© 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 24
6811-6817
Yobing of DNA structure with osmium
s
Alena Kuderova-Krejiova, Alexander M.Poverenny1 and Emil Palecek*
Institute of Biophysics, Czechoslovak Academy of Sciences, Kralovopolska 135, 612 65 Brno,
Czechoslovakia and institute of Medical Radiology, 149 029 Obninsk, USSR
Received September 26, 1991; Revised and Accepted November 19, 1991
ABSTRACT
Antibodies against DNA modified with a single-strand
selective probe, OsO4 in complex with 2,2'-bipyridine
(Os,bipy), were raised in rabbits. These antibodies were
fractionated using affinity column chromatography and
fractions S89-II and S89-III characterized as highly
specific for DNA-Os,bipy adduct with no cross reactivity
to at least 1000-fold excess of unmodified DNA, RNA
and Os.blpy-modified and unmodified proteins. Crossreactivity to Os,bipy-modified RNA was very small.
S89-II showed no cross-reactivity to DNA modified with
OsO4 completed with fetramethylethylenediamine or
with bathophenanthroline disulphonic acid and to DNA
oxidized with KWJnO4. It cross-reacted, however, with
DNA modified with OsO4,1,10-phenanthroline complex. The limit of detection of Immunodot-blot analysis
of extensively Os.bipy-modified DNA was below 0.5 pg.
Small extent of Os,bipy-modificaf ion of supercoiled and
linearized plasmids can be detected by DNA gel
retardation and immunoblotting techniques. E. coli
cells contain DNA regions in which bases are accessible to the single-strand selective probe.
INTRODUCTION
Among the chemical agents single-strand selective probes have
particularly been useful in the in vitro studies of the local
supercoil-stabilized DNA structures (reviewed in 1,2). Osmium
tetroxide in complex with pyridine (Os,py) is one of the most
frequently used single-strand selective probes (reviewed in 1,2).
Few years ago we showed that osmium tetroxide,2,2'-bipyridine
complex (Os.bipy) can be used to probe DNA structure directly
inside a bacterial cell (3). Using this probe existence of lefthanded DNA (3 — 5), cruciform (4,6) and protonated triplex DNA
(1,7) in E. coli cells has been demonstrated.
Os,py and Os,bipy react preferentially with thymine residues
contained in single-stranded and distorted DNA regions (1,2).
In the B-DNA double helix the target C5-C6 double bond of
thymine residue (in the major groove) is not accessible to the
probe (2). Changes in the helix geometry may render this double
bond accessible to the osmium probe (2,8). Such changes may
• To whom correspondence should be addressed
include single-base mismatches and bulges, base unstacking,
distortions in the vicinity of the single-strand interruptions, gaps
and drug binding sites, local changes in twist in (A-T)n sequences,
premelting of AT-rich sequences in supercoiled DNA, etc. The
DNA site-specific osmium modification can be detected in various
ways (1) including detection at single-nucleotide resolution by
means of sequencing techniques (6,7).
Application of these techniques to probing of the DNA structure
in the cell requires isolation and purification of osmium-modified
DNA. This approach is suitable for studies of intracellular
plasmid and viral DNAs (2). On the other hand, technologies
for chemical probing of chromosomal DNAs are not yet available.
In an attempt to extend the possibilities of the detection of osmium
probe-modified DNA and particularly to facilitate studies of
chromosomal DNAs we elicited polyclonal antibodies to Os,py
DNA adducts and characterized them by ELISA and DNA gel
retardation techniques (9). Recently we have found (5) that Os,py
reagent induces early lysis of E. coli cells. No such difficulties
have been observed with Os,bipy which has proved to be suitable
for direct probing of the DNA structure in both prokaryotic
(3,6,7) and eukaryotic cells (10). We therefore attempted to raise
and characterize antibodies against DNA-Os,bipy adducts.
In this paper we show that (i) affinity collumn fractions of these
antibodies are highly specific showing no cross-reactivity to
unmodified DNA and other biomacromolecules, (ii) immunoassay
techniques can be applied for the adduct detection in supercoiled
and linearized plasmids as well as in chromosomal DNA, (iii)
regions containing bases accessible to the chemical probe are
present in chromosomal DNA inside E. coli cells.
MATERIALS AND METHODS
Nucleic acids, proteins, reagents and buffers
Origin and preparation of calf thymus DNA, apurinic acid,
pAT32 DNA (4,11), yeast RNA and bovine serum albumine
(BSA) was described (9). Chicken erythrocytes histone was kindly
donated by Dr. M. Stros. Affinity purified swine anti-rabbit IgGPeroxidase and restriction endonucleases were from USOL
(CSFR). Agarose was from Serva. OsO4 was from Fisher
Scientific Co.
6812 Nucleic Acids Research, Vol. 19, No. 24
Chemical modification
Prior modification calf thymus DNA in SSC/20 was denatured
by heating for 10 min at 100°C followed by rapid cooling in
an ice bath. The extent of modification was measured by UV
absorption spectroscopy (12).
DNA used for immunization. Denatured calf thymus DNA was
modified as described (9).
Nucleic acids and proteins used in ELJSA and immuno-dot blot
assay. The biomacromolecules at a concentration of about 150
or 300 /tg/ml were modified either in 0.05 M sodium phosphate
(pH 7.4) or in TE buffer (pH 7.8) with 2 or 4 mM Os.bipy
overnight at 26°C. DNA was also modified with osmium
tetroxide in complex with other ligands (Os,L): Os,py,
OsO4,l,10-phenanthroline (Os.phe), Os04,bathophenanthroline
disulphonic acid (Os.BPDS) and OsO4,tetramethylethylene
diamine (Os,TMEN). Proteins used in immunodot blotting were
treated in buffer either with or without 8 M urea. The samples
were purified by ethanol precipitation and/or by 24-hour dialysis
against SSC/20.
DNA oxidized with KMnO4 (DNA-ox, 13). DNA at 150 /*g/ml
in SSC was reacted with 1.5 mM KMnO4 for 30 min at 4°C.
The reaction was stopped by ethanol precipitation.
pAT32 DNA used in mapping ofOs,bipy sites. DNA was modified
in supercoiled form at about 50 /ig/ml in 5 mM TE (pH 7.8)
with 1 mM Os.bipy for 60 min at 37°C. pAT32-Os,bipy samples
were purified by ethanol precipitation.
to a nylon membrane filter (MSI Gelman Sciences, USA). The
nitrocellulose or nylon membranes were baked in vacuum at 80
or 78°C, respectively, for 1 - 2 hours and then soaked overnight
at 7°C in a solution of 5% nonfat dried milk in PBS to block
nonspecific binding. After washing three times in PBST, the
primary polyclonal antibody in a dilution of 1:200 (i.e.
approximately 1 /tg/ml) was added for 1 h and a membrane
washed again 4 — 5 times with PBST. Then peroxidase-tagged
anti-rabbit second antibody was added in a 1000-fold dilution
( 4 - 8 /ig/ml) for another 1 h. Incubations were kept at 37°C.
After a final washing 3—4 times the filters were shaken in a
detection 0.1 mM Na-phosphate buffer containing 0.5 mg/ml of
DAB (3,3-diaminobenzidine tetrahydrochloride), 0.02% NiCl2,
0.025% CoCl2 and 0.01% H2O2. The procedure was stopped
by washing the membranes with destiled water several times.
DNA gel retardation technique (15) was performed as described
(9).
Immobilization of separated fragments to a membrane filter
and immunoprobing (25)
The plasmid molecules of pAT32 DNA or their restriction
enzyme digests separated (during overnight gel electrophoresis
in TAE buffer at 25 V at room temperature) and stained in the
gel were slightly hydrolysed by soaking the gel in 0.25 M HC1
for 10 min with gentle agitation. The gel was then soaked in 1.5
M NaCl and 0.25 M NaOH for 30 min followed by constantly
shaking the gel in 3 M sodium acetate (pH 5.5). Then the DNA
in the gel was capillary blotted to a nitrocellulose membrane in
10 X SSC followed by washing the membrane with 5 xSSC and
drying at room temperature. The immunoprobing was carried
out in the same way as mentioned above (immunodot-blot assay).
DNA modification in IE. coliil cells (3). DNA was modified by
incubating 2 mg of dry-weight cells in 0.2 M sodium phosphate
(pH 7.5) with 2 mM Os,bipy in a total volume of 1 ml for 45
min either at 37°C or in an ice bath (0°C). The reaction was
stopped by washing cells twice in ice-cold buffer.
Preparation of antibodies
To produce specific antibodies the immunization procedure
described in (9) was used. Immunoglobulins were purified from
polyclonal antiserum by a combination of ammonium sulphate
precipitation and immunoaffinity purification. Calf thymus DNAOs,bipy was coupled with AN-sepharose-4B (Pharmacia) which
was placed into a column. To bind the specific antibodies the
antiserum (after precipitation) was passed through the column
using a peristaltic pump (LKB, Multiperpex). Three fractions
of the antiserum were obtained in three eluting steps: 1. with
2 M NaCl, 2. with 0.1 M glycine buffer (pH 10.4) and 3. with
0.1 M glycine buffer (pH 2). The fractions were dialysed against
PBS and tested by ELISA. This fractionation completely removed
a weak reaction with unmodified DNA observed in preimmune
sera. Even unfractioned preimmune sera exhibited no staining
with DNA-Os,bipy. For titrations the immunoglobulin
concentrations were calculated assuming that a solution at a
concentration of 1 mg/ml has an A28o = 1.4.
Enzyme-linked immunosorbent assay (ELJSA) was performed
as described (9).
Immunodot-blot assay (14-16)
Aliquots (1 or 2 /tl) of nucleic acids or proteins serially diluted
in PBS were applied to a nitrocellulose (SYNPOR, CSFR) or
0H
(orOsO
Fig, 1. A/ Formation of the adduct between thy mine and osmium tetroxide2,2'-bipyridine complex; .... hydrogen bonding in the Watson-Crick base pair.
B/ Some ligands which can replace bipyridine in the complex: I, 1,10-phenanthroline (phe). II. bathophenanthroline (4,7-diphenyl-1.10-phenanthroline)
disulfonic acid (BPDS) and III. tetramethylethylenediamine (TMEN). Cl Thymine
oxidation with KMnO4 (OsO4).
Nucleic Acids Research, Vol. 19, No. 24 6813
Growth of cells
E. coli (Hr30 strain not harboring any plasmid) was grown at
37°C to an OD550 = 0.8 in 180 ml of LB medium. Half a
volume (90 ml) was withdrawn under sterile conditions and
cooled in an ice bath followed by immediate centriftigation of
the cells. NaCl was added to the rest of the volume to a final
concentration of 0.5 M and the cells then incubated for another
40 min at 26°C. Osmotically shocked cells were collected by
centrifugation and washed with 0.1 M sodium phosphate (pH
7.5) together with the cells not exposed to high salt concentration.
DNA was then modified in situ (see Chemical modification).
After modification, washing and centrifuging the cells were
suspended in ST buffer. NaCl, EDTA and lysozyme (Sigma) in
ST buffer were added to a final concentration of 0.1 M, 0.02
M and 0.25 mg/ml, respectively. The mixture was incubated at
37°C for 1 min, dissolved in TE buffer and after addition of SDS
(to a final concentration of 0.5%) held for 3 min at 55°C and
cooled down in ice. The lysates were sonicated, diluted and dotted
onto a nylon membrane under vacuum by a Bio-Dot
Microfiltration Apparatus (BIO-RAD). The amount of DNA was
estimated provided that one E. coli cell contains 3.1% of DNA
(18).
RESULTS
Specificity of antibodies toward DNA-Os,bipy
To improve the specificity of antiserum S89, this antiserum was
fractioned on the affinity collumn and the fractions tested by
ELISA (Figs.2 and 3). With the antisera absorbance (at 490 nm)
slightly above 1 was obtained at 6400 Xdilution (Fig.2); to obtain
the same absorbance with the fractions 3O-6Ox lower dilutions
were necessary. The results of titration of S89-II and S89-IH
(which showed the best specificity to DNA-Os.bipy) by ELISA
are shown in Fig.3. In both fractions no cross-reactivity to
1000-fold excess of unmodified DNA and RNA, almost no crossreactivity to unmodified histon and small cross-reactivity to RNAOs,bipy was observed. The cross-reaction with Os.bipy-modified
histon in S89-III was lower than with RNA-Os,bipy while in
S89-II it was about the same as with the unmodified histon.
Competition experiments performed with S89-II produced similar
results (not shown).
Immunoblotting techniques have been very useful in nucleic
acid studies (14-17); in this paper we attempted to apply these
techniques to the detection of DNA-Os,bipy adducts. Using the
immunodot analysis we obtained a faint but significant staining
with an amount as low as 0.6 pg of the almost completely
Os.bipy-modified DNA (Fig.4C). 10000-fold excess of
unmodified DNA produced no significant staining with both
S89-n and S89-IH (Fig.4A).
We tested cross-reactivity of S89-II and S89-IH with
unmodified and Os,bipy-modified RNA and proteins also by
means of immunodot analysis to obtain more information about
their specificity. 40 ng of unmodified RNA (Fig.4B) and 60 ng
of unmodified BSA and histon (Fig.4D,E) produced no staining.
The same amounts of RNA-Os,bipy and modified proteins
produced only a very faint staining (Fig.4B,D,E) comparable to
that produced by 10000-fold lower amount of DNA-Os.bipy. The
results of this analysis confirmed the high specificity of both
S89-II and S89-III determined by ELISA (Fig.3) which makes
them applicable to partially purified DNA samples, cell lysates
or histochemistry of eukaryotic cells.
Interaction of S89-II with Os,L adducts
It has been shown (1,2,19) that DNA forms adducts with osmium
tetroxide in complexes with other ligands (Os,L). They include
Os.TMEN, Os,BPDS and Os,phe (Fig.l) which have already
been applied in the DNA structure probing. Osmium tetroxide
itself (20,21) and KMnO4 (13) oxidize DNA (DNA-ox) yielding
mainly cis-thymine glycol (5,6-dihydroxy-5,6-dihydrothymine)
(Fig.l). We were interested whether anti-DNA-Os,bipy cross-
1000
10000
DILUTION
001
0.1
1
10
001
0.1
1
tO
ANTIGEN lug/ml)
Fig. 2. Binding of anti-DNA-Os,bipy (antismim and its fractions) to DNA-Os.bipy
measured by ELISA. Dependence of absorbance at 490 nm on the dilution of
the original antiserum (A), and the first (I), the second (II) or the third (III) fraction
of polyclonal antibodies obtained after immunoaffinity purification in three ehaing
steps: with 2 M NaCl, with 0.1 M glycine-NaOH (pH 10.4) and with 0.1 M
glyrine-HCI (pH 2); the peroxidase tagged swine anti-rabbh antibodies in a dilution
of 1:5000.
Fig. 3. Binding of S89-II (A) and S89-III (B) to various antigens measured by
ELISA. Dependence of absorbance at 490 nm on the antigen concentration. AntiDNA-Os,bipy in a dilution of 1:400; the peroxidase tagged swine anti-rabbit
antibodies in a 30000-fold dilution: Antigens: (1) DNA-Os.bipy, (2) RNA-Os,bipy,
(3) unmodified histon, (4) histon-Os.bipy, (5) unmodified RNA, (6) thermally
denatured unmodified DNA.
6814 Nucleic Acids Research, Vol. 19, No. 24
reacts with any of the above mentioned chemically modified
DNAs. Testing of anti-DNA-Os,bipy by immunodot-blot assay
(Fig.5) did not show any cross-reaction with 10 ng of DNA-ox,
DNA-Os,TMEN or DNA-Os,BPDS, respectively. On the other
hand, 0.6 ng of DNA-Os,phe was sufficient to produce a marked
cross-reaction (Fig.5). High cross-reactivity of anti-DNA-Os,bipy
with DNA-Os,phe makes it possible to apply this antibody for
the detection of the DNA-Os,phe adduct. The antibody produced
very faint staining with 10 ng of DNA-Os,py, which is consistent
with our previous ELISA tests with anti-DNA-Os.bipy (9).
In contrast to Os,bipy (which does not react with intact B-DNA)
Os.phe showed, in addition to the reaction with single-stranded
DNA regions, also reactions with B-DNA (6). Fig.6 shows the
results of the immunodot-blot analysis of supercoiled, linearized
and denatured pAT32 modified with Os.bipy and Os.phe. In
supercoiled pAT32 DNA modified by Os,bipy in 50 mM sodium
phosphate weak staining can be observed (Fig.6, lane 2) due to
modification of few thymines in the cruciform loop (22) compared
to a substantially more intensive staining of the same plasmid
modified at lower ionic strength (Fig.6, lane 3) where
modification of all the thymines in the insert was demonstrated.
Very weak staining of linear DNA treated with Os.bipy at low
ionic strength (Fig.6, lane 4) may be due to modification of the
ends of the molecule and to DNA 'breathing'. In contrast to
Os.bipy the modification of the same plasmid with Os.phe showed
no difference in the staining of supercoiled and linear DNA
1
2
1
3
A
(Fig. 6, lanes 7 and 8) in agreement with our previous results
(5). The lack of structural specificity of Os,phe may be connected
with intercalation of the reagent in DNA (5,23). S89-II can thus
be applied not only for the detection of local DNA structures
hypersensitive to Os.bipy but also for the detection of both doubleand single-stranded DNAs modified with Os.phe.
Immunodetection of DNA fragments separated by gel
elect rophoresis
Supercoiled pAT32 DNA in 5mM Tris-EDTA pH 7.8 was
modified with lmM Os,bipy (60 min at 37°C). Purified DNA
containing Os.bipy adducts was incubated with various
concentrations of S89-II and analysed by agarose gel
electrophoresis (Fig.7). Increasing the antibody concentration
resulted in a stronger retardation of the supercoiled form (Fig.7
lanes 2 - 4 ) . Retardation was observed also in linearized DNA
(Os.bipy-modified supercoiled DNA was linearized with EcoRI).
Bgll digestion of supercoiled Os,bipy-modified DNA produced
two fragments of which only the longer one (1600 bp) containing
the (A-T)|6 insert was strongly retarded. Removal of the insert
from this fragment by Hindm did not eliminate the retardation
2
B
1
•
L,
5
6
6
3-5
7
S 89-IH
1
D
c
1
2
•
2
3
Fig. 5. Immunodot analysis of DNA-Os.L by S89-U. Six serial tenfold dilutions
(columns) of denatured calf thymus DNA modified overnight with osmium
tetroxkte (Os) in 4 mM complex with various ligands (L) (rows, see Abbreviations);
each spot of the first column contains 10 ng of DNA; (UC) unmodified control
(denatured calf thymus DNA); (DNA-ox) denatured DNA oxidized with
KMnO4.
«
3
5
#
F
«
6
1 -3
4-6
S 85-11
1
2
3
4 5
6
7
8
9
7-9
Fig. 4. Immunodot analysis of sensitivity (A,Q and cross-reactions (B,D,E) of
S89-III and S89-II. A/ (1) thermally denatured unmodified DNA in the amount
of 0.02 fig; ( 2 - 6 ) Five serial tenfold dilutions of DNA-Os,bipy of which the
first spot contains 0.02 /ig. B/ Each spot contains about 0.04 ng of unmodified
(1) double stranded DNA, (2) thermally denatured DNA and (3) apurinic acid,
(4) apurinic acid-Os.bipy, (5) DNA-Os,bipy, (6) unmodified RNA and (7) RNAOs,bipy. C/ (1 - 6 ) Six serial tenfold dilutions of DNA-Os,bipy. The first spot
contains 0.06 ^g. D/ 6 ng of (1) DNA-Os,bipy, (2) unmodified thermaUy denatured
DNA, (3) RNA-Os.bipy, (4) unmodified BSA, (5) BSA-Os,bipy, (6) unmodified
RNA. E/ 6 ng of (1) DNA-Os,bipy, unmodified (2) histon, (3) BSA and (4)
thermally denatured calf thymus DNA, (5) histon-Os.bipy, (6) BSA-Os.bipy. (7)
unmodified thermally denatured E. coli DNA, (8) histon-Os.bipy (8 M urea),
(9) BSA-Os,bipy (8 M urea). Unless otherwise stated, calf thymus DNA was
used in the experiments. Nylon filters onto which proteins were dotted (D and
E) were only let getting dry for 1 hour at room temperature.
SC,H
SC
LIN
Os.bipy
DEN
SC/H
•
*
SC
LIN
•
b
DEN
0s.phe
Fig. 6. Os,bipy and Os.phe modification of pAT32 DNA detected by S89-II.
Three serial tenfold dilutions (rows) of different pAT32 DNA samples (columns).
10 ng of the antigens is contained in each spot of the first row. (3,7) supercoiled
(SC), (4.8) cleaved with restriction endonuclease EcoRI (LIN) and (5,9) denatured
(10 min at 100°C followed by rapid cooling in an ice bath) pAT32/EcoRI DNA
(DEN) were prepared prior modification. Compare DNA samples of 3. 4 and
5 with those of 7, 8 and 9, respectively, the former modified with Os.bipy. the
latter modified with Os.phe under the same conditions. pAT32 DNA was also
modified with Os.bipy (2) or Os.phe (6) in supercoiled form at higher ionic strength
of 50 mM sodium phosphate buffer, pH 7.5 (SC/H); (1) unmodified pAT32 DNA.
Nucleic Acids Research, Vol. 19, No. 24 6815
(Fig.7, lane 12) suggesting modification of other bases in addition
to those in the (A-T)i6 insert. No significant retardation was
observed in unmodified DNAs (Fig.7, lanes 14,16).
In addition to gel retardation (9) immunoanalysis of capillary
blotted plasmid molecules and their enzyme digests can be applied
to detect the Os,bipy-modified DNA fragments separated by gel
electrophoresis. Fig.8 flanes 1-9) shows agarose gel
electrophoresis with the similar spectrum of pAT32 fragments
to that shown in Fig.7 and the same gel (lanes 10—18) after
immunodetection of Southern blots by S89-II. In agreement with
the results of the DNA gel retardation technique (Fig.7) the
strongest immuno-blot staining was observed in Os,bipy-modified
supercoiled DNA and its linearized form (Fig.8, lanes 6,7 and
16,17). In DNA fragments obtained by cleavage with Bgll (lane
8) and Bgll plus HindHI (lane 9) only fragments of 1600 bp (lane
18) and 1366 bp (lane 19) in length were stained in agreement
with the results of the DNA gel retardation (Fig.7).
1 2 3A
5 6 7 8
Os,bipy-hypersensitive sites in chromosomal DNA
Presence of local open DNA structures hypersensitive to singlestrand selective chemical probes has reliably been demonstrated
in supercoiled plasmids both in vitro and in E. coli cells (rewiewed
in 2). On the other hand, no evidence of the presence of such
DNA structures in intracellular chromosomes has been published
up to now. A high specificity of S89-II to Os.bipy modified DNA
made us possible to apply this antibody for the analysis of
chromosomal DNA in cell lysates. The strongest staining was
observed in the osmotically shocked E. coli cells treated with
Os,bipy at 37°C (Fig.9). Staining of cells not exposed to the
osmotic shock was weaker. No staining appeared in Os.bipyuntreated control cells. Os.bipy-treated cells showed staining in
several experiments under slightly different experimental
conditions (23), the difference between the staining in osmotically
shocked and unshocked cells shown in this paper (Fig.9) was,
however, poorly reproducible. Experiments to clarify this point
as well as those oriented to a quantification of Os.bipy adducts
in purified DNA samples are under way.
9 10 11 12 13 14 15 16
DISCUSSION
-REL
•1118
Fig. 7. Gel retardation of pAT32 DNA-Os,bipy by S89-II. Ay Dependence on
the S89-II concentration; pAT32 DNA-Os,bipy without (lane 1) or with 0.03
(lane 2), 0.3 (lane 3) and 2.0 /ig (lane 4) of S89-II (after 45-min incubation of
the DNA-antibody mixture at 37°C followed by cooling the samples in an ice
bath). B/ Mapping of the Os,bipy sites on pAT32 DNA molecules; the amount
of 2 /ig of the S89-II was used in each reaction. Compare pAT32-Os,bipy alone
with pAT32 DNA-Os,bipy-IgG complex in following pairs: the whole plasmid
molecules (lane 5 with lane 6), Os.bipy-modified supercoiled (SC) DNA cleaved
with EcoRI (7 with 8), Bgll (9 with 10) and Bgll plus Hindm (11 with 12); similarly
unmodified DNA without (lane 13) and with the antibody (lane 14); Bgll digest
of unmodified pAT32 DNA without Gane 15) and with S89-II (lane 16); (SC)
supercoiled unmodified DNA; (SCJ supercoiled modified DNA, (REL) relaxed
DNA; (LIN) DNA linearized with EcoRI. Size in base pairs for DNA fragments
are indicated.
1
1011 12 13 14 15 16 17 18
2 3 4 5 6
LIN
Occurence of DNA segments with a single-stranded character
in genomes is associated with basic biological processes such as
DNA replication, recombination and RNA transcription (2).
Local DNA structures including cruciforms, triplexes, B-Z
junctions, etc. contain open regions accessible to single-strand
selective chemical probes. Nucleotide sequences from which these
structures may be extruded are frequently, located in biologically
significant sites of the genomes suggesting possible role of these
structures in cellular processes. Studies of DNA single-stranded
and distorted double-stranded regions in vitro stay in the center
of interest of many laboratories at present time (for review see
2). Our knowledge about such DNA structures inside the cell
is, however, very limited mainly due to the lack of suitable
techniques. Recent application of Os.bipy for direct probing of
the DNA structure in the cell (2,3,4,6,7) might represent an
important step toward obtaining more information about the DNA
structure-function relations in the cell. However, further
development of the probing technology is needed to'improve the
sensitivity of the adduct detection and to extend its applicability
to studies of chromosomal DNA.
Antibodies raised against DNA modified with some chemical
carcinogens have successfully been applied for the detection of
low levels of the adducts in DNA (14,24—28). We have shown
1
2
3
V
•
4
5
6
7
1118
Fig. 8. Binding of Os.bipy complex to pAT32 DNA detected by nitrocellulose
immunoprobing. (1-9) the separated plasmids or their restriction enzymes digests
after ethidium bromide staining, (10-18) the same gel after immunodetection
of Southern blots. Supercoiled pAT32 DNA was modified with Os.bipy complex
(lanes 6 - 9 & 15-18) and unmodified DNA used as a control (lanes l - 4 &
10-13). Unmodified or modified samples were cleaved with therestrictionenzyme
EcoRI (lanes 2,7 & 11,16), Bgll (lanes 3,8 & 12,17) or combination of Bgll
and HindDJ (lanes 4,9 & 13,18). The whole plasmids (lanes 1,6 & 10,15) or
their fragments were separated on 1% agarose gel. Samples were Southern
transferred and osmium-binding sites detected by immunoprobing the filter with
S89-II at a dilution of 1:400; (lane 5) 2027 bp, 1904 bp and 1584 bp fragments
of bacteriofage X DNA cleaved with the restriction endonucleases EcoRI and
HindHI; (SC) supercoiled pDNA, (LIN) pDNA linearized with EcoRI.
Fig. 9. Dotblots of E. coli lysates after modification in situ detected by S89-II.
Three serial five fold dilutions (rows) of various samples of E. coli lysates. Each
spot of the first row contains about 0.3 ^g of the chromosomal DNA. The
modification of E. coli cells with Os.bipy was either held in an ice bath (0°C,
columns 2,5) or at 37°C (3,6). In addition cells were modified either without
(5,6) or in the presence of 0.5 M NaCI (2,3). DNA lysates from unmodified
NaCI-shocked (1) and normal (4) cells were used as controls. (7) thermally
denatured extensively modified calf thymus DNA-Os,bipy in the same dilutions.
6816 Nucleic Acids Research, Vol. 19, No. 24
(1,9) that a similar approach can be applied to study adducts
formed in DNA due to its treatment with a single-strand selective
probe in vitro. In this paper we report production and
characterization of antibodies that specifically recognize the
Os,bipy adducts which are formed as a result of DNA structure
probing both in vitro and in situ. These antibodies are highly
specific and can be used in ELJSA, immunoblotting and DNA
gel retardation experiments.
Specificity of inununoassay and antibody binding sites
Antibodies to several adducts of DNA with single-strand selective
probes have been reported including bisulphite.o-methylhydroxylamine mixture (29) and carbodiimide (17,30). These
antibodies showed practically no cross-reactions with unmodified
DNA but in no one of them the absence of cross-reaction with
proteins and RNA modified with the given chemical probe was
demonstrated. Absence of cross-reaction of S89-I1 and S89-IH
with Os,bipy-modified proteins (Figs.3 and 4) as well as only
very weak cross-reaction with RNA-Os,bipy (Figs.3 and 4)
makes it possible to apply these antibodies for the in situ studies
of the DNA structure. High specificity of S89-II to DNA-Os,L
adducts (Fig.5) including the absence of any cross-reaction with
DNA modified with Os,TMEN or Os.BPDS as well as DNA
oxidized with KMnO4 suggest that the ligand forms a part of the
antibody binding site, i.e. the epitope. A substantial cross-reaction
of S89-II with DNA-Os,phe might be explained by the
involvement of the bipyridine rings in the epitope. In DNAOs,BPDS the antibody binding might be prevented due to the
presence of two phenyls and/or the negatively charged sulfonic
groups. Only a little cross-reactivity of S89-II and S89-ITJ with
RNA-Os,bipy (Fig.4) suggests that either the thymine ring or
the sugar moiety may form a part of the epitope. If the bipy rings
and a part of the nucleotide are involved in the specific binding
to the antibody it is not surprising that Os.bipy-modified proteins
show little cross-reactivity. Among the amino acids most reactive
toward Os,bipy only tryptophan was shown (31) to form an
osmate ester (while cystine was oxidized to cysteic acid and
methionine to methione sulfone) but the tryptophan reaction
product was not isolated from proteins. Recently we have
obtained (32) a monoclonal antibody which show no crossreaction with RNA-Os,bipy. Our preliminary results suggest
involvement of the thymine residue in this antibody binding.
Detection of Os,bipy adducts in plasmid DNA
Local supercoil-stabilized DNA structures have been detected in
plasmid and viral DNAs by osmium tetroxide and other chemical
probes (2). Here we show that these structures can also be studied
by antibodies against DNA-Os,bipy and that the adducts can be
detected both in single- and double-stranded DNAs (Fig.5). In
addition application of immunoassay for the detection of bases
available to the chemical probe due to site-unspecific changes
(induced by various agents) as well as due to the structural
dynamics of the DNA molecule may become especially useful
because the so far available methods of the adducts detection
(12,33) are either not sufficiently sensitive or recognize only the
site-specific modification of DNA. The most important
application of die immunoassay of the DNA-Os,bipy adducts may
be in the studies of the structure of chromosomal DNA.
Probing of DNA structure in the cell
At present three single-strand selective chemical probes are
available (Os.bipy, Os.TMEN and KMnO4) to study DNA
structure in the cell (2). Polyclonal antibody raised against DNA
modified with OsO4 alone (21) can be applied for the detection
of DNA oxidized with KMnO4 because the main reduction
product is the same (Fig.l). With this antibody high specificity
necessary for its application in situ has not, however, been
demonstrated. Recently reported (17,30) antibodies against DNA
modified with carbodiimide derivative (CMC) were not tested
for their cross-reactivity with other CMC-treated
biomacromolecules and an ability of the bulky CMC molecule
to penetrate into the cell has not been demonstrated. Antibodies
with a potential ability to detect the reaction products of a singlestrand selective probe with DNA in partially purified DNA
samples, cell extracts or directly in the cell are thus limited to
those reported in this paper. Antibodies against DNA-Os,pyridine
(9) might be applied for the same purpose but the probed cells
have to be carefully chosen because integrity at least of some
of them is disturbed by the Os,py reagent (5).
The results obtained in this paper suggest that chromosomal
DNA in E. coli contains regions in which bases are accessible
to the Os.bipy probe (Fig.9). The amount of the Os,bipy-reacted
DNA cannot be accurately determined in this paper; from the
available data we may only roughly estimate that this amount
might be below 5 %. A more accurate determination or reacted
DNA will require further work.
Earlier it was shown that a certain portion of DNA isolated
from various organisms had a single-stranded character (reviewed
in 34) and in some cases the size of this portion was related to
biological processes in the cell (2). The experiments were,
however, performed with isolated DNA; it was therefore difficult
to exclude the possibility that open DNA regions were formed
secondarilly (e.g. due to nuclease cleavage after the cell
disruption). In this paper we report for the first time the presence
of open DNA regions in chromosomal DNA detected directly
inside the cell by a chemical probe. Our results do not allow us
to say anything about location of these regions in the genome
and about their structural details. The possibility of specific
chemical modification of structurally changed DNA segment of
the genome inside the cell and a specific recognition of the
chemical modification by an antibody represent a new approach
of the DNA structure studies in the cell. We believe that it is
only a question of time to locate the Os.bipy-modified DNA
regions by means of currently available techniques and to relate
the occurrence of open DNA structures to specific biological
processes in the cell.
ACKNOWLEDGEMENT
This paper is dedicated to the memory of Dr V.Doleialovd.
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