477.
22. Phillips.D.R. (1995) Transcription assay for probing molecular aspects of
drug-DNA interactions. In HurIey,L.H. and ChairesJ.B. (eds), Advances
in DNA Sequence Specific Agents. JAI Press, Texas, vol. II, in press.
23. MillardJ.T., Raucher,S. and Hopkins, (1990) Mechloroethamine crosslinks
deoxyguanine residues at 5'-GNC sequence in duplex DNA fragments.
/ Am. Chem. Soc., 112, 2459-2460.
24. Pieper.R.O. and Enckson.L.C. (1990) DNA adenine adducts induced by
nitrogen mustards and their role in transcription termination in vitro.
Carcinogenesis, 11, 1739-1746.
531
A.Masta, PJ.Gray and D.R.Phlllips
25. Pieper.R.O., Fustscher.B.W. and Erickson,L.C. (1989) Transcriptionterminating lesions by bifunctiona] alkylating agents. Carcinogenesis, 10,
1307-1314.
26. Lawley.P.D., LethbridgeJ.H., Edwards.P.A. and Shooter.K.V. (1969)
Inactivation of bactenophage T7 by mono- and difunctional sulphur
mustard in relation to cross-linking and depurination of bactenophage
DNA. J. MoL Biol., 39, 181-198.
27.Masta,A., GrayJ.P. and Phillips,D.R. (1994) Molecular basis of nitrogen
mustard effects on transcription: role of depurination. Nucleic Acids Res.,
22, 3880-3886.
28.Zhou,W. and Doetsch.P. (1993) Efficient bypass and base misinsertion at
abasic sites by prokaryotic RNA polymerases. Proc. Natl Acad. Sci. USA,
90, 6601-6605.
Received on September 22, 1995; revised on December 12, 1995; accepted
on December 13, 1995
532