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(Received for publication, September 26,
1995; and in revised form, December 12, 1995) From the
Adriamycin is known to specifically induce DNA interstrand
cross-links at 5`-GC sequences. Because 5`-GC sequences are a
predominant feature of 5`-untranslated regions (transcription
factor-binding sites, promoter, and enhancer regions), it is likely
that adriamycin adducts at GC sites would affect the binding of
DNA-interacting proteins. Two model systems were chosen for the
analysis: the octamer-binding proteins Oct-1, N-Oct-3 and N-Oct-5,
which bind to ATGCAAAT and TAATGARAT recognition sites, and Escherichia coli RNA polymerase binding to the lac UV5 promoter. Electrophoretic mobility shift studies showed that
adriamycin adducts at GC sites inhibited the binding of octamer
proteins to their consensus motifs at drug levels as low as 1
µM, but no effect was observed with a control sequence
lacking a GC site. Adriamycin adducts at GC sites also inhibited the
binding of RNA polymerase to the lac UV5 promoter. Adriamycin
may therefore function by down-regulating the expression of specific
genes by means of inactivation of short but critical motifs containing
one or more GC sites. The mode of action of the anticancer drug adriamycin has been
examined extensively over the past 20 years. Many different studies
have cited the interaction of the inherently reactive drug with cell
membranes, DNA, proteins, metal ions, and molecular oxygen, leading to
an apparently complex interplay of the mechanism of antitumor action,
the major determinants of which may differ according to the properties
of target cancer cells (Myers et al., 1988). Although the
reactivities of adriamycin with a range of cellular constituents are
well known, the specific cellular mechanisms involved and the ultimate
cause of tumor cell death are still to be elucidated. One possible
physiological action in tumor cells involves the altered regulations of
DNA-binding proteins in actively transcribed DNA (i.e. the
open DNA regions in nuclear matrix attachment sites (Ciejek et
al., 1983)). Covalent attachment of the drug chromophore to
specific DNA consensus sequences required for recognition by
DNA-binding proteins may lead to altered levels and modes of binding by
these proteins. The majority of adriamycin administered to sensitive
tumor cells is known to rapidly localize in the nucleus (Gigli et
al., 1988). The drug has a high affinity for DNA, thus providing
the driving force for further nuclear uptake. It is well known that
intercalation is the immediate form of interaction, and there is an
extensive body of evidence to show that one of the first cellular
responses is the impairment of topoisomerase II activity (see for
example reviews by Liu (l989); Holm et al. (l99l); Capranico
and Zunino (l992); Sinha (l995)). However, upon reductive activation
the drug can also bind covalently to DNA (Moore, 1977; Sinha, 1980).
The sequence specificity of this interaction in vitro is
highly GC selective (Cullinane and Phillips, 1990), and it appears that
an interstrand cross-link occurs at this site (Cullinane et
al., 1994b; Cutts and Phillips, 1995). Adriamycin-induced
cross-links have also been reported in HeLa S Once a DNA-drug adduct is formed, it
is widely accepted that the nature of the interaction impedes cellular
functions that involve DNA (i.e. replication and
transcription), particularly in the event of damage to both strands by
the formation of an interstrand cross-link (Hopkins et al.,
1991). It follows that DNA-binding proteins would be affected to
varying degrees by their modified substrate. Although many
intercalating drugs have been repeatedly shown to disrupt the actions
of topoisomerase II, recently some covalently binding agents have also
been shown to disrupt the binding of transcription factors to their
specific consensus sequences (Broggini and D'Incalci, 1994; Welch et al., 1994; Sun and Hurley, 1994). If these adducts prevent
binding of transcription factors to DNA in tumor cells, then the
sequence selectivity of the particular drug will determine which
transcription factors are affected, and hence which genes are
inhibited. The net effect of this process is that gene-specific
inhibition may occur, depending on the sequence specificity of the
particular drug adducts. The availability of in vitro electrophoretic mobility shift assay (EMSA) ( DNA-dependent polymerases are
also potential targets for inhibition when site-specific DNA adducts
are formed in promoter regions. Simple gel retardation assays for the
detection of binding of eukaryote RNA polymerases are not available.
However, the interaction between Escherichia coli RNA
polymerase and the lac UV5 promoter has been extensively
studied, and a simple assay for this interaction is well documented
(Straney and Crothers, 1985; Gray and Phillips, 1993). Although there
is a high frequency of GC sites in this region, they do not appear to
play a role in direct contact with the RNA polymerase (von Hippel et al., 1984; Brodolin et al., 1993). This system
therefore provides an opportunity to study the indirect effect of
adducts on protein binding within the promoter region. In this study
we show that the covalent binding of low concentrations of adriamycin
to GC sequences interferes with the ability of octamer proteins to bind
at these sites. The binding of prokaryote RNA polymerase was also
inhibited in its binding to the lac UV5 promoter.
Fig. 1shows that
there is an adriamycin concentration-dependent inhibition of octamer
binding with the N-Oct-3, N-Oct-5, and Oct-1 proteins, with all
following similar inhibition patterns. At 10 µM adriamycin, the binding of Oct-1, N-Oct-3, and N-Oct-5 is
inhibited by approximately 25, 40, and 30% respectively (Fig. 1B). To confirm that this effect was due to
covalent binding of the drug chromophore to the consensus sequence (or
whether it was merely an effect of drug intercalation), 10 µM of adriamycin was reacted with the DNA probe for increasing times
prior to exposure to the nuclear extract (Fig. 2, A and B). The yield of adriamycin-DNA adducts is known to be optimal
after a reaction time of approximately 40 h with a half-life of
formation of adducts being about 15 h (Cullinane et al.,
1994b), and this effect is consistent with that shown in Fig. 2B where inhibition of protein binding is maximal
after 60-80 h with a half-life of inhibition of all three octamer
proteins being less than 20 h. Because the percentage inhibition of
protein binding increased with drug-DNA reaction time, this confirms
that the inhibition is due to specific adriamycin-induced adducts
rather than other nonspecific effects such as intercalation. All three
octamer-binding proteins were also inhibited (but to a lesser extent)
by exposure of the DNA to 1 µM adriamycin for 40 h (Fig. 2, C and D.).
Figure 1:
Gel retardation assay of inhibition of
binding by octamer proteins with increasing adriamycin concentration. A, the H2B probe was exposed to 0.5-100 µM adriamycin in the presence of 20 µM FeCl
Figure 2:
Inhibition of octamer protein binding as a
function of drug reaction time. The H2B probe was reacted for
0-96 h with 10 µM adriamycin and 20 µM FeCl
The extent of formation
of adriamycin-induced adducts with DNA is known to be catalyzed by the
presence of FeCl
Figure 3:
Octamer protein binding to the alternative
motif TAATGAATT. A, the OA25 probe was reacted with increasing
concentrations of adriamycin (0.5-100 µM) in the
presence of 20 µM FeCl
Figure 4:
Lambda exonuclease digestion of OA25 and
H2B probes following reaction with adriamycin. Exonuclease digestion
was performed after reaction with 1 µM adriamycin (lanes 5), 1 µM adriamycin and 20 µM FeCl
Figure 5:
Quantitative analysis of lambda
exonuclease blockage sites in OA25 (A) and H2B (B)
probes. The histograms show the results for lane 7 of Fig. 4(10 µM adriamycin). Only bands representing
greater than 2% of the mole fraction of total radioactivity in the lane
are shown. The boxed sequences represent the complementary
strand to the consensus sequence of the alternative motif (A)
and the sequence of the wild-type consensus motif (B). The
numbering scheme employed is arbitrary.
The time dependence of formation of
adriamycin adducts (prior to exposure to polymerase) under these
conditions is shown in Fig. 6A. There is a decreasing
amount of the DNA-RNA polymerase complex with increasing time of
reaction of adriamycin with the DNA fragment (Fig. 6A)
or with increasing adriamycin concentration (Fig. 7A),
and the relative amount of DNA existing in the complexed form is shown
in Fig. 6B and 7B, respectively. The extent of
binding is reduced 5-fold with saturation at 10 µM drug
concentration. To confirm that the promoter region was being alkylated,
the DNA was probed by digestion with lambda exonuclease for DNA reacted
for increasing times with 5 µM adriamycin.
Sequence-specific blockages are evident in the image shown in Fig. 8, and the location of the blockages are shown in Fig. 9. Every potential adriamycin GC cross-linking site is
alkylated, as indicated by inhibition of digestion several nucleotides
5` to each GC site. More importantly, adducts are evident at every GC
site in the RNA polymerase-binding site.
Figure 6:
Gel retardation assay of the inhibition of
formation of the RNA polymerase-promoter complex by adriamycin. A, end-labeled 188-bp fragment containing the lac UV5
promoter was incubated with 5 µM adriamycin and 40
µM FeCl
Figure 7:
Adriamycin concentration-dependent
inhibition of binding of RNA polymerase to the lac UV5
promoter. A, the 188-bp fragment was reacted with 0.5-50
µM adriamycin in the presence of 40 µM FeCl
Figure 8:
Lambda exonuclease digestion of adriamycin
reacted lac UV5 promoter region. The 188-bp fragment was
reacted with 5 µM adriamycin and 40 µM FeCl
Figure 9:
Location of adducts in the lac UV5 promoter region. Only bands comprising greater than 1% of the
mole fraction of lane 4 of Fig. 8are shown. The boxed sequence section represents the region covered by RNA
polymerase when bound to the promoter in the open complex (Carpousis
and Gralla, 1985).
In
the TAATGARAT motif, chemical modification and mutation analyses
suggest that the Oct-1 POU-specific domain contacts the 3`-GARAT
sequence (R indicates purine), whereas the 5`-TAAT sequence is
contacted by the Oct-1 POU homeodomain. As the exonuclease digestion
pattern shows (Fig. 5A), there are no adducts in this
region to cause direct disruption of binding. However, DEPC alkylation
(which forms adducts with adenine and guanine bases) does result in
disruption of the interaction between the Oct-1 protein and the
TAATGARAT binding sequence (Cleary and Herr, 1995). These results
collectively show that DEPC alkylation of the alternative motif
probably interferes with octamer binding in a similar manner to
adriamycin inhibition of octamer binding to ATGCAAAT. However, because
the exonuclease digestion indicates that adriamycin has no capacity to
form adducts at the TAATGAATT sequence, the adriamycin-induced
inhibition must be directly due to adducts at the 5`-GC sequence.
Previous studies of the characteristics of adriamycin-induced DNA
adducts show that the nature of this sequence specificity to be due to
an interstrand cross-link (Cutts and Phillips, 1995). Overall, the
results from this study indicate that the covalent adducts induced by
adriamycin in DNA do have a potential inhibitory role in vivo with respect to protein binding to sequences containing 5`-GC
sites. Because the octamer-binding proteins are able to recognize a
variety of motifs with varying affinities of binding, it is likely that
a selective drug-induced inhibition of some octamer proteins would also
arise in vivo. It is questionable whether the extent of
inhibition of octamer protein binding facilitated by adriamycin would
be significant enough to lead to detectably altered regulation patterns
of specific cellular proteins, but it is possible that the effects of
adducts could be further magnified when the complex interactions of
proteins required in transcription initiation are considered.
It is
significant that many genes in the mammalian genome (and especially
those genes such as protooncogenes associated with proliferation) have
a high occurrence of GC runs in their 5` regions (Hartley et
al., 1988). For example the c-Ha-ras oncogene contains
seven GC runs in the 5`-flanking region, some of these runs being
contained in Sp1 binding enhancer regions (Mattes et al.,
1988). GC modifying drugs such as adriamycin are likely to favor these
regions due to the sequence content and accessibility of these regions
(the tight packaging of DNA in nucleosome structures appears to become
relaxed and accessible when assembled on nuclear matix attachment
regions for transcriptional processes to occur) (Workman and Buchman,
1993; Krajewska, 1992; Wolffe, 1992; Ciejek et al., 1983).
There are a number of transcription factors that require a GC in their
consensus DNA sequences, including CTF/NF1, AP2, SP1, and Oct-1 and
Oct-2 proteins (Wingender, 1990; Williams and Tijian, 1991), and all of
these sequences are therefore potential targets for adriamycin
alkylation. It is now important to establish if there is a
correlation between adriamycin treatment and selective targeting of
genes that are high in GC content. Although adriamycin has been a major
inclusion in chemotherapy regimes for 20 years and appears to act as a
topoisomerase II inhibitor, a major research effort is still required
directed toward elucidating its basic modes of action in tumor tissue.
It has recently been shown that the ability of a range of adriamycin
derivatives to induce interstrand cross-links correlated well with
their cytotoxicity (Skladanowski and Konopa, 1994b), and it has
generally been assumed that this effect is related essentially to the
impairment of the processive movement of DNA polymerase and RNA
polymerase. The present results suggest that interstrand cross-links
may well exert a separate earlier effect in inhibiting the binding of
proteins such as transcription factors and RNA polymerase to the
modified DNA and because of the short sequences (e.g. octamer)
involved in promoter activity may confer gene specificity to adriamycin
action.
Volume 271,
Number 10,
Issue of March 8, 1996 pp. 5422-5429
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
cells
(Skladanowski and Konopa, 1994a), but it remains to be seen whether the
same in vitro sequence selectivity applies, although studies
with other sequence-specific damaging agents suggest that this is
likely (Murray and Martin, 1985; Hartley et al., 1992;
Cullinane and Phillips, 1994) .
)systems
for the octamer family of transcription factors provides a model system
for the study of adriamycin-induced inhibition of protein-DNA binding
and avoids the denaturation, heating, or alkaline treatments required
in other types of DNA damage detection, all of which contribute to
instability of adriamycin adducts (van Rosmalen et al., 1995).
The consensus recognition sequence for these proteins is an 8-bp
ATGCAAAT element and involves a single GC dinucleotide within the
octamer element that participates directly in binding of the Oct
proteins through the POU-specific region of the POU DNA-binding domain
(Klemm et al.(1994); reviewed in Herr and Cleary(1995)). An
alternative recognition sequence for some octamer-binding proteins is
the TAATGARAT motif present in herpes simplex virus immediate early
gene promoters (O'Hare and Goding, 1988; Preston et al.,
1988; Baumruker et al., 1988), but because this site does not
include a GC dinucleotide in the binding site, it serves as an
excellent control to assess the significance of adriamycin-induced
interstrand cross-links at GC sites.
Materials
Adriamycin hydrochloride was a gift
from Farmitalia Carlo Erba, Milan, Italy. The Klenow fragment of DNA
polymerase I and the radiochemicals
[
-P]dATP and dGTP were purchased from
Amersham Corp. E. coli RNA polymerase and DNase free bovine
serum albumin were obtained from Pharmacia Biotech Inc., and the
restriction enzymes EcoRI, HindIII, and PvuII were from New England Biolabs. Phenol was obtained as a
solid from International Biotechnologies Incorporated, lambda
exonuclease was from Life Technologies, Inc., and calf thymus DNA was
from Worthington Biochemical Corporation.
Plasmids and Nuclear Extracts for EMSA
The
plasmids pUC119 H2B-box+ and pBSOA25 have been described
previously (Baumruker et al., 1988; Bendall et al.,
1993). These plasmids were digested with EcoRI and HindIII to release restriction fragments containing the
following motifs: ATGCAAAT, the wild-type H2B gene octamer-binding
sequence, and TAATGAATT, the OA25 high affinity binding site for the
N-Oct-3- and N-Oct-5-binding proteins. The fragments were purified by
electrophoresis through 2% agarose and then electroeluted in a biotrap
apparatus (Schleicher & Schuell). The fragments were labeled with
[-P]dATP using the Klenow fragment of DNA
polymerase I and separated from unincorporated nucleotides and proteins
using Nensorb 20 cartridges (DuPont NEN). Nuclear extracts from the
secondary malignant melanoma cell line A2058 were prepared as described
previously (Sturm et al., 1991), and the protein content was
determined by the Bradford assay (Bio-Rad) as containing 2-4
µg of protein/µl.
Adriamycin Reactions
Drug-DNA reactions were
performed in a volume of 5 µl. These reactions typically contained
7 mM dithiothrietol, approximately 500 cpm of labeled DNA
fragment, 25 µM bp linearized pSP64 plasmid DNA, 20
µM FeCl, and 10 µM adriamycin in
a transcription buffer consisting of 40 mM Tris, 3 mM MgCl
, 100 mM KCl, 0.1 mM EDTA, pH
8.0. The reactions were supplemented with pSP64 at the optimal
concentration (25 µM bp) for the formation of adriamycin
adducts with DNA (Cullinane et al., 1994a), and the amount of
labeled DNA fragment added was assumed to be negligible. Reactions were
allowed to proceed for up to 96 h. Samples were either used immediately
in the EMSA assay or stored at -20 °C for up to 1 week. The
EMSA assay was performed following the conditions outlined elsewhere
(Sturm et al., 1991), and the samples were characterized by
nondenaturing polyacrylamide gel electrophoresis.Exonuclease Assay
The OA25 and H2B fragments were
3` end-labeled at the HindIII site by using
[-P]dGTP following standard procedures.
Drug reactions were set up in 10-µl volumes and included the
components as described previously. The reaction was allowed to proceed
for 40 h, after which samples were made up to 30 µl with
transcription buffer. The DNA was then precipitated with ethanol and
resuspended in 5 µl of a solution containing 0.36 units/µl
lambda exonuclease, 50 µg/µl bovine serum albumin, 67 mM glycine-KOH, 2.5 mM MgCl
, pH 9.4. Exonuclease
digestion was allowed to proceed for 1 h at 37 °C, and then an
equal volume of 90% formamide loading buffer was added and samples were
heat denatured at 90 °C for 5 min before being subjected to
electrophoresis using a 12% denaturing polyacrylamide sequencing gel.
Maxam-Gilbert G sequencing lanes were performed as described elsewhere
(Maxam and Gilbert, 1977).RNA Polymerase Assay
A 188-bp fragment containing
the lac UV5 promoter was isolated from the plasmid pCC1
(Cullinane and Phillips, 1993) using the restriction enzymes EcoRI and PvuII. The DNA was purified and 3` end
labeled using [-P]dATP. After
lyophilization, the fragment was resuspended in Tris-EDTA buffer, and
calf thymus DNA was added to generate a 175 µM bp stock of
DNA. Drug reactions were performed as described for the octamer assay.
After the reaction, 40 µl of transcription buffer was added and the
samples were extracted twice with an equal volume of Tris-saturated
phenol and once with chloroform. Samples were precipitated with ethanol
using glycogen as an inert carrier of the DNA, and samples were
resuspended in 5 µl of transcription buffer. The samples were then
exposed to RNA polymerase as described previously (Gray and Phillips,
1993) using a final concentration of 615 nM RNA polymerase,
125 µg/µl bovine serum albumin, 10 mM dithiothrietol.
This solution was incubated for 15 min at 37 °C, and heparin was
subsequently added for a further 5 min to quench nonspecific binding of
the RNA polymerase. An equal volume of loading buffer (50% glycerol, 20
mM dithiothrietol) was added to samples prior to
characterization by nondenaturing electrophoresis through a 5%
Tris-glycine gel.
Adriamycin Adducts with the ATGCAAAT Octamer Consensus
Motif
Initial studies were performed to establish at what levels
adriamycin-induced adducts would prevent octamer proteins binding to
their consensus motif. The crystal structure of Oct-1 bound to ATGCAAAT
has revealed major contacts of the POU specific domain of Oct-1
directly to the GC within this sequence (Klemm et al., 1994).
It was therefore anticipated that the tetracyclic structure of
adriamycin covalently bound at a GC site would pose a direct blockage
to this interaction. A2058 human melanoma cells were chosen as the
source of nuclear extract because they contain three different octamer
proteins that produce markedly different mobilities in an EMSA assay.
The octamer motif is important for promoter activation mediated through
the generally expressed Oct-1 protein, such as the histone H2B and
snRNA genes (reviewed by Herr(1992)). The octamer element is also a
target-binding site for the N-Oct-3 and N-Oct-5 octamer factors
(Schreiber et al., 1990; Sturm et al., 1991) encoded
by the Brn-2 POU domain gene (Schreiber et al., 1993; Thomson et al., 1995). N-Oct-3 and N-Oct-5 are commonly present in
neuroectodermal derived tissues (Schreiber et al., 1992;
Thomson et al., 1994) including all human melanoma cell lines
tested (Thomson et al., 1993).
for an incubation period of 24 h. Control samples were incubated
in the presence of 20 µM FeCl (denoted Fe) or in the absence of both FeCl and adriamycin
(denoted C). The reacted probe was exposed to nuclear extract
from A2058 cells. Electrophoretically retarded bands denote protein-DNA
complexes due to Oct-1, N-Oct-3, and N-Oct-5 proteins present in the
nuclear extract. B, PhosphorImager quantitation of the Oct-1
(
), N-Oct-3 (), and N-Oct-5 (
) complexes show the
trends of protein binding with increasing adriamycin concentration.
Band intensities were calculated relative to the Oct-1 band in the 0.5
µM adriamycin lane.
(A), 1 µM adriamycin and 20
µM FeCl (C), or 10 µM adriamycin in the absence of FeCl before exposure to
the A2058 nuclear extract (E). Band intensities were
quantitated for A, C, and E as a percentage
of the total band intensity (i.e. total end-labeled probe) in
each lane, and the results are shown in B, D, and F, respectively. The time-dependent inhibition was fitted to a
single exponential decay.
with approximately 5-fold more adducts
being formed when DNA is reacted with adriamycin for 50 h in the
presence of 10 µM FeCl compared with the
absence of added FeCl (Cullinane et al., 1994b).
This catalytic role is shown in Fig. 2E where Oct-1,
N-Oct-3, and N-Oct-5 proteins were scarcely affected when FeCl was absent but resulted in extensive inhibition of binding
capacity of all three proteins when FeCl was present during
the reaction of drug with DNA (Fig. 2, A and B). Overall, the inhibition of binding of octamer proteins
correlates well with the known characteristics of formation of
adriamycin-induced adducts. Although these trends are quite clear and
highly reproducible, there is some variation in the absolute level of
formation of octamer protein-DNA complexes (5-17% in Fig. 2, B, D, and F), and this is due
dominantly to the varying concentration of transcription factors
present in individual nuclear extracts (Thomson et al., l993). Adriamycin Treatment of a TAATGARAT-like Octamer-binding
Site
Adriamycin cross-links (which comprise a large proportion
of total adducts) occur almost exclusively at 5`-GC dinucleotide sites
(Cullinane and Phillips, 1990). To confirm the effect of adducts at the
GC position of the wild-type octamer sequence, an alternative binding
motif was utilized that lacked the central GC site. The OA25 site
containing the TAATGARAT-like motif TAATGAATT was isolated in a binding
site selection protocol using antibodies to the Oct-1 protein (Bendall et al., 1993) and shows higher affinity for the Brn-2-derived
proteins N-Oct-3 and N-Oct-5 than Oct-1. Moreover, these proteins show
greater affinity for this site than the wild-type octamer sequence
(Thomson et al., 1994). Fig. 3shows the results of a
concentration-dependent reaction of adriamycin with the recognition
fragment before exposure to nuclear extract. There was no discernable
effect of increasing drug concentration up to 50 µM (the
apparent decrease of binding in the 100 µM adriamycin lane
is due to extensive aggregation of the DNA, which is then largely
trapped in the loading well).
prior to exposure to
the A2058 nuclear extract. B, the quantitation is
shown.
DNA Sequence Specificity of Adducts in Octamer-binding
Sequences
To ensure that adducts were located specifically
within the octamer recognition site and not in an alternative motif,
the location of adducts in the H2B and OA25 fragments were probed by
digestion with lambda exonuclease. Fig. 4shows the blockages to
exonuclease digestion of the drug-treated DNA fragments (three drug
levels, lanes 6-8) for both the GC containing octamer
sequence (H2B) and the control octamer sequence lacking the GC site
(OA25). The location of these blockages (with respect to GC sites and
the recognition sequences) is shown in Fig. 5for the DNA
reacted with 1 µM adriamycin. Digestion of DNA by lambda
exonuclease is typically stalled in a staggered manner 1-3
nucleotides prior to adduct sites. A major adduct site is clearly
identified in Fig. 5directly within the octamer-binding site of
the wild-type motif and is located at the GC position. However, within
the TAATGARAT-like motif (lacking a GC site), no adducts are evident,
and this result is consistent with the documented GC specificity of
adriamycin-induced adducts. With increasing adriamycin concentration
and also in the presence of 20 µM FeCl (Fig. 4, lane 8) exonuclease digestion was
limited due to the presence of a greater amount of adducts on both DNA
fragments, and this results in all downstream adduct sites being
greatly underestimated. However, in lane 8 of Fig. 4,
the amount of radiolabel associated with the first adduct site is a
good indication of the extent of adducts present at all other similar
(GC) sites.
(lane 6), 10 µM adriamycin (lane 7), 10 µM adriamycin and 20 µM FeCl (lane 8), in the absence of adriamycin
and FeCl (lane 3), and with only 20 µM FeCl (lane 4). Lanes 1 and 2 denote samples reacted in the presence and the absence of
FeCl, respectively, but not subjected to lambda exonuclease
digestion. Maxam-Gilbert G sequencing lanes G and octamer
binding sequences are shown for each probe.
Effect of Adriamycin Adducts in the lac UV5
Promoter
When E. coli RNA polymerase binds to its
recognition sequence, it has been shown by DNase I footprinting studies
that it covers the -50 to +20 nucleotide region when in the
open promoter complex form (Carpousis and Gralla, 1985). Adriamycin
5`-GC adduct sites are well represented in this sequence but not at
known direct contact sites. It was therefore of interest to establish
if adducts at these sites would affect the interaction between RNA
polymerase and the promoter region. Initial studies were conducted by
exposing DNA to adriamycin for various times followed by the addition
of the RNA polymerase. This resulted in complete inhibition of the
ability of RNA polymerase to bind, probably due to unwinding of the
promoter region by the presence of intercalated drug. Therefore, to
probe the more specific effect of adriamycin adducts on formation of
the transcription process, a rigorous phenol (2
) followed by a
chloroform cleanup of the DNA adducts was employed. The extent of
complex formed between E. coli RNA polymerase and end-labeled
DNA fragment (containing the lac UV5 promoter) was probed by a
gel retardation assay.
for 0-48 h (denoted by +Adr) before a phenol cleanup and exposure to RNA
polymerase. Samples reacted in the absence of adriamycin (C)
for 0 and 48 h are also shown. DNA probe not exposed to RNA polymerase
is indicated by P. B, the percentage of labeled probe
associated with the RNA polymerase-promoter complex with increasing
time of reaction with adriamycin is shown.
prior to the addition of E. coli RNA
polymerase. The DNA fragment was then subjected to electrophoresis, and
the retarded DNA-RNA polymerase band was detected by PhosphorImager
analysis. B, quantitation of the percentage RNA polymerase
bound is shown.
for 0-48 h, and the DNA was extracted twice
with phenol and once with chloroform prior to ethanol precipitation.
Samples were then digested with lambda exonuclease and characterized by
electrophoresis through an 8% denaturing polyacrylamide gel.
Maxam-Gilbert sequencing lanes are denoted as G.
Adriamycin Facilitated Disruption of Octamer
Protein-DNA Interactions
From the gel shift assays shown in Fig. 1Fig. 2Fig. 3, it is clear that micromolar
levels of adriamycin form a sufficient number of adducts with the
wild-type octamer sequence to inhibit the binding of N-Oct-3, N-Oct-5
and Oct-1 proteins. Although there is no structural information
available on the interaction of the N-Oct-3 and N-Oct-5 proteins with
the octamer site, an identical methylation interference pattern is seen
on the wild-type octamer sequence with all three of these proteins
(Schreiber et al., 1990). The interaction of the Oct-1 POU
domain with the octamer element is now well defined, with the crystal
structure of the complex being resolved at 3.0 Å resolution
(Klemm et al., 1994). From this structure it is evident that
the Oct-1 POU specific domain contacts the 5` half of the site (ATGC),
whereas the POU-homeo domain contacts the 3` half, with the domains
binding on opposite sides of the DNA duplex. The adriamycin GC
cross-link site in the motif plays a crucial role in the binding mode
of the POU-specific domain as Arg-49 of the POU-specific domain forms a
hydrogen bond with the O-6 of guanine and also makes contact with the
O-6 and N-7 positions of the guanine on the opposite strand. Because
these guanine bases are integral components of the cross-link as well
as for Oct-1 protein binding, it is highly likely that there will be
steric constraints for protein binding when an adriamycin cross-link
(or adduct) is present. These contact sites have been directly
demonstrated with the use of a variety of other chemical protection and
interference assays, with dimethyl sulfate methylation and
diethylpyrocarbonate carbethoxylation modification (Sturm et
al., 1987; Baumruker et al., 1988; Pruijn et
al., 1988; Verrijzer et al., 1990) of the guanine
nucleotides or ethylation of the phosphate backbone (Pruijn et
al., 1988) of either strand each preventing Oct-1 DNA-protein
interactions.Importance of the GC Specificity of Adriamycin
Adducts
Interestingly, the GC site of an octamer motif does not
provide critical contacts in the recognition of DNA-binding sites by
octamer-binding proteins. As has been discussed previously by Baumruker et al.(1988), flanking sequences may be crucial in binding
site recognition with possibly few obligatory contact nucleotides
within a defined site. Mutation analyses have shown that the GC site
can be altered to TT and still retain good binding capacity. However,
the Arg-49 that makes contacts with the site seems to be important in
making flexible contacts with the DNA region (Cleary and Herr, 1995),
and this site therefore has been established as integral to the binding
process. The bulky adduct caused by an agent such as adriamycin is
unlikely to be able to be accommodated in such an interaction.Inhibition of RNA Polymerase
von Hippel(1984) has
proposed that RNA polymerase can be seen as a complementary surface to
that exposed by the promoter region of DNA and that recognition is
determined predominantly by hydrogen bonding, whereas the stability may
be determined by the electrostatic interaction between basic residues
of the protein and the DNA phosphate backbone, as well as interaction
between hydrophobic groups of protein and methyl groups of thymine.
Although there are five potential adriamycin adduct sites within the
DNA sequence recognized by RNA polymerase, they do not interfere with
the major contacts in the -10 and -35 regions. Formaldehyde
cross-linking studies of the RNA polymerase bound to the lac UV5 promoter have detected DNA-protein interactions at the
-50 to -49, -5 to -10, +5 to +8, and
+18 to +21 regions of the DNA sequence (Brodolin et
al., 1993). Adriamycin cross-links or monoadducts are not present
at any of these positions but are within 2 nucleotides of the +5
to +8 interaction, and this may affect the extent of contact at
this site. The adducts are also likely to influence nonspecific
interactions of the polymerase with its recognition sequence and to
inhibit transition to the open complex. Because significant bending of
the DNA seems to be required for polymerase-promoter interaction
(Cartenberg and Crothers, 1991), it is also possible that adriamycin
adducts disturb the DNA bending potential in the promoter region.Significance of Adducts
In order for transcription
to proceed, transcription factors and RNA polymerase must be able to
recognize and bind to their target sequences, and additional processes
that involve large regions of DNA being bent bring distant components
of the transcription machinery together (van der Vlet and Verrijzer,
1993). Therefore it is conceivable that adducts could inhibit gene
transcription on three different levels: 1) by inhibiting DNA-binding
proteins through steric constraints, rendering DNA unrecognizable due
to adduct induced bending, or through subsequent mutation of the DNA
bases required in the consensus sequence (Broggini and D'Incalci,
1994); 2) by inhibiting the potential of the DNA to bend into the
required conformation to bring distant regions together and initiate
transcription (van der Vlet and Verrijzer, 1993); and 3) by posing a
direct blockage to the path of RNA polymerase (Cullinane and Phillips,
1990). Overall, the extent of inhibition of transcription facilitated
by DNA adducts is potentially much greater than that predicted by the
simple in vitro assays outlined in this study.
)
We thank Farmitalia Carlo Erba for the supply of
adriamycin.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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