Deoxyribonuclease I footprinting reveals different DNA
binding modes of bifunctional platinum complexes
Kater
ˇina Chva
´lova
´
1,
*, Jana Kas
ˇpa
´rkova
´
1,
*, Nicholas Farrell
2
and Viktor Brabec
1
1 Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
2 Department of Chemistry, Virginia Commonwealth University, Richmond, USA
DNA interactions of small molecules are frequently
crucial events underlying their biological effects so that
examinations of these interactions are of great interest.
The specificity of these interactions varies over an
enormously wide range. A number of ‘footprinting’
methods have been developed for determining the
sequence-specific binding of small molecules to DNA.
Some of these methods are based on the ability of
DNA binding molecules to protect DNA from enzy-
matic or chemical cleavage at their binding sites. The
cleavage patterns of regions to which a sequence-select-
ive agent is bound are altered in comparison to free,
nonmodified regions in DNA.
One subclass of footprinting agents that has been
developed comprises enzymes such as deoxyribo-
nuclease I (DNase I) [1]. DNase I is an endonuclease
(monomeric glycoprotein of molecular mass 30.4 kDa)
that specifically cleaves the O3¢–P bond of the phos-
phodiester backbone of the double-helical DNA sub-
strate. It binds to the minor groove of B-DNA and
cleaves each strand independently [2,3]. The nuclease
binds asymmetrically to the minor groove of DNA
contacting two phosphates on each side of the cleaved
bond and two phosphates on the complementary
strand across the minor groove, opposite the phos-
phates contacted on the 5¢side of the cleaved bond
(Fig. 1A). In addition, binding of the enzyme to DNA
induces a widening of the minor groove by 0.3 nm
coupled with a 21bend towards the major groove.
Thus, the local conformation of the DNA at the sites
of the contacts of DNase I with DNA phosphates,
in particular the minor groove width and depth,
Keywords
cross-link; conformation; DNA; DNase I;
footprinting; platinum complex
Correspondence
V. Brabec, Institute of Biophysics, Academy
of Sciences of the Czech Republic,
Kra
´lovopolska
´135, CZ-61265 Brno,
Czech Republic
Fax: +420 51412499
Tel: +420 541517148
E-mail: brabec@ibp.cz
Website: http://www.ibp.cz/labs/BNAIAD
*These authors contributed equally to this
work.
(Received 25 April 2006, revised 1 June
2006, accepted 1 June 2006)
doi:10.1111/j.1742-4658.2006.05356.x
Deoxyribonuclease I (DNase I) footprinting methodology was used to
analyze oligodeoxyribonucleotide duplexes containing unique and single,
site-specific adducts of trinuclear bifunctional platinum compound, [{trans-
PtCl(NH
3
)
2
}
2
l-trans-Pt(NH
3
)
2
{H
2
N(CH
2
)
6
NH
2
}
2
]
4+
(BBR3464) and the
results were compared with DNase I footprints of some adducts of con-
ventional mononuclear cis-diamminedichloroplatinum(II) (cisplatin). These
examinations took into account the fact that the local conformation of the
DNA at the sites of the contacts of DNase I with DNA phosphates, such
as the minor groove width and depth, sequence-dependent flexibility and
bendability of the double helix, are important determinants of sequence-
dependent binding to and cutting of DNA by DNase I. It was shown that
various conformational perturbations induced by platinum binding in the
major groove translated into the minor groove, allowing their detection by
DNase I probing. The results also demonstrate the very high sensitivity of
DNase I to DNA conformational alterations induced by platinum com-
plexes so that the platinum adducts which induce specific local conforma-
tional alterations in DNA are differently recognized by DNase I.
Abbreviations
BBR3464, [{trans-PtCl(NH
3
)
2
}
2
’-trans-Pt(NH
3
)
2
{H
2
N(CH
2
)
6
NH
2
}
2
]
4+
; cisplatin, cis-diamminedichloroplatinum(II); CL, cross-link; DMS, dimethyl
sulfate; DNase I, deoxyribonuclease I; FAAS, flameless atomic absorption spectrophotometry.
FEBS Journal 273 (2006) 3467–3478 ª2006 The Authors Journal compilation ª2006 FEBS 3467
sequence-dependent flexibility and bendability of the
double helix, are very important, indirect determinants
of sequence-dependent binding to and cutting of DNA
by DNase I [4]. Although DNase I cuts all phosphodi-
ester bonds, the cleavage pattern of nonmodified or
noninteracting B-DNA is very uneven since the geom-
etry of the minor groove is sequence-dependent. Also
importantly, DNA segments displaying an A- or
Z-type conformation are resistant to DNase I digestion
[4,5]. Steric hindrance from ligand bound to DNA is
another obvious factor decreasing affinity and cleavage
efficiency of DNase I. In this case, suitably placed
groups of the DNA-bound ligand located in the minor
groove may physically impede the approach of
DNase I to the phosphodiester backbone and in this
way directly prevent cleavage. All this information
supplies a rational basis on which the results of
DNase I footprinting can be interpreted.
DNase I footprinting has been already used to char-
acterize the DNA binding of a number of small mole-
cules of biological significance [1,6–15], including
antitumor cis-diamminedichloroplatinum(II) (cisplatin)
(Fig. 1B) and its clinically ineffective trans isomer [16].
The bifunctional platinum complexes, such as cisplatin
and its analogues, form various types of adducts
on DNA [17,18]. Recently, a trinuclear bifunctional
platinum compound, [{trans-PtCl(NH
3
)
2
}
2
l-trans-
Pt(NH
3
)
2
{H
2
N(CH
2
)
6
NH
2
}
2
]
4+
(BBR3464, Fig. 1B)
has entered clinical trials as a potential new antitumor
drug, which binds to DNA in a way which is funda-
mentally different from that of cisplatin [19–24]. The
conformational alterations induced in DNA by various
bifunctional adducts of BBR3464 have been probed by
a number of methods. Among the unique properties of
BBR3464 are the capability to form long-range cross-
links (CLs), where the platinated sites in DNA are sep-
arated by several intervening base pairs [19]. Hence,
the footprints of BBR3464 when it binds to DNA may
be larger than those of mononuclear platinum com-
pounds, such as cisplatin. BBR3464 and cisplatin allow
preparation of a broad spectrum of DNA adducts
which differ significantly in the extent and character of
the resulting conformational alterations induced in
DNA [19]. To expand the database of DNase I foot-
prints, we compared in the present work DNA
duplexes containing unique and single, site-specific
adducts of trinuclear platinum complex BBR3464 and
mononuclear cisplatin.
Results
DNase I cleavage of duplexes containing
intrastrand CLs of BBR3464 or cisplatin
The 20 bp duplexes containing the single, site-specific
adduct of BBR3464 or cisplatin were prepared as
A
B
C
Fig. 1. (A) Schematic representation of the
contacts made by DNase I with DNA (adap-
ted from ref [34]). The contacts are plotted
on a cylindrical projection of a DNA double
helix. The base pairs (short vertical lines) are
drawn across the minor groove. The grey-
shaded area represents the approximate
diameter of DNase I (it is not meant to
accurately represent the projected three-
dimensional shape of the protein). The fig-
ure shows the phosphate contacts made by
DNase I at a cleavage site, taken from the
structure of DNase I ⁄DNA complex [35,36].
An arrow marks the DNase I cleavage site.
The filled spots (d) indicate the phosphate
groups contacted by DNase I to cleave at
the site indicated. (B) Structures of cisplatin
(left) and BBR3464 (right). (C) Sequences of
the deoxyribonucleotide duplexes. The top
and bottom strands of each pair in Fig. 1C
are designated ’top’ and ’bottom’, respect-
ively, throughout. The bold letters in the top
and bottom strands of the duplexes indicate
the platinated residues.
DNase I cleavage of platinated DNA K. Chva
´lova
´et al.
3468 FEBS Journal 273 (2006) 3467–3478 ª2006 The Authors Journal compilation ª2006 FEBS
described previously [20,21,25] with a radioactive label
at the 5¢end of the top or bottom strand. Nonmodi-
fied and platinated duplexes were subjected to limited
DNase I cleavage [20,21,25]. The digestion products
were treated with 0.2 mNaCN at pH 11 to remove
all platinum [26] to eliminate alterations in electro-
phoretic mobility caused by the charged platinum
moiety and then these products were resolved on
sequencing gels.
BBR3464 forms in natural DNA 80% intrastrand
CLs of different length between guanine residues [19].
In the present work, we analyzed by DNase I footprint-
ing methodology the duplexes 1,3-IAC and 1,5-IAC
(Fig. 1B) containing the 1,3- and 1,5-intrastrand CLs
of BBR3464, respectively. Platinated guanines in the
top strands of these duplexes were separated by one or
three intervening bases, respectively. The susceptibility
to DNase I is more pronounced in the platinated
strand than in the complementary strand (Figs 2 and
3). The protected cleavage sites span approximately
four and nine consecutive bonds in the 1,3- and
1,5-intrastrand duplexes, respectively. The DNase I
footprints of both intrastrand CLs of BBR3464 do not
differ only in the length of the footprint. In the 1,3
intrastrand adduct, protection from cleavage occurs
from the 5¢-platinated guanine and thymine on its 3¢
side and the three sites on the 5¢side (Fig. 2). In addi-
tion, the markedly enhanced cleavage by DNase I of
the bond between two thymidines on the 3¢side of the
adduct in the top strand is another interesting conse-
quence of the formation of 1,3-intrastrand CL of
BBR3464. In the 1,5-intrastrand adduct, the nine clea-
vage sites include bases both 5¢and 3¢to the platinated
guanines (Fig. 3). However, no strong enhancement of
the cleavage occurs, such as that observed for the clea-
vage of the duplex containing 1,3-intrastrand CL of
BBR3464 on its 3¢side. Hence, the patterns of cleavage
by DNase I of the duplexes containing either 1,3- or
1,5-intrastrand CL of BBR3464 are different, but a
strong protection of the cleavage sites in the top strand
on the 5¢side of the adduct is the common feature of
both patterns.
AB
C
Fig. 2. DNase I cleavage of control (non-
modified) DNA duplex 1,3-IAC and the
duplex 1,3-IAC containing single, site
specific 1,3-intrastrand CL of BBR3464. (A)
Phosphorimage of a 24% denaturing poly-
acrylamide gel. Lanes: 1: nonmodified
duplex, 5¢-end-labelled top strand; 2: plati-
nated duplex, 5¢-end-labelled top strand; 3:
nonmodified duplex, 5¢-end-labelled bottom
strand; 4: platinated duplex, 5¢-end-labelled
bottom strand. (B) Quantitative representa-
tion of DNase I cleavage of the nonmodified
and platinated duplexes. The bars represent
the amount of radioactivity associated with
the bands. The white bars represent the
cleavage in the nonmodified duplex and the
black bars the cleavage in the platinated
duplex. (C) The differential histogram; verti-
cal scales are in units of ln[(fa) ⁄(fc)], where
fa is the fractional cleavage at any bond in
the presence of the platinum drug and fcis
the fractional cleavage of the same bond in
the control, nonmodified duplex, given clo-
sely similar extents of overall digestion.
Negative values correspond to the adduct-
protected site and positive values represent
enhanced cleavage. Data shown in
Figs 2B,C are compiled from quantitative
analysis of three sequencing gels (including
the gel shown in Fig. 2A) and must be con-
sidered a set of averaged values.
K. Chva
´lova
´et al. DNase I cleavage of platinated DNA
FEBS Journal 273 (2006) 3467–3478 ª2006 The Authors Journal compilation ª2006 FEBS 3469
Cisplatin can form minor 1,3-intrastrand CLs in
DNA and it is interesting to compare the results of
DNase I cleavage of the duplexes containing 1,3-intra-
strand CLs of trinuclear BBR3464 and mononuclear
cisplatin [22]. To date this type of cisplatin adduct has
not been analyzed by DNase I cleavage assay. The
structure of this CL is known [27] so that a concrete
basis for interpreting DNase I footprinting data is
available. The protected cleavage sites span eight or
nine consecutive bonds in both the top and bottom
strands, respectively, which is considerably more than
the amount of protected sites in the duplex containing
1,3-intrastrand CL of BBR3464 (Fig. 4). Protection
from the cleavage of the sites between the platinated
guanines and the six sites in the top strand on the 5¢
side of this adduct and of the sites at complementary
nucleosides in the bottom strand represent major chan-
ges in the cleavage due to formation of the 1,3-intra-
strand adduct of cisplatin. No markedly enhanced
cleavage by DNase I in the cisplatin-1,3-intrastrand
CL is observed in contrast to that observed for the
analogous adduct of BBR3464. Thus, DNase I foot-
printing data obtained for the duplexes containing the
intrastrand CLs of mononuclear cisplatin and trinu-
clear BBR3464 are distinctly different.
DNase I cleavage of duplexes containing
interstrand CL of cisplatin or BBR3464
BBR3464 and cisplatin also form in DNA interstrand
CLs. BBR3464 forms interstrand CLs with much
higher frequency than cisplatin (20% vs. 6%) [19]. The
unique properties of interstrand CLs of BBR3464
and resulting conformational alterations in DNA are
considered critical consequences for its antitumor
effects [21]. In contrast, contradictory data have been
published on the correlation of DNA interstrand
cross-linking by cisplatin and its cytotoxic effects so
that it is unclear whether these DNA adducts play a
decisive role in the mechanism of antitumor efficiency
of cisplatin [17,28].
BBR3464 forms in DNA long-range interstrand CLs
between guanine residues in both directions, i.e. in the
3¢)3¢and 5¢)5¢direction [21]. The orientation of the
1,4-interstrand CL (where the platinated guanine resi-
dues are separated by two base pairs) in the 3¢)3¢or
AB
C
Fig. 3. DNase I cleavage of control (non-
modified) DNA duplex 1,5-IAC and the
duplex 1,5-IAC containing single, site speci-
fic 1,5-intrastrand CL of BBR3464. See
Fig. 2 for other details.
DNase I cleavage of platinated DNA K. Chva
´lova
´et al.
3470 FEBS Journal 273 (2006) 3467–3478 ª2006 The Authors Journal compilation ª2006 FEBS
5¢)5¢direction can be explained with the aid of the
sequences of duplexes 1,4-IEC-3¢)3¢or 1,4-IEC-5¢)5¢
(for their sequences, see Fig. 1B). For instance, the
1,4-GG interstrand CL oriented in the 3¢)3¢direction
is that formed in duplex 1,4-IEC-3¢)3¢between the
central G residue in the top strand and G7 in the bot-
tom strand, whereas the same CL oriented in the
opposite, 5¢)5¢direction is that formed in duplex 1,4-
IEC-5¢)5¢between the central G residue in the top
strand and G14 in the bottom strand. 1,4-Interstrand
CLs are formed in both directions with approximately
the same frequency [21]. The duplexes 1,4-IEC-3¢)3¢
and 1,4-IEC-5¢)5¢were prepared which contained the
single and central, site specific 1,4-interstrand CL
between guanine residues in the 3¢)3¢and 5¢)5¢direc-
tion, respectively. The duplexes interstrand cross-linked
by BBR3464 are protected from DNase I cleavage at
the sites in both strands, although the protection of
the cleavage sites in the bottom strand is somewhat
less pronounced (Figs 5 and 6). The protection is
always observed at the sites contained in the sequences
covered by the adduct. Additional protection from the
cleavage is observed for the sites contained in the
sequences not covered by the adduct, namely for two
sites on the 3¢side of the platinated guanines in the
3¢)3¢adduct in each strand (Fig. 5). In contrast, this
additional protection in the duplex 1,4-IEC in the
5¢)5¢direction is more extensive (Fig. 6). The latter
additional protection is observed for two sites on the
3¢side of the platinated guanine in each strand and for
four or five sites on the 5¢side of this adduct in the
top or bottom strand, respectively. Taken together the
protection spans, respectively, approximately eight or
12 base pairs if the interstrand CL is formed in DNA
in the 3¢)3¢or 5¢)5¢direction by BBR3464. No
enhancement of cleavage was seen.
We also prepared the 1,2-IEC duplex (Fig. 1B)
which contained the single and central, site-specific
AB
C
Fig. 4. DNase I cleavage of control (non-
modified) DNA duplex 1,3-IAC and the
duplex 1,3-IAC containing single, site speci-
fic 1,3-intrastrand CL of cisplatin. See Fig. 2
for other details.
K. Chva
´lova
´et al. DNase I cleavage of platinated DNA
FEBS Journal 273 (2006) 3467–3478 ª2006 The Authors Journal compilation ª2006 FEBS 3471