A 1,2-d(GpG) cisplatin intrastrand cross-link influences the rotational and translational setting of DNA in nucleosomes

Article abstract of DOI:10.1021/ja710220x

The mechanism of action of platinum-based anticancer drugs such as cis-diamminedichloro-platinum(II), or cisplatin, involves three early steps: cell entry, drug activation, and target binding. A major target in the cell, responsible for the anticancer activity, is nuclear DNA, which is packaged in nucleosomes that comprise chromatin. It is important to understand the nature of platinum-DNA interactions at the level of the nucleosome. The cis-{Pt(NH ₃)₂}²⁺ 1,2-d(GpG) intrastrand cross-link is the DNA lesion most commonly encountered following cisplatin treatment. We therefore assembled two site-specifically platinated nucleosomes using synthetic DNA containing defined cis-{Pt(NH₃)₂}²⁺ 1,2-d(GpG) cross-links and core histones from HeLa-S3 cancer cells. The structures of these complexes were investigated by hydroxyl radical footprinting and exonuclease III mapping. Our experiments demonstrate that the 1,2-d(GpG) cross-link alters the rotational setting of the DNA on the histone octamer core such that the lesion faces inward, with disposition angles of the major groove relative to the core of ξ ≈ -20° and ξ ≈ 40°. We observe increased solvent accessibility of the platinated DNA strand, which may be caused by a structural perturbation in proximity of the 1,2-d(GpG) cisplatin lesion. The effect of the 1,2-d(GpG) cisplatin adduct on the translational setting of the nucleosomal DNA depends strongly on the position of the adduct within the sequence. If the cross-link is located at a site that is in phase with the preferred rotational setting of the unplatinated nucleosomal DNA, the effect on the translational position is negligible. Minor exonuclease III digestion products in this substrate indicate that the cisplatin adduct permits only those translational settings that differ from one another by integral numbers of DNA helical turns. If the lesion is located out of phase with the preferred rotational setting, the translational position of the main conformation was shifted by 5 bp. Additionally, a fraction of platinated nucleosomes with widely distributed translational positions was observed, suggesting increased nucleosome sliding relative to platinated nucleosomes containing the 1,3-intrastrand d(GpTpG) cross-link investigated previously (Ober, M.; Lippard, S. J. J. Am. Chem. Soc. 2007, 129, 6278-6286).

Full text of DOI:10.1021/ja710220x

Published on Web 02/13/2008
A 1,2-d(GpG) Cisplatin Intrastrand Cross-Link Influences the
Rotational and Translational Setting of DNA in Nucleosomes
Matthias Ober and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
Received November 11, 2007; E-mail: lippard@mit.edu
Abstract: The mechanism of action of platinum-based anticancer drugs such as cis-diamminedichloro-
platinum(II), or cisplatin, involves three early steps: cell entry, drug activation, and target binding. A major
target in the cell, responsible for the anticancer activity, is nuclear DNA, which is packaged in nucleosomes
that comprise chromatin. It is important to understand the nature of platinum-DNA interactions at the level
of the nucleosome. The cis-{Pt(NH3)2}2+ 1,2-d(GpG) intrastrand cross-link is the DNA lesion most commonly
encountered following cisplatin treatment. We therefore assembled two site-specifically platinated nucleo-
somes using synthetic DNA containing defined cis-{Pt(NH3)2}2+ 1,2-d(GpG) cross-links and core histones
from HeLa-S3 cancer cells. The structures of these complexes were investigated by hydroxyl radical
footprinting and exonuclease III mapping. Our experiments demonstrate that the 1,2-d(GpG) cross-link
alters the rotational setting of the DNA on the histone octamer core such that the lesion faces inward, with
disposition angles of the major groove relative to the core of ê ≈ -20° and ê ≈ 40°. We observe increased
solvent accessibility of the platinated DNA strand, which may be caused by a structural perturbation in
proximity of the 1,2-d(GpG) cisplatin lesion. The effect of the 1,2-d(GpG) cisplatin adduct on the translational
setting of the nucleosomal DNA depends strongly on the position of the adduct within the sequence. If the
cross-link is located at a site that is in phase with the preferred rotational setting of the unplatinated
nucleosomal DNA, the effect on the translational position is negligible. Minor exonuclease III digestion
products in this substrate indicate that the cisplatin adduct permits only those translational settings that
differ from one another by integral numbers of DNA helical turns. If the lesion is located out of phase with
the preferred rotational setting, the translational position of the main conformation was shifted by 5 bp.
Additionally, a fraction of platinated nucleosomes with widely distributed translational positions was observed,
suggesting increased nucleosome sliding relative to platinated nucleosomes containing the 1,3-intrastrand
d(GpTpG) cross-link investigated previously (Ober, M.; Lippard, S. J. J. Am. Chem. Soc. 2007, 129, 6278-
6286).
Introduction
reaction of activated cisplatin with DNA is the 1,2-d(GpG) cis-
diammineplatinum(II) intrastrand cross-link, with 1,2-d(ApG)
and 1,3-d(GpNpG) intrastrand cross-links being formed to a
lesser extent.8
Platinum-based drugs such as cisplatin [cis-diamminedichlo-
roplatinum(II)] and related agents including carboplatin or
oxaliplatin are successfully used to treat various types of cancer,
including testicular, ovarian, head, neck, certain types of lung,
and colorectal cancer.1-4 The drugs enter the cell by diffusion
or with the aid of a transporter, become activated by forming
aqua complexes, and bind to nuclear DNA, which in eukaryotes
is packaged in highly condensed chromatin structures comprising
nucleosome building blocks. Cisplatin reacts well with both free
and nucleosomal DNA,5 showing a slight preference for the
linker region of the latter.6 Because 75-90% of genomic DNA
is wrapped in nucleosomes,7 intact nucleosomes are the more
likely targets in ViVo. The most common adduct formed by
By employing hydroxyl radical footprinting, we recently
discovered that the 1,3-d(GpTpG) platinum adduct in nucleo-
somal DNA induces a well-defined rotational setting of the DNA
with respect to the histone core.9 The structural effect of the
platinum cross-link is strong enough to override the rotational
preference as defined by a strong nucleosome positioning
sequence.10 Whereas the properties of nucleosomes containing
the d(GpTpG) platinum adduct and other covalent DNA lesions
including N-methylpurines11 and the TT cyclobutane pyrimidine
(7) Segal, E.; Fondufe-Mittendorf, Y.; Chen, L. Y.; Thåstro¨m, A.; Field, Y.;
Moore, I. K.; Wang, J. P. Z.; Widom, J. Nature 2006, 442, 772-778.
(8) Blommaert, F. A.; van Dijk-Knijnenburg, H. C. M.; Dijt, F. J.; den Engelse,
L.; Baan, R. A.; Berends, F.; Fichtinger-Schepman, A. M. J. Biochemistry
1995, 34, 8474-8480.
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A R T I C L E S
Ober and Lippard
dimer12-16 have been previously investigated, the structural
influence of the most abundant 1,2-d(GpG) cisplatin cross-link
within a nucleosome remained to be determined.
A key question is whether the 1,2-d(GpG) cisplatin cross-
link, like the 1,3-d(GpTpG) intrastrand adduct, would be able
to influence the position of the DNA relative to the histone core.
Recent studies suggest that such positioning of the nucleosomes
on the genome may control important regulatory properties
within the cell, both structural and functional.7,17-19 Determining
where cisplatin cross-links are located within the nucleosome
structure represents an important step in understanding their
cellular recognition and processing and may contribute to the
working mechanism of the drug.
In order to answer the question of how a site-specific cisplatin
intrastrand 1,2-(dGpG) cross-link would affect the properties
of the nucleosome, we constructed a substrate containing the
adduct in a DNA duplex wrapped around core histones obtained
from HeLa-S3 cancer cell chromatin. We studied these sample
nucleosomes by hydroxyl radical footprinting20 and exonuclease
III mapping21 to determine the rotational and translational setting
of the DNA in these reconstituted particles.
Figure 1. Product distribution after histone transfer reactions onto the
sample DNA duplexes. Lanes 1-4: free DNA. The platinated DNA samples
migrate more slowly than the corresponding unplatinated samples. Lanes
5-6: after completion of transfer reaction at 4 °C and heat equilibration at
45 °C, 2 h. The platinated samples show a side product that migrates more
rapidly than the main product. Lane 1, D1; lane 2, D1-Pt; lane 3, D2; lane
4, D2-Pt; lane 5, nD1; lane 6, nD1-Pt; lane 7, nD2; lane 8, nD2-Pt.
Results and Discussion
Design and Synthesis of DNA Duplexes. For our experi-
ments, we chose to study DNA that has a moderate affinity for
histone octamers and forms weakly positioned nucleosomes. We
modified a nucleosome sequence, used previously in our
laboratory,9 in which a single d(GpG) dinucleotide is positioned
in the central region to allow for site-specific platination.
In order to construct radioactively labeled duplex samples,
we employed a two-step protocol. In the first step, we ligated
six oligonucleotides of which one contained the site-specific
cis-{Pt(NH3)2}2+ 1,2-d(GpG) cross-link to generate a 171-/173-
mer DNA duplex, R1-Pt. The DNA sequence was designed
with orthogonal blunt-end restriction sites close to the 3′
(EcoRV) and 5′ (AfeI) termini of the duplex. The 32P label was
introduced in the second step. In order to mark exclusively the
platinated coding strand of the duplex, the 3′ end was first
truncated with EcoRV and dephosphorylated. Next, the coding
and template strand were 5′-end-labeled with γ-32P-ATP. A
second digestion with AfeI finally truncated the duplex at the
5′ end, thereby removing the label from the template strand.
The remaining, single-end-labeled duplex, D1-Ptc, had a length
of 146 bp (Scheme S1, Supporting Information) and contained
a single, site-specific cis-{Pt(NH3)2}2+ 1,2-d(GpG) intrastrand
cross-link within the coding strand. In order to label exclusively
the template strands of the duplex for separate experiments, the
sequence of the EcoRV and AfeI digestion steps was inter-
changed to afford D1-Ptt as the product.
Six additional labeled duplexes were constructed in the same
fashion. D2-Ptc and D2-Ptt were obtained from R2-Pt, which
has a sequence identical to that in D1-Ptc and D2-Ptt except
that the platinated d(GpG) dinucleotide was shifted by six base
pairs from position 71.72 to position 77.78. Duplexes D1c, D1t,
D2c, and D2t were unplatinated reference probes. The sequences
of the oligonucleotide components and final strands are provided
in Scheme S2 (Supporting Information).
Construction of Platinated Nucleosomes. We prepared
the coding or template strand labeled nucleosomes nD1c/t,
nD1-Ptc/t, nD2c/t, and nD2-Ptc/t from the respective DNA
duplexes D1c/t, D1-Ptc/t, D2c/t, and D2-Ptc/t by using a histone
transfer protocol,22 followed by heat equilibration. The donor
chromatin used in the histone transfer reactions was prepared
from HeLa-S3 cancer cells10 and had an average length of
450 bp.
Analysis of the reactions by non-denaturing polyacrylamide
gel electrophoresis showed a single band for nucleosomes
containing unplatinated DNA. The platinated nucleosomes
reproducibly contained, in addition to this main band, a faster
migrating side product with an intensity of approximately 20-
25% that of the main band (Figure 1). Although we have not
been able conclusively to confirm the identity of this side
product, there are several factors that may result in multiple
bands in reconstituted nucleosome samples. First, differences
in the translational setting of the DNA wrapped around the
nucleosome octamer core could contribute to measurable
differences in mobility during gel electrophoresis. Nucleosomes
with translational settings that are asymmetric exhibit higher
mobility than those with symmetrical settings.23 By comparing
data from exonuclease III footprinting (see below) of unplati-
nated nucleosome samples nD1c/t with those for the platinated
samples nD1-Ptc/t, however, we were unable to detect significant
(12) Kosmoski, J. V.; Ackerman, E. J.; Smerdon, M. J. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 10113-10118.
(13) Svedruzˇic´, Zˇ. M.; Wang, C.; Kosmoski, J. V.; Smerdon, M. J. J. Biol. Chem.
2005, 280, 40051-40057.
(14) Thoma, F. EMBO J. 1999, 18, 6585-6598.
(15) Schieferstein, U.; Thoma, F. EMBO J. 1998, 17, 306-316.
(16) Schieferstein, U.; Thoma, F. Biochemistry 1996, 35, 7705-7714.
(17) Ioshikhes, I.; Bolshoy, A.; Derenshteyn, K.; Borodovsky, M.; Trifonov, E.
N. J. Mol. Biol. 1996, 262, 129-139.
(18) Kornberg, R. D. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12955-12961.
(19) Lee, W.; Tillo, D.; Bray, N.; Morse, R. H.; Davis, R. W.; Hughes, T. R.;
Nislow, C. Nat. Genet. 2007, 39, 1235-1244.
(20) Balasubramanian, B.; Pogozelski, W. K.; Tullius, T. D. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 9738-9743.
(22) Utley, R. T.; Owen-Hughes, T. A.; Juan, L. J.; Coˆte´, J.; Adams, C. C.;
Workman, J. L. Methods Enzymol. 1996, 274, 276-291.
(23) Pennings, S. Methods Enzymol. 1999, 304, 298-312.
(21) Tullius, T. D.; Lippard, S. J. J. Am. Chem. Soc. 1981, 103, 4620-4622.
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Effect of a Cisplatin Cross-Link on Nucleosome Structure
A R T I C L E S
Figure 2. Autoradiographs of hydroxyl radical footprinting experiments. (a) The coding strands of the duplexes were 5′-labeled with 32P. The positions of
the platinum adduct are highlighted by red arrows. Lane 1, D1c; lane 2, D1-Ptc; lane 3, D2c; lane 4, D2-Ptc; lane 5, nD1c; lane 6, nD1-Ptc; lane 7, nD2c;
lane 8, nD2-Ptc (main product); lane 9, nD2-Ptc (side product); lanes M1, Maxam-Gilbert A/G reaction of D1c; lane M2, Maxam-Gilbert A/G reaction of
D1-Ptc; lane M3, Maxam-Gilbert A/G reaction of D2c; lane M4, Maxam-Gilbert A/G reaction of D2-Ptc. (b) The template strands of the duplexes were
5′-labeled. The positions of the d(CpC) dinucleotide opposite the platinum adduct are highlighted by red arrows. Lane 1, D1t; lane 2, D1-Ptt; lane 3, D2t;
lane 4, D2-Ptt; lane 5, nD1t; lane 6, nD1-Ptt; lane 7, nD2t; lane 8, nD2-Ptt (main product); lane 9, nD2-Ptt (side product); lanes M1, Maxam-Gilbert A/G
reaction of D1t; lane M2, Maxam-Gilbert A/G reaction of D1-Ptt; lane M3, Maxam-Gilbert A/G reaction of D2t; lane M4, Maxam-Gilbert A/G reaction
of D2-Ptt.
differences in the distribution of translational settings that would
support this possibility.
to the histone core.26,27 After reaction with hydroxyl radicals,
the nucleosome samples were subsequently purified by native
polyacrylamide gel electrophoresis. The resolution of this
purification step was sufficiently high to separate the faster
migrating side band from the main product of the platinated
nucleosome samples, nD2-Ptc/t. Consequently, we were able
to examine the structures of the DNA from particles isolated
from two bands individually (Figure S1, Supporting Informa-
tion). For nucleosome samples nD1-Ptc/t there was insufficient
material, and we were only able to isolate and study of material
from the main band. Apart from analyzing the nucleosome
samples, we examined as a point of reference the labeled coding
and template strands of the free DNA duplexes D1c/t, D1-Ptc/t,
D2c/t, and D2-Ptc/t. Autoradiographs from these experiments are
presented in Figure 2, and enlargements of the areas of interest
are provided in Figure S2 (Supporting Information).
Second, it is possible that the lower band contains particles
with incomplete histone cores. The interactions of H2a/H2b
histone heterodimers with DNA are weaker than the interaction
of the H32H42 tetramer. Association of the H2a/H2b het-
erodimers during salt-gradient dialysis, which was used to
reconstitute the nucleosomes, occurs at a lower ionic strength
than association of the H32H42 tetramer with DNA.24 Dissocia-
tion of H2a/H2b heterodimers from the core tetramer can also
be induced by various cellular mechanisms. Incomplete particles
lacking one or two H2a/H2b histone heterodimers, hexasomes
or tetrasomes, also migrate more rapidly during electrophore-
sis.25 It is possible that the structural distortions induced by the
cisplatin 1,2-intrastrand cross-link obstruct the formation of
complete histone octamers. Further experiments are needed to
verify this hypothesis, however.
We aligned each band with the corresponding DNA sequence
with the aid of A/G reference lanes from a Maxam-Gilbert
gel analysis. Aligned densitometric traces of every lane together
with sequence assignments are presented in Figures S3 and S4
(Supporting Information). The positions of the cisplatin adduct
Hydroxyl Radical Footprinting of Platinated Nucleosomes.
We used hydroxyl radical footprinting of the substrates described
above to reveal the rotational setting of both the coding and
the template strand of all nucleosomal DNA samples relative
(26) Dixon, W. J.; Hayes, J. J.; Levin, J. R.; Weidner, M. F.; Dombroski, B.
A.; Tullius, T. D. Methods Enzymol. 1991, 208, 380-413.
(27) Vitolo, J. M.; Thiriet, C.; Hayes, J. J. Curr. Protoc. Mol. Biol. 1999,
21.4.21-21.4.29.
(24) Widom, J. Q. ReV. Biophys. 2001, 34, 269-324.
(25) Kireeva, M. L.; Walter, W.; Tchernajenko, V.; Bondarenko, V.; Kashlev,
M.; Studitsky, V. M. Mol. Cell 2002, 9, 541-552.
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were revealed by diminished double bands in the Maxam-
Gilbert density traces upon comparison of the traces of D1c with
D1-Ptc and D2c with D2-Ptc.
Lanes 1-4 in Figure 2a,b represent the footprinting patterns
of the platinated DNA duplexes in the absence of a histone core.
As expected, smooth fragmentation patterns were produced
because the backbones of these strands were isotropically
exposed to hydroxyl radicals. Lanes 5-8 show the fragmentation
patterns of the reconstituted nucleosomes that were isolated as
the main product (major band, see above). Lane 9 in both
autoradiographs portrays the hydroxyl radical footprint of the
side products of nD2-Ptc and nD2-Ptt that were isolated from
the lower band (see above).
The pattern in lanes 5-9 exhibits a periodic modulation of
intensity that reflects the helical structure of the DNA packed
against the histone octamer core. Hydroxyl radicals react
preferentially with the C5′- and, to a lesser extent, the C4′-
hydrogen atoms of the sugar-phosphate backbone.20 The
hydroxyl radical footprinting pattern correlates fairly well with
the solvent accessibility of these backbone atoms. Preferential
cleavage and darker bands are observed in areas where the
backbone faces away from the histone core. Reduced cleavage
and lighter bands are produced in areas where the backbone is
packed against the histone octamer, which reduces the acces-
sibility to hydroxyl radical cleavage.
By plotting the deconvoluted intensity of each band against
its position in the sequence, we obtained hydroxyl radical
cleavage curves revealing the solvent accessibility of the
backbone at each nucleotide. Detailed structural information
about the site-specifically platinated nucleosomal DNA was
derived by overlaying the cleavage curves of both strands in a
duplex. We were able to identify the disposition of the major
and minor grooves of the nucleosomal DNA relative to the
histone core in every sample. This disposition is described by
the angle ê, defined previously.10 It approaches 0° in areas where
the major groove faces toward the histone core and 180° where
it points directly toward the solvent, away from the surface of
the core octamer.
Influence of the Cisplatin Cross-Link on the Rotational
Setting. We obtained sinusoidal hydroxyl radical cleavage
curves for all samples. The position of the platinum lesion
strongly influences the rotational setting of the nucleosomal
DNA and overrides the slight rotational preference induced by
the DNA sequence.
In order to investigate the extent to which the existence and
position of the cisplatin adduct, rather than the position of the
d(GpG) dinucleotide itself, are responsible for this effect, we
superimposed the hydroxyl radical cleavage curves of the
reference nucleosomes nD1 and nD2 (Figure 3a). Shifting the
unplatinated d(GpG) dinucleotide by six base pairs within our
DNA sequence has almost no effect on the rotational setting of
the nucleosomal DNA. The cleavage curves exhibit a phase
difference of <1 bp (30 ( 6°) in the vicinity of the d(GpG)
dinucleotide, between positions 71.72 and 77.78. The phase
difference becomes negligible in the adjacent helix turns, and
the curves superimpose almost perfectly at sites further remote
from the dinucleotide.
Figure 3. Superposition of the cleavage curves of (a) unplatinated and (b)
platinated nucleosomes. (a) The DNA sequences of the unplatinated
nucleosomes nD1c/t (blue) and nD2c/t (green) are identical except for the
fact that a d(GpG) dinucleotide within the bottom strand was shifted by 6
bp from position 71.72 to position 77.78. The corresponding cleavage curves
are nearly congruent. (b) The platinated nucleosomes nD1-Ptc/t (red) and
nD2-Ptc/t (orange/brown) have identical sequences except for the fact that
the 1,2-d(GpG) cisplatin adduct, symbolized by a black rectangle, was
shifted by 6 bp. The corresponding cleavage curves are out of phase by
2-4 bp. The phasing difference is approximately 125° near the cisplatin
adduct.
base pairs to the 3′-direction within our nucleosomal DNA
sequence has a profound effect on its rotational setting. We
observed a phase difference in the cleavage curves of ap-
proximately 3-4 bp (125 ( 20°) in immediate proximity of
the lesion that gradually decreases to a phase difference of less
than a quarter turn (71 ( 6°) at regions further remote from
the adduct.
This finding represents the first example in which it has been
possible to demonstrate that a structural difference between
platinated nucleosome particles, nD1-Pt and nD2-Pt in the
present case, is exclusively caused by the position of the cisplatin
adduct within the sequence. It is not a consequence of the slight
sequence difference between nD1-Pt and nD2-Pt that was
necessary in order to accommodate the lesion at different
locations along the duplex.
In order to develop a structural model to account for the
rotational setting of DNA in our nucleosomes, we superimposed
the cleavage curves of the coding and template strands for all
nucleosome samples. The cis-{Pt(NH3)2}2+ 1,2-d(GpG) adduct,
which is located in the major groove, rotates the duplex in both
samples in such a manner that it faces more toward than away
from the histone core proteins.
Within the unplatinated nucleosome nD1, the major groove
faces approximately toward the histone core with ê ≈ -50° at
the position of the d(GpG) cisplatin target dinucleotide (Figure
4a). After platination to form nD1-Pt, the structure changes
slightly such that the cisplatin adduct points directly toward the
histone core (ê ≈ -20°, Figure 4b). A structural model of this
conformational change is presented in Figure S5a (Supporting
Information).
Next, we superimposed the cleavage curves of the platinated
nucleosomes nD1-Pt and nD2-Pt (Figure 3b). Shifting the
position of the cis-{Pt(NH3)2}2+ 1,2-d(GpG) cross-link by six
The major groove of the d(GpG) dinucleotide in nD2 faces
approximately toward the solvent with ê ≈ 120°. Introduction
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Effect of a Cisplatin Cross-Link on Nucleosome Structure
A R T I C L E S
Figure 4. The cleavage curves reveal the rotational setting of the nucleosomal DNA duplexes nD1c/t, nD1-Ptc/t, nD2c/t, and nD2-Ptc/t in the nucleosome.
Gray bars indicate areas in which the minor groove is exposed to the solvent and the major groove faces inward toward the nucleosome. The position of the
unplatinated and platinated d(GpG) dinucleotide is indicated. Exonuclease III stop sites of each sample are presented underneath the respective hydroxyl
radical cleavage curve. (a) The major groove of the unplatinated d(GpG) trinucleotide points approximately toward the histone core. (b) After platination,
the major groove and the platinum adduct face completely toward the histone core. (c) The major groove of the d(GpG) dinucleotide points sideways toward
the solvent with ê ≈ 120°. (d) After platination, the major groove faces approximately toward the histone core with ê ≈ 40°. The cis-{Pt(NH3)2}2+ moiety,
which binds to guanine in the major groove of DNA, is in both cases buried inside the nucleosome, inaccessible to the solvent.
of the cisplatin cross-link in nD2-Pt changes the orientation of
the major groove in such a manner that it faces approximately
toward the histone core (ê ≈ 40°). A model is again presented,
in Figure S5b (Supporting Information). The structural flexibility
of the cis-{Pt(NH3)2}2+ 1,2-d(GpG) cross-link with respect to
possible rotational settings seems to be greater than that of the
cis-{Pt(NH3)2}2+ 1,3-d(GpTpG) cross-link described previ-
ously.10 The deviation of the rotational settings of nD1-Pt and
nD2-Pt is approximately 60°, measured from the midpoint of
the cross-link. By comparison, for the cis-{Pt(NH3)2}2+ 1,3-
d(GpTpG), we typically observed values very close to ê ≈ 0°
with a deviation of <15°, even in the presence of a strong
nucleosome positioning sequence.9,10
difference of 120-180° (3-6 bp) compared to that of the
unplatinated samples nD2c/t (Figure S7a, Supporting Informa-
tion). The cisplatin adduct assumes a position of ê ≈ 15° with
the major groove, again pointing away from the solvent. (Figure
S7b-d, Supporting Information).
As we show below, the DNA in samples nD1c/t, nD2c/t, and
nD1-Ptc/t adopts a preferred single translational position with
respect to the histone core. Because of the weak nucleosome
positioning capability of the underlying DNA sequence, we
observe additional particle conformations with other translational
settings. It is not possible to separate these constructs from those
having the major conformation. We wish to stress that these
differently positioned nucleosomes will influence the intensity
modulation pattern of our footprinting data only to a small
extent. The most affected are the nD2-Ptc/t samples, which
contain an increased fraction of particles with less defined
translational positions.
The footprinting patterns of the coding strands nD1-Ptc and
nD2-Ptc display significantly darker bands in direct proximity
to the 1,2 cisplatin adduct compared to those of the unplatinated
coding strands nD1c and nD2c at the same positions (Figures
S2a and S6, Supporting Information). This preferred cleavage
of the coding strand is probably a consequence of the local
structural perturbation induced by the cisplatin adduct, resulting
in increased solvent accessibility. A chemical effect, such as
cisplatin-induced DNA nicking, can be ruled out, because no
such increased cleavage is observed in the proximity of the
adduct for the free DNA strands D1-Ptc and D2-Ptc. Densito-
metric scans of the respective free DNA strands are presented
in Figure S3, rows 1-4 (Supporting Information).
We also investigated the structure of the DNA in the faster
migrating side product that was isolated during the construction
of nucleosome samples nD2-Ptc/t. This nucleosomal DNA also
displays a sinusoidal cleavage pattern that persists throughout
the entire DNA sequence. The side product contains DNA
that is therefore most likely wrapped around histone core
proteins s the footprinting patterns of the product isolated from
the main and side bands are quite similar. The intensity
modulation of the footprinting pattern has a global phase
Exonuclease III Footprinting of Platinated Nucleosomes.
We studied the translational positions of the nucleosomal DNA
by partial digestion of the nucleosome with exonuclease III.28
This enzyme digests double-stranded DNA in a stepwise manner
from the 3′ termini. DNA shielded by the histone core is digested
more slowly, resulting in darker bands. This method reveals
the approximate translational boundaries of the DNA that
interacts with the histone core. As a reference, the coding or
template strand-labeled free DNA duplexes D1c/t, D1-Ptc/t, D2c/t,
and D2-Ptc/t were similarly analyzed. Autoradiographs of these
experiments are presented in Figure 5. Again, it was possible
to assign each individual band to the corresponding nucleotides
in the DNA sequence with the help of Maxam-Gilbert A/G
reference lanes.
Lanes 1-4 in Figure 5a,b show the digestion patterns of the
unbound DNA duplexes. The cisplatin cross-link significantly
(28) Prunell, A. Biochemistry 1983, 22, 4887-4894.
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Figure 5. Autoradiographs of exonuclease III mapping experiments. (a) The coding strands of the duplexes were 5′-labeled with 32P. The positions of the
platinum adduct are highlighted by red arrows. Lane 1, D1c; lane 2, D1-Ptc; lane 3, D2c; lane 4, D2-Ptc; lane 5, nD1c; lane 6, nD1-Ptc; lane 7, nD2c; lane
8, nD2-Ptc; lane M1, Maxam-Gilbert A/G reaction of D1c; lane M2, Maxam-Gilbert A/G reaction of D1-Ptc; lane M3, Maxam-Gilbert A/G reaction of
D2c. (b) The template strands of the duplexes were 5′-labeled. The positions of the d(CpC) dinucleotide opposite the platinum adduct are highlighted by red
arrows. Lane 1, D1t; lane 2, D1-Ptt; lane 3, D2t; lane 4, D2-Ptt; lane 5, nD1t; lane 6, nD1-Ptt; lane 7, nD2t; lane 8, nD2-Ptt; lane M1, Maxam-Gilbert A/G
reaction of D1t; lane M2, Maxam-Gilbert A/G reaction of D1-Ptt; lane M3, Maxam-Gilbert A/G reaction of D2t.
decelerates digestion of the coding strand by exonuclease III,
as revealed by a dark triple band toward the 3′-direction of the
adduct (lanes 2 and 4, Figure 5a). This behavior is consistent
with previous work from our laboratory.21 Cisplatin does not
interfere with digestion of the template strand opposite the
adduct (lanes 2 and 4, Figure 5b). Unlike the results for the
coding strand, the template strands exhibits several sequence-
dependent stop sites for exonuclease III, appearing as dark bands
in lanes 1-4, Figure 5b.
Lanes 5-8 display the digestion pattern of the reconstituted
nucleosomes. The DNA-histone interaction causes the exonu-
clease III to pause, an effect that results in dark bands toward
the top of the gel indicating the translational position of the
histone core relative to the DNA duplex. These bands occur at
locations different from any of the sequence-dependent stop
sites. The cisplatin cross-link-induced exonuclease III pause sites
are significantly diminished in the reconstituted nucleosomes
because only a small fraction of the enzyme progresses that far
along the DNA.
Influence of the Cisplatin Cross-Link on the Translational
Setting. We used the data from the exonuclease III mapping
experiments to reconstruct the translational position of the
nucleosomal DNA. All nucleosome samples were positioned
asymmetrically with respect to the histone core.
Exonuclear digestion of unplatinated samples nD1c and nD2c
with a labeled coding strand generated a main band at position
133 (Figure 6a), indicating that DNA in the majority of the
nucleosome particles is bound to the histone core, as designated
by the blue and green ovals in Figure 6c. Digestion of the
template strand is largely blocked by the histone core, as
revealed when the experiment was repeated for samples nD1t
and nD2t. This asymmetric translational setting protects the 3′-
ends of the template strands. In addition to the main stop sites,
additional bands were generated at positions 146, 128, and 125
in the coding strands as well as 5 and 15 in the template strand
of both unplatinated nucleosome samples. The translational
positions corresponding to these additional stop sites are
demarcated by dotted ovals in Figure 6c. The minor difference
in sequence for the nucleosome samples nD1c/t and nD2c/t has
no influence on the respective distribution of their translational
settings. The platinated samples nD1-Ptc/t display a digestion
pattern similar to those of the unplatinated nucleosomes. The
major stop site in the coding strand again corresponds to position
133, and in the majority of the particles the template strand is
protected from digestion. Additional stop sites occur at positions
11 and 20, whereas the bands at sites 5 and 128 are significantly
reduced in intensity (Figure 6b, red trace). Overall, the effect
of the cisplatin adduct on the translational setting of the DNA
in nD1-Pt is minor. The major fraction of the nucleosome core
particles has the same position as in the unplatinated nucleo-
somes (Figure 6c). The distances between the stop sites in the
samples nD1-Ptc/t are roughly multiples of 10. The unplatinated
nucleosomes show a less regular pattern in comparison. The
cisplatin adduct thus appears to hold the nucleosomal DNA
tightly, in a well-defined rotational position, thereby permitting
only translational settings that differ from one another by integral
numbers of complete DNA helical turns.
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Effect of a Cisplatin Cross-Link on Nucleosome Structure
A R T I C L E S
abrogate to some degree a sequence-inherent preference for
specific translational settings, resulting in the increased structural
variability. Second, the cisplatin adduct may reduce the strength
of the DNA-histone interaction and facilitate “nucleosome
sliding”,29 increasing the possibility that the histone core may
be translocated by the action of exonuclease III. Third, reduced
interaction of the oligonucleotide duplex ends with the histone
core may alter the association/dissociation dynamics of DNA
and the histone core.30 This behavior could increase the
probability that exonuclease III would digest DNA into the
particle, rather than dissociating at the border of the nucleo-
some.28 Experiments beyond the scope of the present study, such
as site-directed hydroxyl radical footprinting,31 might provide
further information about these observations.
In summary, depending on its position within the nucleosome,
a cisplatin adduct can have the following effects on the
distribution of translational settings. When located in phase, that
is, at a site that does not require any adjustment of the rotational
setting of the DNA, the cis-{Pt(NH3)2}2+ 1,2-intrastrand d(GpG)
cross-link locks the DNA tightly into that rotational position.
As a consequence, there is only a narrow cluster of possible
translational settings that are separated from each other by
multiples of 10 bp. Alternatively, if the cross-link is positioned
out of phase with the natural rotational setting of the DNA, we
observe a shift in the translational setting by half a helical turn
and a strongly increased variability of possible translational
settings.
Structural Differences between 1,2- and 1,3-Intrastrand
Cisplatin Cross-Links and Their Implications. We previously
investigated in detail the effect on the nucleosome structure of
cis-{Pt(NH3)2}2+ 1,3-d(GpTpG) intrastrand cross-links and
learned that it defines the rotational setting and strongly affects
the translational setting of DNA in a nucleosome particle. The
present results clearly demonstrate that the more abundant cis-
{Pt(NH3)2}2+ 1,2-d(GpG) intrastrand cross-link also influences
the DNA conformation within a nucleosome, but in a manner
that depends on its position within the DNA sequence.
Both the 1,2- and 1,3-intrastrand cross-links enforce a DNA
rotational setting such that the lesion, positioned in the major
groove, faces toward the histone core. The structural flexibility
of nucleosomes having such a site-specific 1,3-intrastrand cross-
link is less than that observed for the 1,2-intrastrand cross-link,
however. Nucleosomes with a 1,3-adduct have rotational settings
very close to ê ≈ 0°, irrespective of where it is located within
the sequence. In these nucleosomes, the deviation in rotational
orientations of the cisplatin lesion is <15°, even when a strong,
counteracting positioning sequence was employed. In the present
study, the rotational settings of nD1-Pt and nD2-Pt were
determined to differ by approximately 60° at the respective
platinum adduct locations.
Figure 6. Exonuclease III stop sites and derived translational setting of
the nucleosomal DNA. Superposition of the stop sites of (a) unplatinated
and (b) platinated nucleosomes. (a) The slight sequence difference of the
nucleosome samples nD1c/t (blue) and nD2c/t (green) has no effect on the
exonuclease III digestion pattern. (b) The platinated nucleosomes nD1-
Ptc/t (red) and nD2-Ptc/t (orange/brown) have significantly different stop
positions. (c) Reconstructed translational setting of DNA in the nucleosomes
nD1 (blue, top), nD1-Pt (red), nD2 (green), and nD2-Pt (brown, bottom).
The DNA is represented as graduated horizontal bars. The major conforma-
tions in each sample are indicated by colored ovals, and minor conformations
by noncolored ovals. The positions of the cisplatin adducts are indicated
by red rectangles. In addition, the approximate pseudodyad position of the
major conformation in each sample is indicated. The translational setting
of the main conformation in nD2-Pt is shifted by 5 bp from that in all
other nucleosome samples. An increased fraction of particles with widely
distributed translational positions is evident.
The data obtained for samples nD2-Ptc/t are different. The
main stop site in the coding strand was shifted by 5 bp to
position 128, and a second strong band appeared at position
118, one complete helical turn away. Correspondingly, in a
significant fraction of the particles, the template strand is
protected against digestion (Figure 6b, orange/brown trace). The
related translational conformations are shown as solid-bordered
ovals in Figure 6c. Additional minor bands appeared at positions
146, 133, and 108 in the coding strands and at 5, 15, 23-26,
and 36-37 in the template strands. Overall, there is a greater
distribution of translational settings than observed in the other
nucleosome samples.
Two synergistic effects were responsible for the overall
structural effect of the 1,3-d(GpTpG) intrastrand cross-link. The
position of the unplatinated d(GpTpG) trinucleotide in the
oligonucletide sequence itself had an influence on the rotational
setting of the DNA. The 1,3-d(GpTpG) cisplatin cross-link
significantly enhanced this effect. In the present case, however,
(29) Becker, P. B. EMBO J. 2002, 21, 4749-4753.
(30) Li, G.; Levitus, M.; Bustamante, C.; Widom, J. Nat. Struct. Mol. Biol. 2005,
12, 46-53.
(31) Flaus, A.; Luger, K.; Tan, S.; Richmond, T. J. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 1370-1375.
The following factors may contribute to this effect. First, the
position of the cisplatin adduct in nD2-Pt at position 77.78 may
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A R T I C L E S
Ober and Lippard
the 1,2-d(GpG) cisplatin adduct is exclusively responsible for
the observed structural changes. A positional shift of the
unplatinated dinucleotide has virtually no effect on the nucleo-
some structure.
The 1,2-intrastrand d(GpG) lesion unwinds the nucleosomal
DNA only very slightly in the proximity of the lesion. This
small effect is compensated in the adjacent helical turns and
does not induce global unwinding of nucleosomal DNA, as we
observed for the 1,3-intrastrand d(GpTpG) cisplatin adduct.10
This finding parallels that in previous studies with free DNA,
in which we determined an unwinding angle of just 13° for the
1,2-intrastrand d(GpG) cisplatin cross-link compared to a value
of approximately 23° for a 1,3-intrastrand lesion.32
binding by the various proteins of the repair complex as well
as their specific functions, are not completely clear at present.
According to a current model, the first step involves recognition
of a structural distortion at the undamaged strand, probably by
XPC. This initial binding apparently aids in location of the actual
site of damage.34 Damage recognition induces then chromatin
remodeling, dissociation of the DNA from the histone core, and
excision of the damaged DNA.35
In light of our current results, there are at least three possible
factors that might account for the significant disparity in the
ability of a nucleosome to inhibit the repair of a 1,2-d(GpG)
cisplatin cross-link compared to the 1,3-d(GpTpG) cisplatin
adduct. First, the greater structural flexibility observed for the
nucleosomal 1,2 cisplatin adduct could improve access to the
first response protein of the NER pathway, presumably XPC.
Second, the local structural perturbation of the nucleosomal
DNA induced only by the 1,2 cisplatin adduct could enable
preferred recognition by the NER machinery. Finally, a possible
reduction in the stability of the histone octamer core triggered
by the 1,2 lesion may further assist nucleosome remodeling
needed for NER.
According to recent studies, the majority of the genomic
nucleosomes are well-positioned,7,19,36 only a subset of which
are directly determined by specific nucleosome positioning
sequences.37 Such sequences are encountered in promoter
regions and at translation start sites.7,17,38 These strongly
positioned nucleosomes indirectly determine the translational
location of other nucleosomes by effects like steric hin-
drance.7,17,38 Positioned nucleosomes perform many important
regulatory functions, such as attenuating or increasing gene
expression,17 and they fulfill structural tasks, such as protecting
the centromere.7 The strong positioning effects of the major
cisplatin 1,2-d(GpG) and 1,3-d(GpTpG) lesions that we observe
at the single nucleosome level may influence regulatory
mechanisms in the cell that are linked to chromatin structure
and may even disrupt the local geometry in proximity to the
lesion.
The ability of DNA to position itself on the histones is
achieved by curvature of the double helix that predefines the
binding face that interacts with the octamer core. The intrinsic
DNA sequence can promote DNA bending and preferred
rotational and translational settings of the nucleosome by subtle
additive effects of dinucleotide steps at specific positions.7,39
The 1,2-d(GpG) cisplatin adduct induces, in a manner similar
to that previously encountered for the 1,3-d(GpTpG) adduct,
a kink toward the major groove with an angle of approxi-
mately 30°.32 This structural motif has a dramatic effect on the
overall curvature of the DNA and explains why these platinum
adducts have such a strong influence on the nucleosome
structure.
The accessibility of nucleosomes to hydroxyl radical cleavage
on the coding strand near the 1,2-intrastrand d(GpG) cisplatin
cross-link is significantly enhanced by comparison to results
for the unplatinated reference nucleosomes at the same position.
This increased accessibility most likely reflects a local structural
perturbation of the nucleosomal DNA induced by the cisplatin
adduct. Possibly, the 1,2-intrastrand cisplatin cross-link cannot
be as well accommodated within the nucleosome structure as
the 1,3-intrastrand cross-link. Moreover, the structural distortion
induced by the 1,2 cisplatin lesion appears to obstruct the
assembly of complete histone octamers, resulting in formation
of a side product with a greater gel electrophoretic mobility
than that of the main product. We did not observe such an effect
either with unplatinated DNA or with DNA carrying a site-
specific 1,3-intrastrand cisplatin cross-link.
The 1,2-d(GpG) cisplatin intrastrand cross-link has a less
profound effect on the translational setting at position 71.72
than the 1,3-d(GpTpG) adduct at position 71.73 in an otherwise
identical context.9 Whereas the former alters the distribution of
translational positions only slightly, the latter induces formation
of two main nucleosome structures with translational settings
that differ from those of the respective unplatinated nucleo-
somes. On the other hand, the 1,2-d(GpG) intrastrand cisplatin
cross-link at position 77.78 and the 1,3-d(GpTpG) adduct at
position 77.79 both strongly affect the translational settings and
position the DNA in a highly asymmetric fashion relative to
the histone octamer core. The 1,2-cross-link at site 77.78
generates two main nucleosome settings with DNA overhangs,
starting from either position, of 118 or 128 bp, in addition to a
variety of minor conformations, suggesting increased “nucleo-
some sliding” and reduced histone-DNA interactions. The 1,3-
adduct, by comparison, locks the DNA mainly into a single
asymmetric translational setting.
The 1,2-d(GpG) and 1,3-d(GpNpG) cisplatin intrastrand
cross-links are recognized and repaired via the nucleotide
excision repair (NER) pathway. The nucleosome inhibits NER
of both DNA lesions significantly by comparison to free DNA
bearing the same platinated sequence. Whereas the nucleosome
reduces the repair rate of a 1,3-d(GpTpG) cisplatin adduct by
about a factor of 10, a nucleosome sample containing a 1,2-
d(GpG) cross-link is repaired only approximately 3 times less
efficiently than the corresponding free DNA.33
Summary and Outlook. In this work, we investigated the
effect of a 1,2-d(GpG) cisplatin intrastrand cross-link on the
rotational and translational position of nucleosomal DNA by
(34) Buterin, T.; Meyer, C.; Giese, B.; Naegeli, H. Chem. Biol. 2005, 12, 913-
922.
The details by which the NER machinery restores nucleo-
somal DNA that has been damaged, including the sequence of
(35) Thoma, F. EMBO J. 1999, 18, 6585-6598.
(36) Yuan, G. C.; Liu, Y. J.; Dion, M. F.; Slack, M. D.; Wu, L. F.; Altschuler,
S. J.; Rando, O. J. Science 2005, 309, 626-630.
(37) Peckham, H. E.; Thurman, R. E.; Fu, Y.; Stamatoyannopoulos, J. A.; Noble,
W. S.; Struhl, K.; Weng, Z. Genome Res. 2007, 17, 1170-1177.
(38) Ercan, S.; Lieb, J. D. Nat. Genet. 2006, 38, 1104-1105.
(39) Satchwell, S. C.; Drew, H. R.; Travers, A. A. J. Mol. Biol. 1986, 191,
659-675.
(32) Bellon, S. F.; Coleman, J. H.; Lippard, S. J. Biochemistry 1991, 30, 8026-
8035.
(33) Wang, D.; Hara, R.; Singh, G.; Sancar, A.; Lippard, S. J. Biochemistry
2003, 42, 6747-6753.
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Effect of a Cisplatin Cross-Link on Nucleosome Structure
A R T I C L E S
proteins, such as chromatin isolation, purification, and histone transfer,
were carried out at 4 °C or on ice if not otherwise noted. Dialysis
membranes were obtained from Spectra/Por and treated with hot 50
mM EDTA solution, followed by treatment with 10 mM mercapto-
ethanol and several washes with high-purity water with a conductivity
of >12 MΩ before use to remove trace divalent cations. Denaturing
DNA polyacrylamide gel electrophoresis (PAGE) was performed on a
Life Technologies S2 sequencing gel electrophoresis apparatus. For
native DNA and nucleosome gel electrophoresis, a Protean II xi cell
from BioRad was used. Fluorescent agarose electrophoresis gels were
documented on a BioRad Fluor-S MultiImager. Electrophoresis gels
with radioactive samples were dried and documented on a Storm 840
Phosphorimager system from Amersham or exposed wet to a Kodak
Biomax MS film. Radioactive samples were quantified on a Beckman
LS 6500 scintillation counter. Fast protein liquid chromatography was
performed with a system from Amersham (P1 peristaltic pump and a
programmable Frac-100 fraction collector).
hydroxyl radical footprinting and exonuclease III mapping. In
comparison to structural studies of nucleosomes containing the
related 1,3-d(GpTpG) cisplatin cross-link, we identified some
similarities but also major differences. Most interesting is the
hyper-reactivity of the DNA toward hydroxyl radicals near the
d(GpG) cross-link which, together with greater structural
flexibility, indicates a distortion of the DNA that was not evident
for the 1,3-d(GpTpG) cross-link. The two different cisplatin
adducts also influence the translational setting in a different
fashion.
Our nucleosome studies to date have been focused on cis-
{Pt(NH3)2}2+ cross-links that are induced by the drugs cisplatin
or carboplatin. The more recently approved platinum-based
anticancer drugs oxaliplatin and tetraplatin, as well as com-
pounds currently under development, react with DNA to form
adducts that carry pendant ligands different from ammonia. The
effect of these so-called “carrier ligands”, which are present in
cis-{Pt(R,R-DACH)}2+ cross-links, where R,R-DACH is the
R,R-stereoisomer of 1,2-diaminocyclohexane present in oxali-
platin, or in DNA adducts of monofunctional complexes such
as {Ptpy(NH3)2)}2+, on the nucleosome structure is unknown
and will be the subject of future research. Additional studies
will also endeavor to elucidate the biological consequences of
nucleosomal platination, including damage recognition, repair,
and interference with transcription.
Synthesis of Platinated Oligonucleotides. First, eight oligonucle-
otides, a, b1, b2, c, d, e1, e2, and f, the sequences of which are provided
in Scheme S2 (Supporting Information), were chemically synthesized
and purified by preparative PAGE in the presence of 7.5 M urea. The
oligonucleotides were ethanol precipitated and desalted with SepPak
C18 cartridges. The purified strands were characterized by MALDI-
TOF mass spectrometry and quantified by UV/vis spectroscopy. The
two 21-mer oligonucleotides b1 and b2 were converted into b1-Pt
and b2-Pt by introduction of specific cis-{Pt(NH3)2}2+ 1,2-d(GpG)
intrastrand cross-links according to published procedures.43 The
platinated strands were purified by semipreparative HPLC, and the
quality of the fractions was monitored by analytical HPLC. The pure
fractions were pooled and concentrated by ethanol precipitation. The
rb values of the 21-mers b1-Pt and b2-Pt were determined to be 0.98/
21 and 1.05/21, respectively, corresponding to one cisplatin adduct per
oligonucleotide.
Experimental Section
Materials and Methods. Chemical reagents and solvents were
purchased from commercial sources. Cisplatin was synthesized from
K2[PtCl4] according to a published procedure.40 γ-32P-ATP having a
specific activity of at least 6000 Ci/mmol was obtained from Perkin-
Elmer (Boston, MA) and used on the day of arrival. DNA synthesis
was conducted with an Applied Biosystems 392 DNA/RNA synthesizer
on a 1 µmol scale by using standard phosphoramidite chemistry. The
oligonucleotides were deprotected overnight with a saturated ammonium
hydroxide solution at 60 °C and dried with an Eppendorf Vacufuge.
Chromatographic analysis and preparative HPLC purification of oli-
gonucleotides were performed on a Varian Star HPLC system with a
Hydopore-5-AX 10 × 100 mm SAX column. Buffer A contained 5 M
urea and 20 mM aqueous sodium phosphate, pH 6.5, and buffer B
contained 5 M urea and 1 M (NH4)2SO4 in 20 mM aqueous sodium
phosphate buffer. The rb values, moles of Pt bound per nucleotide
strand, were determined by quantification of platinum using flameless
atomic absorption spectrophotometry (Perkin-Elmer AAnalyst 300
system) and of DNA by UV/vis spectroscopy (Varian Cary 1E
spectrometer). The specific extinction coefficient of each oligonucleotide
was estimated as the sum of the individual extinction coefficients of
the nucleotides in the sequence.41,42 MALDI measurements were
conducted on a Bruker Omniflex mass spectrometer at the Department
of Chemistry Instrumentation Facility (DCIF), Massachusetts Institute
of Technology (MIT), or on an Applied Biosystems Model Voyager
DE-STR mass spectrometer at the MIT CCR Biopolymers Laboratory.
Enzymes were obtained from New England Biolabs (Ipswich, MA)
unless otherwise specified. Enzymatic reactions were carried out in
reaction buffers provided by the supplier unless otherwise noted.
Pelleted HeLa-S3 cells were obtained from the National Cell Culture
Center (Minneapolis, MN). Beckmann-Coulter Avanti J-25 and Optima
L centrifuges were used for chromatin preparation. For chromatin
sonication, a Branson digital sonifier was utilized. All steps involving
Preparation of DNA Duplexes. The oligonucleotides b1, b1-Pt,
b2, b2-Pt, c, d, e1, and e2 were 5′-phosphorylated with T4 polynucle-
otide kinase under standard conditions. A 500 pmol quantity of each
appropriate phosphorylated strand, 1000 pmol of either 21-mer b1, b1-
Pt, b2, or b2-Pt, and oligonucleotides a (500 pmol) and e (1000 pmol)
were annealed in buffer (100 mM NaCl, 70 mM Tris/HCl, pH 7.5, 10
mM MgCl2) by applying a temperature gradient from 90 °C to 4 °C
over 3 h and ligated in situ (50 mM NaCl, 60 mM Tris/HCl, 10 mM
MgCl2, 10 mM DTT, 1.5 mM ATP, 25 µg/mL BSA, 10 U/µL T4 DNA
ligase, 48 h, 16 °C) to give the 171-/173-mer duplex DNA strands R1,
R1-Pt, R2, and R2-Pt as detailed in Scheme S1 (Supporting Informa-
tion). The sample strands were then phenol extracted, ether extracted,
ethanol precipitated, and purified on a 6% denaturing polyacrylamide
gel. The gel pieces were extracted with 50 mM NaCl, 10 mM Tris/
HCl, and 1 mM EDTA, ethanol precipitated, and further purified by
gel filtration chromatography (Probequant G-50 cartridges, GE Health-
care). The eluted DNA was brought to 150 mM NaCl, annealed by
applying a temperature gradient from 90 °C to 4 °C over 3 h, and
concentrated with Nanosep MWCO 10kD cartridges (Pall Corp., East
Hills, NY).
In order to label exclusively the coding strands of the duplexes, 100
pmol samples of all duplexes were overdigested with EcoRV (20 U)
overnight in separate reactions. The strands were dephosphorylated with
Antarctic phosphatase (10 U, 1 h), and the enzymes were removed by
phenol extraction and ethanol precipitation. Next, 20 pmol of the
truncated strands was phosphorylated with 250 µCi γ-32P-ATP per
reaction by using T4 polynucleotide kinase (20 U, 37 °C, 2 h). The
reactions were brought to 20 mM EDTA, the enzyme was heat
deactivated, and the EDTA was removed by gel filtration (Microspin
G-25 cartridges, GE Healthcare). Finally, the strands were overdigested
(40) Dhara, S. C. Indian J. Chem. 1970, 8, 193-194.
(41) Cantor, C. R.; Warshaw, M. M.; Shapiro, H. Biopolymers 1970, 9, 1059-
1077.
(42) Fasman, G. D. Optical Properties of Nucleic Acids, Absorption, and Circular
Dichroism Spectra, 3rd ed.; CRC Press: Cleveland, OH, 1975; Vol. 1.
(43) Wei, M.; Cohen, S. M.; Silverman, A. P.; Lippard, S. J. J. Biol. Chem.
2001, 276, 38774-38780.
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with AfeI (20 U, 4 h), and the reactions were brought to 20 mM EDTA
and purified with Micropure EZ cartridges (Millipore), ethanol pre-
cipitation, and subsequent denaturing PAGE (8%, 20:1 acrylamide/
bisacrylamide). Single-end radiolabeled (2.8 × 106-4.2 × 106 cpm)
146-mer duplexes (D1c, D1-Ptc, D2c, and D2-Ptc, sequences in Scheme
S2, Supporting Information) were obtained, which were stored frozen
at concentrations no higher than 2 × 104 cpm/µL to reduce nicking by
autoradiolysis. In order to label exclusively the primer strands of the
duplexes, the sequence of the EcoRV and AfeI digestions in the protocol
above was swapped to yield the sequence-identical duplexes D1t, D1-
Ptt, D2t, and D2-Ptt in similar quantities.
separated from the main band and worked up individually (Figure S1,
Supporting Information).
The nucleosomes were isolated from the gel by electroelution with
a Millipore Centrilutor Microeluter system into Centricon 3 K micro-
filtration cartridges (0.3 × TBE, 200 V, 2 h). The sample volumes
were reduced, and the buffer was exchanged against 300 µL of TE
buffer by centrifugation. The histones were digested afterward by
treatment with 200 µg/mL of Proteinase K and 0.1% (w/v) SDS for
3 h at 50 °C and removed by two subsequent phenol extractions.
Residual phenol was then removed by ether extraction followed by
ethanol precipitation. The pellets were dissolved in 40 µL of 0.2 M
KCN and shaken at 50 °C overnight to remove the platinum adduct.
The samples were then ethanol precipitated twice, and the pellet was
rinsed with 70% ethanol. The purified DNA was finally taken up in
Characterization of DNA Duplexes. The resulting duplexes were
analyzed by Maxam-Gilbert A/G-reactions44 to confirm the sequence
and position of the cisplatin adducts. Before electrophoretic analysis
of the Maxam-Gilbert fragments, the samples were treated with
0.2 M KCN at 50 °C overnight to remove the platinum adducts.
Residual KCN was separated by ethanol precipitation and redissolution
of the DNA. This procedure removed the DNA-bound cis-diammine-
platinum(II) moiety completely, resulting in significantly increased
resolution of the sequencing reactions. Aligned profiles of the Maxam-
Gilbert traces were generated with the program SAFA V1.1,45 which
is available free of charge from https://simtk.org/home/safa/. Ap-
proximately 120 bands were resolved that corresponded to the designed
sequence. The position of the cisplatin lesion was identified by the
missing double bands in the platinated samples when compared to the
corresponding unplatinated reference strands.
Nucleosome Reconstitution. A 7 × 105-1 × 106 cpm quantity of
D1c, D1-Ptc, D2c, and D2-Ptc or D1t, D1-Ptt, D2t, and D2-Ptt was
combined with donor chromatin from HeLa-S3 cells10,22 having an
average length of 2-3 nucleosomes. The reaction was brought to
50 µL volume in 10 mM Tris, 2 M NaCl, 5 mM DTT, 0.5 mM PMSF,
0.2 mM EDTA, 0.01% (v/v) Nonidet-P40, pH 7.5, and dialyzed for 2
days in microdialysis buttons that were constructed from the cap of a
500 µL PCR reaction tube and a 1 cm2 Spectra/Por MWCO 6-8 kDa
dialysis membrane.46 The membrane was treated with EDTA and soaked
in a 2 M NaCl solution prior to use. The buttons were first dialyzed at
4 °C for 1 h against 20 mL of a high-salt buffer TB-1 containing
10 mM Tris, 2 M NaCl, 1 mM DTT, 0.5 mM PMSF, and 0.01% (v/v)
Nonidet-P40 at pH 7.5. The buffer TB-1 was transferred together with
the buttons into a secondary dialysis tube (Spectra/Por MWCO 6-8
kDa). The secondary tube was dialyzed against 1.5 L of a low-salt
buffer TB-2 (10 mM Tris, 0.2 mM PMSF, pH 7.5, 4 °C) overnight.
The coding strand-labeled nucleosome samples, nD1c, nD1-Ptc, nD2c,
and nD2-Ptc, or template strand-labeled nucleosome samples, nD1t,
nD1-Ptt, nD2t, and nD2-Ptt, were recovered from the dialysis buttons
and equilibrated at 45 °C for 2 h.
TE buffer and stored at 5 °C. Typically, 8 × 10
4-2 × 105 cpm of
radiolabeled DNA was recovered. As a reference, we conducted
hydroxyl radical footprinting with all unbound sample duplexes, D1c,
D1-Ptc, D2c, and D2-Ptc and D1t, D1-Ptt, D2t, and D2-Ptt, as previously
described.10,27
A 3.5 × 104 cpm aliquot of each footprinting sample was pipetted
into new reaction tubes and completely dried by vacuum centrifugation.
The dried samples were taken up in 2.5 µL of freshly deionized
formamide containing 10 mM EDTA and heated to 90 °C for 120 s.
Immediately after heating, the samples were cooled on ice and loaded
onto an 8% denaturing sequencing gel (0.4 mm thickness, 25:1
acrylamide/bisacrylamide, 7.5 M urea, 1 × TBE). The electrophoresis
was run for 2-3 h at 65 W until the bromphenol blue marker (run in
an extra lane) was about 4 cm above the bottom edge of the gel. Gels
were blotted onto Whatman paper, dried, and exposed for 3 days to a
phosphor screen.
Exonuclease III Digestion Analysis of Nucleosomes. A 5 µL
aliquot of each nucleosome sample (approximately 4 × 104 cpm) was
diluted to 50 µL and brought to 10 mM 1,3-bis(tris(hydroxymethyl)-
methylamino)propane‚HCl, pH 7.0, 10 mM MgCl2, and 1 mM
dithiothreithol. Exonucleoase III (1 U) was added, and the mixture was
incubated for 300 s at 25 °C. The reaction was quenched by addition
of 50 µL of a solution containing 40 mM EDTA-Na2, 0.8% (w/w) SDS,
50 µg/mL salmon sperm DNA, and 5.5 mg/mL proteinase K. The
samples were heated to 50 °C and ethanol precipitated. The pellets
were dissolved in 45 µL of 0.2 M KCN, shaken at 50 °C overnight to
remove the platinum adduct, ethanol precipitated twice, washed with
70% (v/v) ethanol, finally taken up in TE buffer, and stored at 5 °C.
Analysis by 8% denaturing sequencing gel electrophoresis was
performed as described above.
Data Collection and Processing. The gel images were processed
by the footprinting analysis software SAFA V1.1.45 First, the gel was
horizontally and vertically aligned. Next, each band of the Maxam-
Gilbert reference lane was aligned with the known DNA sequence.
The program uses this information as a starting guess to conduct a
least-squares fit of a sum of Lorentzians to the intensity profiles derived
from the footprinting data. Each identified band was assigned a relative
value derived by integration of the fitted Lorentzian. The individual
values for hydroxyl and exonuclease footprinting experiments are shown
in Tables S1 and S2 (Supporting Information).
Hydroxyl Radical Footprinting of Nucleosomes. Hydroxyl radical
footprinting27 and nucleosome purification were performed as follows.
A 7.5 µL aliquot of 10 mM sodium ascorbate, 7.5 µL of a solution
containing 1 mM Fe(NH4)2(SO4)2‚6H2O and 2 mM EDTA, and 7.5 µL
of a 0.12% (w/w) H2O2 solution were premixed and added within 5 s
to 52.5 µL of the nucleosome sample (approximately 5 × 105 cpm).
The reaction was incubated for 120 s at room temperature and stopped
by addition of 8 µL of 50% (v/v) glycerol and 2 µL of a 500 mM
EDTA solution. The samples were immediately loaded onto a cooled
4.5% native gel (37.5:1 acrylamide/bisacrylamide, thickness 1.6 mm,
0.3 × TBE) and run for 3 h at 200 V. The gel was wrapped in plastic
and exposed to a Biomax MS film for 25 min. The bands were cut out
using the exposed film as a template. An additional band with an
intensity of ca. 25% that of the main band, that migrated faster than
the main band, was visible for samples nD2-Ptc and nD2-Ptt. It was
Hydroxyl radical cleavage curves were calculated from these values
by applying a moving average with a window of 5 bp and normalized.
The areas between nearer maxima of the hydroxyl radical cleavage
curves of the complementary strands of a DNA duplex indicate where
the minor groove was exposed to solvent on the nucleosome surface.
At the remaining locations, the major groove is solvent-exposed. The
resulting exonuclease cleavage curves were normalized. The aligned
densitometric profiles (Figures S3 and S4, Supporting Information) were
also determined with SAFA. All traces were consistently color-coded
according their sequence as follows: (n)D1, blue; (n)D1-Pt, red; (n)-
D2, green; (n)D2-Pt, orange/brown.
(44) Ambrose, B. J. B.; Pless, R. C. Methods Enzymol. 1987, 152, 522-538.
(45) Das, R.; Laederach, A.; Pearlman, S. M.; Herschlag, D.; Altman, R. B.
RNA 2005, 11, 344-354.
(46) Thåstro¨m, A.; Lowary, P. T.; Widom, J. Methods 2004, 33, 33-44.
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2860 J. AM. CHEM. SOC. VOL. 130, NO. 9, 2008

Effect of a Cisplatin Cross-Link on Nucleosome Structure
A R T I C L E S
Acknowledgment. This work was generously supported by
NIH grant CA34992. Part of the experimental work was
performed at the University of Michigan, Department of
Chemistry. M.O. thanks Prof. Nils G. Walter for the opportunity
to work in his laboratory as a visiting scholar and the Alexander
von Humboldt Foundation, Germany, for support through a
Feodor-Lynen Fellowship.
purification gels, enlargement of autoradiographs, densitometric
scans of footprinting reactions and sequence alignment, struc-
tural models of the nucleosome samples, deconvoluted 32P
signals of hydroxyl radical, and exonuclease footprinting
(tables). This material is available free of charge via the Internet
at http://pubs.acs.org.
Supporting Information Available: Sequences and assembly
of sample duplexes, autoradiographs of preparative nucleosome
JA710220X
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