BIOCHEMICAL
Vol. 169, No. 1, 1990
AND BIOPHYSKAL
RESEARCH COMMUNICATIONS Pages 87-94
May 31, 1990
PRBFGRRNTIAL
IIODIFICATION
DTMRTHYLDLOXIRANE
R.
Jeremy
AND
H.
Division
of
D.
16,
R.
and
of
Boyd’,
of
Chemistry, BT9
TO
DNA
DNA
BY
SlZQURNCclNG
Kumarl,
Stevenson’
Biology
and
Queen’s
78L,
IN
Shiv
Clarke
School
School
8A3BS
APPLICATION
Sharma’
Belfast
March
CDANINB
Derek
Biochemistry, 2
and
Received
IT3
Davies’,
Rarain 1
OF
Biochemistry,
University,
Northern
Treland
1990
32 Susasary: From gel sequencing experiments with P-end-labelled oligodeoxyribonucleotides. it is shown that treatment of DNA with the powerful oxidant dimethyldioxirane, followed by heating in piperidine, causes selective strand scission at the sites of guanine bases. The same specificity for cleavage at guanine was observed with a 45-mer labelled at either the 3’or S.-end and with a single and double stranded 34-mer. On account of its speed and operational simplicity, modification with dimethyldioxirane is proposed as a practicable alternative to conventional chemical sequencing procedures for locating guanine bases in DNA. 01990 Academic Press, Inc.
0 Dimetbyldioxirane powerful
(CH3j2
oxidising
a wide
range
agent
which
of
organic
conditions
(1.2).
It
compounds,
hydroxylamines
or
Product
isolation
nitrones
(3),
dioxirane the
and
biochemistry
(4-7).
of
is
modified DNA
by with
predominantly
to
map
DNA the
is
for
capable
is
relation
to
to
information
the
dimethyldioxirane
produces
position
sites
of
piperidine of
guanine causes
guanine
Dimethyldioxirane foxone) to at room temperature,
DNA
has of
this
bases. selective in
AND
sequencing
Although studied
radiation
and
its we
unknown
(81,
components
show
that
are treatment
structure) heating
cleavage
of which
the can
be
used
experiments.
METHODS
was prepared by adding potassium a mixture of acetone and aqueous as described by Nurray and
87
into
dimethyl-
ionising
Subsequent chain
into
nitro
volatile. extensively
DNA
(of
to imines
both
communication, lesions
atom reaction
amines
highly
how
oxygen
converts
been
effects
residues
MTRRTALS klaterials: monoperoxysulphate bicarbonate,
are
concerning In
the
and because
acetone
extremely
neutral
oxidise (2)
simplified
damage
an
mild.
cleanly
&oxides
and
inserting
under
example, amine
versatile,
of
groups
dimethyldioxirane.
with
a new,
by-product
in no
at
modified
can,
oxidative
currently
is
functional
reaction
particularly
there
of
its
C
of
dimethyldioxirane
was treated
dG and dT are converted
S-methyldeoxycytidine
reactivity
22OC for 1 h following which was estimated by HPLC analysis.
The HPLC profiles
indicate
relative
deoryribonucleoside
dimethyldioxirane deoxyribonucleoside the
the
products
been determined
increase
in
derived
from
but
is under
Vol.
BIOCHEMICAL
169, No. 1, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(bl
1.5
$
dc
10
2
0.5
m5dC
(Cl
Cd1
_A-
I 0
cA 20 0 Ehtion Tie (mid
b
10
20
Fir. 1. HPLC elution profiles (at 254 nm) of 2'-deoxyrihonucleosides with so equimolsr quantity of dimethyldioxirene: (a) deoxyguanosine (b) thy&dine (dT). (c) deoxycytidiae (dC). (d) S-methyldeoxycytidiae Details of the HPLC system sre given under Hethods.
The modification explored
by gel
of
nucleotide
sequencing
residues
experiments
in DNA by dimethyldioxirane 32 P-end-labelled synthetic
witb
An approximately
oligonucleotides containing 34 or 45 bases. of carrier DNA was mixed with the labelled with mol
dimetbyldioxirane. per mol
allowed
of
An amount
total
to react
nucleotide
for
2 min
residual
dimetbyldioxirane.
presence
of frank
gels
with
reactions.
reference
chain
of
before
to fragments
or base produced
in the
was added beat3ng
Tbe oligonucleotides breaks
before
dimetbyldioxirane,
labile
to
each
to SS°C to were
cleavage
by conventional
was
lo-fold
oligonucleotide
phosphate,
at 20°C
treated (dC), (a'dc).
tben sites
excess treating
range
sample
0.3
it to
and
decompose analysed
for
the
on DM~ sequencing chemical
sequencing
3
Vol.
169, No. 1, 1990
As
evidenced
in
dimethyldioxirane scission
Fig.
under of
DNA
with
the
fragments
the
BIOCHEMICAL
AND BIOPHYSICAL
2
and
(lanes
the
10
conditions
caused
produced
standard
guanine-specific
obtained
far
both
1
did
backbone. selective
having
the
single
C A ?CdG 2 3 4 5
at
same mobility
the
not
stranded
DNA
cause
appreciable
of
the
results (Fig.
treated
C;uanine
generated
Similar 34-mer
to
heating
sites
as those
procedure.
and double
of
However,
cleavage
Uaxam-Gilbert
the
exposure
employed
deoxyrlbose-phosphate
piperidine
151,
RESEARCH COMMUNICATIONS
bases,
by the were
2, lanes
7 to
G 6
7 6
9
10
11 12
13
14
15 16
FiK. 2. Autorsdiogropb of [5S-32Pl end-lsbelled single and double stranded 34-mer treated with dimetbyldiozirane and plperidine. Lanes 1 sod 2: uncut 34-mer before and after besting witb piperfdine; lanes 3 to 6 and 16: products of HaxamGilbert sequencing reactions (13) ss indicated; lanes 7 to 9: single stranded 34-mer (20 plol) plus carrier DNA (4 ug) reacted witb 7, 14 and 22 nmol of dimetbyldioxirane respectively then heated with piperidine; lanes 11 (20 ~1) plus carrier iMA (4 vg) reacted with to 14: double stranded 34-mer 7, 14, 22 end 36 nmol of dimetbyldiosirane respectively then bested with piperidine; lanes 10 and 15: same as lanes 9 and 14 but without plperidine treatment.
91
9
Vol.
169,
No.
1, 1990
and 11 to 14) dioxirane At the
with
a single
On treatment material strong
the
the
(gel 3'-end preference
not
that
4S-mer
labelled for
(lanes chain.
labelled
Identically
molecule
1
are at
c f
with
RESEARCH
concentration of
C
the
4
G
shown sites
G
234567
COMMUNICATIONS
of
cleavage
dimethylwith
the
9 and 14)
there
was a bias
cleavage
sites
were
Experiments
were
32P at either
and piperidine,
behaved
cleavage
the
multiple
dimethyldioxirane shown)
BIOPHYSICAL
same extent
oligonucleotide
stranded with
the used
indicating
into
twice
to effect
concentrations
framnts
AND
approximately
was required
introduced
for
though
hi&hest
shorter
BIOCHEMICAL
to the in Fig.
also
S.-end
34-mer
(Fig.
3 and likewise
of guanine
bases.
towards
being
its
the
duplex.
S'-
conducted or 3'-end.
labelled 2).
Results
illustrate The even
8 9101? G A*,G CGT G AC TGG C G ET GT T G
I.
ii G G
,
C T G ,.,
C A C A G T A G G
Fix. 3. Autoradiograph Of single stranded /3'-32Pl end-labelled 45-mer treated with dimetbyldioxirame and piperidine. Lanes 1 and 2: uncut 45-r lanes 3 to 6 and 11: products of before and after beating with piperidiee; Waxsm-Cilbert sequencing reactions as indicated; leaes 7 to 10, 45-mer (20 DNA (4 ~6) reacted with 4, 7. 14 and 22 nmol of pool) plus carrier dimethyldioxirane respectively then heated with piperidine.
92
a
Vol.
169, No. 1, 1990
density
of
context
has
the
bands
to
those
are
modified
that,
in
it
is
to
some
on The
sulphate
(17).
offers
several entails
the
or to
solutions.
totally
reagent
(9)
has
which
a half-life Other
DNA
at
acetone
is
stable 48
methods
guanine
photolysis
that
addition
can
be
laser
found
for
have
chemical DNA
by
at
proteins
and
potential
chemical
uncharged
248
of
has small
Acknowledgments: Dr R.R. RcGuckin
molecule
no
necessity
the
sample
by
of
dimethyl
dimethyldioxirane fewer for
is
ethanol
is
is
because
and
oxygen,
noteworthy
prepared stored
waste
avoided
acetone It
without
supernatant
agent
volatile
reagents
achieved
radioactive
when
reported
to
treatment blue
nm
at
and
that
and
inexpensive
-7OOC;
at
the
To
sequence with
bases the
with
diethylpyrocarbonate
2S°C,
it
these
secondary
structure
in
DNA-protein
or
was
to
be
properties readily
and
93
this
of
the
by SERC preparation
not
used
as
a
recognition
of
towards respect.
As
local
complexes,
being in
the
but
reactivity in
DNA-ligand of
soluble
supported with the
DNA widely
its
useful
or intensity have
the
upon
DNA
is
of
investigating
Depending prove
high
approaches
is
of (la),
(20)
with
determination
for
cleavage
methylamine
date,
also
which
predominant
excitation
may
desirable
This work for assistance
to
dimethylsulphate,
reagent
of
lead
(19),
and
(22).
molecules.
probe guanine
months
(21).
cosxnon
other
dimethyldioxirane
use
is
a readily
and labelled
a
requires
heating.
methylene
‘footprinting’
proteins,
dimethyldioxirane
many
a simple
DNA
of
to
gentle
its
in in
or
chain
chemical
a quenching
is
been
of
application
probe
environment
of
dication radiation
routine
to
terminally provides
the the
of
decomposed
include
diethylpyrocarbonate,
methods,
generation
by
it
time-consuming,
solution
dimethyldiazaperipyrenium pulsed
such.
of
There
or
in
guanine--specific
less
the
presence
the
appear rise
h.
residues in
thymine,
would give
affords
bases
As
recovery
simply
in
of
than
deoxyribo-
it
not
clearly
guanine
these
is
salt
removed,
dimethyldioxirane
DNA
especially
so, do
modification
with
added
dimethyldioxirane be
above
steps.
Furthermore,
excess
If
sequence.
quantitative
of
DNA,
be
of
individual
in
By
should
fragments
the
lesions
conventional
It
that
it
longer of
of
utilise
operational
so
complications
thus
the
comparison
fewer
sequence
guanines.
concentration for
resultant
unknown
advantages.
precipitation
base
individual
nucleobases
positions
which
In
that
of
described
the
known
procedures
suggests
direthyldioxirane. the
RESEARCH COMMUNICATIONS
piperidine.
alternative
sequencing
gel
reactivity
other
by
mapping
of
practicable
the
protocol
for
fragments
of that
with
the reactivity
conditions
guanine,
experimental
method
view
degree
beating
on
dimethyldioxirane
probable
to
AND BIOPHYSICAL
the
cleavage
In
contrast
scission
the
the
here.
nucleosides,
lanes on
of
optimise
studied
and
the
influence
adjustment
feasible
DNA
in
little
appropriate
rapid
BIOCHEMICAL
a highly aqueous
grant of
reactive, mediaatneutralpH
GR/B 48060. dimethyldioxirane.
We
thank
a
Vol.
169,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERRNCES 1. 2. 3. 4.
5. 6. 7. a. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Adam, W.. Curci, R., and Bdwards. J.O. (1989) Act. Chem. Res. 22, 205-211. Murray, R-W. (1989) Chem. Rev. 89, 1187-1201. Boyd. D.R.. Coulter, P.B., HcGuckin, R.R., Sharma, N.D., Jennings, U.B., and Wilson, V.I. (1990) J. Chem. Sot. Perkin Trans. I, in press, Ames, B.N., Saul, R.L., Schwiers, 1.. Adelman. R., and Cathcart, R. (1985) In Molecular Biology of Aging: Gene Stability and Gene Expression (R.S. Sohal, L.S. Birnbaum and R.G. Cutler, Ids.), pp. 137-144. Raven, New York. Sies, H. (1986) Angeu. Cbem. Int. Rd. Engl. 25, 1058-1071. Storx. G., Christman, R.F., Sles, H., and Ames, B.N. (1987) Proc. Natl. Acad. Sci. USA 84, 8917-8921. Teebor, G.W.. Boorstein, R.J., and Cadet, J. (1988) Int. J. Radiat. Biol. 54, 131-150. von Sonntag, C. (1987) The Chemical Basis of Radiation Biology, Taylor 6 Francis, London. Murray, R.V.. and Jeyaraman, R. (1985) J. Org. Chem. 50. 2847-2853. Adam. W., Chan, Y-Y., Cremer, D., Gauss, J., Scheutzow, D., and Schindler, H. (1987) J. Org. Chem. 52, 2800-2803. Haniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory. Smith, H.O. (1980) Methods 8nxymol. 65, 371-380. Raxam, A.H., and Gilbert, W. (1980) Methods Bntymol. 65. 499-560. Yousaf, S-1.. Carroll, A.R., and Clarke, B.E. (1984) Gene 27, 309-313. Kumar, S., and Davies. R.J.H. 11987) Photochem. Photobiol. 45, 571-579. Banasxuk, A.M., Deugau, K.V., Sherwood, J.. Hichalak, H., and Glick, B.R. (1983) Anal. Biochem. 128, 281-286. Ambrose, B.J.B., and Pless, R.C. (1987) Methods Rneymol. 152. 522-538. Krayev, A-S. (1981) FBBS Lett. 130, 19-22. Friedmann, T., and Brown, D.#f. (1978) Nucleic Acids Res. 5, 615-622. Saito, I., Sugiyama, H.. Hatsuura, T., Ueda, K., and Komano, T. (1984) Nucleic Acids Res. 12, 2879-2885. Slama-Schwok, A., Jarwinski, J., Bere, A., Uontenay-Garestier, T., Rougee, H., He'lene, C., and Lehn, J.-M. (1989) Biochemistry 28, 3227-3234. Blau, U., Croke, D-T., Kelly, J.R., McConnell, D.J., OhUigin, C., and Van der Putten. U.J.R. (1987) J. Chem. Sot. Chem. Cornnun. 751-752,
94