High
resolution
footprinting
of
EcoRI
and,
distamycin
with
Rh(phi)2(bpy)3+,
a
new
photofootprinting
reagent
Kiyoshi
Uchida+,
Anna
Marie
Pyle§,
Takashi
MoriiO
and
Jacqueline
K.Barton*O
Department
of
Chemistry,
Columbia
University,
New
York,
NY
10027,
USA
Received
September
18,
1989;
Revised
and
Accepted
November
8,
1989
ABSTRACT
The
complex
bis(phenanthrenequinone
diimine)(bipyridyl)rhodium(lI),
Rh(phi)2(bpy)3
,
cleaves
DNA
efficiently
in
a
sequence-neutral
fashion
upon
photoactivation
so
as
to
provide
a
novel,
high
resolution,
chemical
photofootprinting
reagent.
Photofootprinting
of
two
crystallographically
characterized
DNA-binding
agents,
distamycin,
a
small
natural
product
which
binds
to
DNA
in
the
minor
groove,
and
the
endonuclease
EcoRI,
which
binds
in
the
major
groove,
gave
respectively
a
5
-7
base
pair
footprint
for
the
drug
at
its
A6
binding
site
and
a
10-12
base
pair
footprint
for
the
enzyme
centered
at
its
recognition
site
(5'-GAATTC-3').
Both
footprints
agree
closely
with
the
crystallographic
results.
The
photocleavage
reaction
can
be performed
using
either
a
high
intensity
lamp
or,
conveniently,
a simple
transilluminator
box,
and
the
photoreaction
is
not
inhibited
by
moderate
concentrations
of
reagents
which
are
sometimes
required
for
examining
interactions
of
molecules
with
DNA.
When
compared
with
other
popular
footprinting
agents,
the
rhodium
complex
shows
a
number
of
distinct
advantages:
sequence-neutrality,
high
resolution,
ability
to
footprint
major
as
well
as
minor
groove-binding
ligands,
applicability
in
the
presence
of
additives
such
as
Mg2+
or
glycerol,
ease
of
handling,
and
a
sharply
footprinted
pattern.
Light
activated
footprinting
reactions
furthermore
offer
the
possibility
of
examining
DNA-binding
interactions
with
time
resolution
and
within
the
cell.
INTRODUCTION
The
technique
of
DNA
footprinting
has
been
used
extensively
to
observe
the
site-specific
binding
of
proteins,
peptides,
and
drugs
to
DNA
(1-4).
Using
a
variety
of
chemical
and
enzymatic
footprinting
agents,
it
has
been
possible
to
determine
the
relative
binding
site
sizes
and
locations
for
a
large
variety
of
DNA-binding
proteins.
Subtle
molecular
interactions
between
DNA
and
transcription
factors,
repressors,
and
other
constituents
of
the
transcriptional
apparatus
are
being
actively
explored
using
DNA
footprinting
(5).
Given
the
power
of
this
methodology,
extensive
efforts
to
find
new,
high
resolution
reagents
for
footprinting
are
underway.
The
most
popular
and
the
original
footprinting
reagent
is
DNase
I,
a
large
nuclease
which
cleaves
with
some
preference
for
sequences
of
intermediate
groove
widths
(6).
This
level
of
sequence-neutrality
is
sufficient
for
determining
the
binding
sites
of
large
DNA
binding
proteins.
However,
small
peptides
or
proteins
which
bind
to
sequences
insensitive
to
attack
by
DNase
I
can
be
difficult
to
visualize.
Many
synthetic
footprinting
reagents
such
as
Cu(phen)2+
and
metalloporphyrins
share
this
inherent
problem
(7,8).
In
order
to
examine
DNA
binding
interactions
at
higher
resolution,
many
workers
have
turned
to
MPE-Fe(LI),
the
first
synthetic
footprinting
reagent
and
a
remarkable
tool
with
respect
to
its
sequence
neutrality.
An
intercalating
dye
tethered
to
an
Fe(EDTA)2-
moiety,
MPE-Fe(fl)
has
been
useful
in
elucidating
the
binding
sites
and
sizes
of
small
natural
products
as
well
as
proteins
(9-12).
More
recently,
the
clever
Nucleic
Acids
Research
Volume
17
Number
24
1989
1
0259
(D
IRL
Press
Nucleic
Acids
Research
application
to
footprinting
of
Fe(EDTA)2-
itself,
without
a
tethered
DNA-binding
moiety,
has
been
made
(3,13).
Both
for
Fe(EDTA)2-
and
MPE-Fe(II),
cleavage
results
from
the
diffusion
to
the
DNA
helix
of
hydroxyl
radicals,
generated
in
the
presence
of
peroxide
and
a
reducing
agent
(3,9,13).
Fe(EDTA)2-,
which
as
an
anionic
species
generates
the
radicals
far
from
the
DNA
surface,
also
shows
a
high
level
of
sequence
neutrality,
but
since
the
radical
generator
does
not
bind
to
the
DNA,
high
concentrations
of
reagents
are
required.
Additionally
a
drawback
with
respect
to
both
complexes
has
been
their
sensitivity
to
the
presence
of
various
common
additives,
such
as
glycerol
or
Mg>,
and
their
requirements
for
high
concentrations
of
chemical
activators.
Some
techniques
of
photofootprinting
have
also
been
developed.
An
advantage
of
this
method
is
that
the
activation
of
the
DNA
cleavage
reaction
is
controlled
by
light,
eliminating
the
need
for
adding
other
chemicals
to
the
protein
solution.
These
techniques
include
ultraviolet
footprinting
(14),
photofootprinting
in
the
presence
of
uranyl
salts
(15),
and
that
in
the
presence
of
psoralen
or
its
analogs
(16).
Ultraviolet
light
photofootprinting
has
been
applied
in
vivo
as
well
as
in
vitro.
This
technique
requires
chemical
treatment
after
the
photocleavage
reaction,
however,
and
the
results
obtained
are
sometimes
complicated
because
of
differential
enhancements
due
to
DNA-protein
crosslinking.
The
second
technique,
using
uranyl
salts,
shows
excellent
sequence
neutrality
but
high
concentrations
of
the
uranyl
salts
are
required,
which
may
perturb
the
protein
interactions
with
the
DNA
or
the
DNA
structure
itself.
The
cleavage
pattern
with
psoralen
shows
sequence
preferences
(16).
Owing
to
these
difficulties,
despite
the
inherent
advantages
of
light
activation,
these
photofootprinting
reagents
have
not
been
widely
applied.
Recently,
coordinatively
saturated
phenanthrenequinone
diimine
complexes
of
rhodium(III)
have
been
reported
to
cleave
DNA
efficiently
upon
irradiation
with
long-
wavelength
ultraviolet
light
(17).
Photocleavage
with
Rh(phi)2(bpy)3
+
yields
sharp,
sequence-neutral
cleavage
of
linear
DNA
fragments.
The
addition
of
free
metal
ions,
chelators,
or
oxidizing
agents
is
not
necessary
in
this
system
because
the
Rh(phi)2(bpy)3+
complex
is
fully
assembled
and
requires
activation
only
by
light.
The
structure
of
Rh(phi)2(bpy)3
+
is
schematically
illustrated
below.
Here
we
report
the
development
of
Rh(phi)2(bpy)3+
as
a
high-resolution
photofootprinting
reagent
which
successfully
maps
the
precise
binding
locations
and
site
sizes
of
distamycin-A
and
the
restriction
endonuclease
EcoRI.
This
is
the
first
report
of
a
footprint
for
EcoRI
by
a
synthetic
footprinting
reagent
and
the
first
example
of
a
footprint
which
reflects
the
proper
site
size
(18).
Rh(phi)2(bpy)3
+
is
able
to
detect
both
EcoRI
bound
in
the
major
10260
Nucleic
Acids
Research
groove
of
DNA
and
the
small
peptide
distamycin,
bound
in
the
minor
groove.
Footprinting
with
Rh(phi)2(bpy)3+
is
not
inhibited
by
moderate
concentrations
of
salts,
EDTA,
glycerol,
or
reducing
agents,
many
of
which
are
sometimes
necessary
to
obtain
a
native
interaction
of
DNA
with
protein.
The
complex
is
easy
to
handle,
being
very
stable
under
ordinary
conditions
and
requiring
no
complicated
reaction
conditions.
Activation
with
low
energy
light
from
a
lamp
or
transilluminator
permits
excellent
experimental
control
over
Rh(phi)2(bpy)3+
footprinting,
an
absolute
requirement
for
application
in
vivo.
EXPERIMENTAL
Materials
[Rh(phi)2(bpy)]C13
was
synthesized
as
described
previously
(17)
and
MPE
was
kindly
provided
by
Prof.
P.B.
Dervan.
Distamycin
A,
alkaline
phosphatase,
bovine
serum
albumin,
and
2,9-dimethyl-1,10-phenanthroline
were
obtained
from
Sigma;
lyophilized
EcoR
I,
HindU,
Pvu
II,
terminal
deoxynucleotidyl
transferase
and
T4
polynucleotide
kinase
from
BRL;
DNase
I
from
Boehringer
Mannheim;
dithiothreitol,
3-mercaptopropionic
acid,
and
1,
10-phenanthroline
from
Aldrich;
and
at-32P-3'-dATP
and
-y-32P-ATP
from
NEN.
Tris-
acetate
buffer
for
irradiations
with
Rh(phi)2(bpy)3+
contained
the
following
unless
specified
otherwise:
50
mM
tris,
20
mM
sodium
acetate,
18
mM
NaCl,
pH
7.
Loading
buffer
for
electrophoresis
on
a
denaturing
polyacrylamide
gel
contained
80
%
formamide,
50
mM
tris
borate
buffer
(pH
8),
0.1
%
xylene
cyanol,
0.1
%
bromophenol
blue,
0.1
N
NaOH,
and
1
mM
EDTA.
Polyacrylamide
Gel-Mix
8
from
BRL
was
used
for
pouring
denaturing
polyacrylamide
gels.
Preparation
of
Labeled
DNA
fragment:
Plasmid
pJT18-T6
was
obtained
by
inserting
an
18
base
pair
oligonucleotide
(5'-ATATGCAAAAAAGCATAT-3')
by
blunt-end
ligation
into
the
Sma
I
site
of
plasmid
pUC
18
and
amplifying
in
E.
coli
(JM109)
cells
by
culture.
The
plasmid
was
isolated
according
to
methods
published
in
the
literature
(19).
The
plasmid
thus
obtained
was
first
digested
with
restriction
enzyme
Hind
Ill
and
purified
by
ethanol
precipitation.
To
obtain
the
3'
end-labeled
fragment,
32P-ax-3'-dATP
and
terminal
deoxynucleotidyl
transferase
were
reacted
with
linearized
DNA.
This
was
followed
by
a
second
digestion
with
Pvu
H,
yielding
a
245 bp
DNA
fragment
containing
the
insert
which
was
purified
on
a
5%
polyacrylamide
gel
and
isolated
by
subsequent
electrophoretic
elution.
The
DNA
fragment
was
then
ethanol
precipitated
in
the
presence
of
sodium
acetate
and
washed
with
EtOH.
After
lyophilization,
the
DNA
fragment
was
dissolved
in
1/10
dilution
tris-acetate
buffer
containing
0.
1mM
EDTA
for
use
or
storage
at
4°C.
To
obtain
the
5'
end-labeled
fragment,
linearized
DNA
was
treated
with
alkaline
phosphatase
and
labeled
with
'y-32P-ATP
in
the
presence
of
polynucleotide
kinase.
Fragment
isolation
and
purification
were
performed
as
described
for
the
3'
end-labeled
fragment.
If
32P-labeled
samples
were
stored
for
appreciable
time,
some
background
damage
was
evident
in
the
autoradiograms
of
the
unreacted
fragment.
DNA
Cleavage
by
Rh(Phi)2(bpy)3+
A
typical
procedure
for
carrying
out
DNA
photocleavage
with
Rh(phi)2(bpy)3+
in
the
absence
of
any
other
DNA-binding
molecule
was
as
follows:
50
tdI
of
a
reaction
mixture
containing
32P-end
labeled
DNA
fragment
(5
uM
bp
and
approx
30,000
cpm,
concentration
adjusted
with
calf
thymus
carrier
DNA),
and
5
/tM
[Rh(phi)2(bpy)]Cl3
(E350
=
23,600
M-
'cm-')
in
tris-acetate
buffer
was
added
to
a
1.7
ml
siliconized
polypropylene
tube.
Open
reaction
tubes
were
fixed
such
that
the
reaction
mixture
was
10261