From the
The interaction between the Cu(I)
Organisms have evolved homeostatic mechanisms to respond to
external conditions such as heat shock, starvation, or toxic levels of
metal ions. In yeast, elevated internal copper concentrations result in
the repression of biosynthesis of the copper transporter,
CTR1, and activation of biosynthesis of metallothionein
(1, 2, 3, 4) . Metallothionein
sequesters Cu(I) ions, thereby lowering the potentially toxic free
Cu(I) concentration
(1, 5) . In yeast, metallothionein
biosynthesis is regulated at the transcription level by a trans-acting
factor, called ACE1
(2, 6) or CUP2
(7, 8) . ACE1 is constitutively expressed and exists in
an inactive basal state. Upon binding Cu(I) within a polycopper
cluster, the ACE1 molecule is activated for DNA binding
(2, 9, 10) . The Cu(I) cluster forms in the
NH
The Cu(I)
The
NH
CuACE1 is known to preferentially bind the left half-site as opposed
to the right half-site of the UAS
In the present work we assessed the
base-specific interactions between CuACE1 and a chemically modified
CuACE1 with the left half-site of the UAS
Incubations of modified
CuACE1 with the CUP1 UAS
In initial studies with
unmodified CuACE1 the top strand proximal region and the A-rich middle
site showed continuous interferences between G
The missing contact interference experiments
also resulted in a more accurate picture of the minimal DNA binding
site than the previous DNase I footprint assays
(20, 23) . Modified CuACE1 showed in the missing contact
interference assay a decrease in interference between the
In contrast, the ability of the modified CuACE1
to bind to DNA was unchanged by left
Right
Hydroxyl radical protection of the
sugar-phosphate backbone was reported at the same region
(23) .
The lack of observed base-specific contacts within this region
suggested protein-mediated sugar-phosphate backbone interactions toward
the dyad symmetry center. The weak missing contact interference found
only on the bottom strand at positions
Studies of the modified CuACE1 and right
Previous experiments showed that Cu(I) binding
protects ACE1 against degradation by low concentrations of trypsin
(2, 11) . It was also found that DNA binding enhanced
the protection and resulted in a highly trypsin-resistant protein core
with a different electrophoretic mobility
(11) . This allowed us
to use trypsin to probe the ability of CuACE1 to bind to the truncated
UAS
Based on
the results of the mobility shift assays, protein protection, missing
contact interference experiments, and in vivo studies
(2) , we deduced that the minimal CuACE1 binding site on the
CUP1 UAS
The interaction of CuACE1 and CUP1 UAS
The first aspect of the model is the
combination of major groove and minor groove contacts. Methylation
interference experiments provided the main evidence for the major
groove interactions at positions G
The second
feature of the model is that CuACE1 binding to the proximal site is
stronger than binding to the distal site. This was demonstrated by
studying a series of deletion and substitution mutants that
independently altered each site. The experiment showed that mutation or
deletion of the middle or proximal binding site resulted in the
complete loss of binding. By contrast, deletion or mutation of the
distal region resulted in a reduced but still detectable binding.
Based on the DNA mutation analysis, we proposed the minimal DNA
binding sequence for CuACE1 to be 5`-GCGTCTTTTCCGCTGA-3`. This minimal
DNA sequence was shown to bind CuACE1. Previously, Thiele
(34) used evolutionary comparisons to propose a different,
shorter consensus binding site for ACE1 and its homologues. However,
the functionality of the proposed sequence was not tested.
The third
feature of the model is that distinct regions of CuACE1 are involved in
binding to the different regions of the UAS
Our biochemical experiments are
supported by several previous results. The bases within the UAS
Our experiments suggest that the CuACE1
DNA-binding domain consists of two subdomains that contact distinct
regions of the double helix. Although studies carried out presently
utilized ACE1 reconstituted with 6 mol eq of Cu(I), ACE1 isolated from
Escherichia coli after expression of the ACE1 gene encoding
the NH
ACE1 (CuACE1)
transcription factor and its DNA binding site in the yeast
metallothionein gene was studied by systematically altering the DNA
sequence through base substitution, modification, and deletions as well
as by altering the protein structure through chemical modification. We
show here that CuACE1 is comprised of two distinct domains that contact
DNA through minor groove interactions located between two major groove
interaction sites. The minor groove interactions are shown to be
critical for formation of a stable CuACE1
DNA complex. The
NH
-terminal segment of ACE1 is shown to contact the 5`-most
distal major groove site.
-terminal half of ACE1, which is also the domain
responsible for DNA binding
(2) . Previous experiments with the
ACE1 mutant proteins revealed that the DNA-binding residues of the ACE1
amino-terminal domain (1-122) are interdigitated with the
cysteine residues involved in copper cluster formation
(11) .
ACE1 complex
(2, 9, 10) shares
structural similarities with the molecule (metallothionein) whose
synthesis ACE1 regulates
(12, 13, 14) . Both
ACE1 and metallothionein exhibit an abundance of Cys- X-Cys and
Cys- X- X-Cys sequence motifs and a paucity of
hydrophobic residues. The two molecules form polycopper clusters with a
number of similar properties
(14) .
-terminal half of ACE1 is homologous to the
copper-responsive transcription factor in Candida glabrata,
AMT1
(15, 16, 17) . A second Saccharomyces
cerevisiae transcription factor, MAC1, exhibits limited
sequence homology to ACE1 and AMT1
(18) . Characterization
of the NH
-terminal domains of ACE1 and AMT1 expressed in
bacteria revealed that both factors contain a tetracopper cluster and a
single Zn(II) ion
(19) . The tetracopper centers in ACE1 and
AMT1 form in an all-or-nothing manner that stabilizes a conformation in
each molecule capable of specific interaction with upstream activator
sequences (UAS)
(
) in the 5`-sequences of
metallothionein genes and the AMT1 gene itself
(19, 20) . As a consequence, CuACE1 and
Cu(I)
AMT1 induce metallothionein expression in
vivo. CuACE1 also regulates the Cu,Zn-superoxide dismutase gene,
which contains a related regulatory sequence
(21, 22) .
palindrome in the
CUP1 metallothionein gene
(11) . Each half-site
consists of nearly 20 nucleotides and thus spans two turns of the DNA
double helix
(23, 24) . Previous footprinting studies
suggested that CuACE1 crosses a minor groove to make major groove
contacts at each end of the half-site
(23) . Studies of an
ace1-1 mutant protein with a C11Y mutation indicated that DNA
interaction was restricted to the innermost portion of the UAS
half-site
(23) .
sequence. In
addition to the known major groove contacts at each end of the
half-site, we demonstrate that CuACE1 makes specific minor groove
contacts that are important for stabilization of the CuACE1
DNA
complex. The chemically modified CuACE1 was found to be incapable of
binding to the distal major groove site of the UAS
half-site, whereas the minor and proximal major groove
interactions remained unchanged.
Preparation of CuACE1
The bacterial expression
and purification of CuACE1(1-122) have been described
(9) . The CuACE1 samples used in the present studies were
prepared by in vitro reconstitution from apoACE1. ApoACE1 was
prepared by removal of Cu(I) from ACE1 in two stages. Initially, the
protein was incubated with 20 mM KCN at pH 6.5 prior to
ultrafiltration on an Amicon PM-10 membrane. The retained protein was
acidified to pH 0.2 with HCl and gel-filtered on Sephadex G-25
equilibrated in 25 mM HCl. The metal-free ACE1 was recovered
and concentrated by lyophilization. The dried material was dissolved in
2 ml of 0.2 M Tris-HCl, pH 8.3, containing 6 M
guanidine HCl, 150 mM dithiothreitol, and 10 mM EDTA
and incubated at 65 °C for 1 h to achieve reduction of any oxidized
cysteinyl residues. The reduced protein was recovered by gel filtration
on a Sephadex G-25 column equilibrated in 25 mM HCl. The ACE1
protein concentration was determined by amino acid analysis. The extent
of reduction was assessed by quantitation of thiols with
dithiodipyridine. Only ACE1 preparations that were >97% reduced were
used in subsequent experiments. Cu(I) reconstitution of apoACE1 was
performed anaerobically as described previously
(9) . The final
Cu(I)/ACE1 stoichiometry was verified by copper analysis (by atomic
absorption spectroscopy) and protein quantitation (by amino acid
analysis).
Modification of CuACE1 with Iodoacetic
Acid
Reconstituted CuACE1 was modified under anaerobic
conditions by incubating the protein with a 5-fold molar excess of
iodoacetate with respect to the cysteine concentration in ACE1. After
the incubation at 37 °C for 3 h, the reaction was quenched by the
addition of -mercaptoethanol to 5 mM. The positions of
the modified carboxymethylcysteinyl residues were determined by Edman
sequence analysis.
Protein Characterization
Amino acid analysis was
performed to quantify ACE1 protein levels after hydrolysis of samples
in 5.7 M HCl at 110 °C for 24 h on a Beckman model 6300
analyzer. Edman degradation was carried out on an Applied Biosystems
model 475 sequencer with on-line high performance liquid chromatography
analysis of phenylthiohydantoin-derivatives. Metal analysis was made by
atomic absorption spectroscopy on a Perkin-Elmer model 305A instrument.
Methylation Interference Assay
The interference
assays were performed on synthetic oligonucleotide probes (see
Fig. 1
). The oligonucleotides were labeled at the 5`-end with
[-
P]ATP and polynucleotide kinase
(25) . Annealing was performed by heating the mixture of the
complementary oligonucleotides to 65 °C for 35 min and cooling to
room temperature for 20 min in the labeling buffer. Three pmol of probe
(7
10
cpm) was modified with 1.5 µl of dimethyl
sulfate in a final volume of 100 µl in the presence of 50
mM sodium cacodylate, pH 7.0, 1 mM EDTA at 23 °C
for 15 min. The reaction was quenched by the addition of 25 µl of
dimethyl sulfate stop solution (DuPont), and the DNA was precipitated
with 400 µl of ethanol in the presence of 3 µg of glycogen.
After centrifugation the DNA pellet was washed with 1 ml of 70%
ethanol, dried under vacuum, and resuspended in binding buffer (12%
glycerol, 12 mM HEPES-NaOH, pH 7.9, 60 mM KCl, 5
mM MgCl
, 4 mM Tris-HCl, pH 7.9, 0.6
mM EDTA, and 0.6 mM dithiothreitol)
(25) in a
total volume of 100 µl. The binding reaction contained 500,000 cpm
of probe and 25 ng of the nonmodified or 250 ng of the modified protein
(determined by a titration carried out before the actual experiment) in
the binding buffer in a total volume of 50 µl. After an incubation
on ice for 10 min, the mixtures were loaded onto an 8% polyacrylamide
gel (acrylamide/bisacrylamide; 40:1) and electrophoresed at 4 watts
constant power for 3 h at 23 °C in 0.5
TBE buffer (45mM Tris-HCl, 45mM boric acid, 1mM EDTA (pH 8.0)).
Wet gels were autoradiographed, and subsequently the bound and free
fractions were excised and eluted by soaking overnight at 37 °C in
500 µl of elution buffer (0.5 M NH
OAc, 0.1%
SDS, 2 mM EDTA, 10% methanol). The samples were extracted with
phenol/chloroform and precipitated with ethanol twice in the presence
of 3 µg of glycogen. Pellets were washed with 70% ethanol, dried,
and resuspended in 100 µl of 10% (v/v) piperidine. Samples were
incubated at 90 °C for 30 min to cleave the probe at the methylated
guanine residues
(26) . Samples were lyophilized, dissolved in
20 µl of H
O, lyophilized again, and then resuspended in
H
O and adjusted to 1000 cpm/µl. Samples of 3000 cpm
were mixed with 2 µl of 90% formamide and dyes, heated at 90 °C
for 3 min, and loaded onto a 20% polyacrylamide, 7 M urea, TBE
sequencing gel. The samples were electrophoresed at 33 watts constant
power for 2 h. The gel was autoradiographed at
80 °C
overnight with Kodak X-Omat AR film.
Figure 1:
Sequence of the
synthetic oligonucleotides used in the
experiments.
Missing Contact Footprints
The experiments were
carried out as described by Brunelle and Schleif
(27) . The
oligonucleotide probes (see Fig. 1) were premodified for the A
+ G and C + T reactions as described
(26) . For the A
+ G reaction, 2 µl (1.5 pmol) of probe (3.5 10
cpm) was mixed with 4 µl of 1 M piperidine formate
in the presence of 4 µg of sonicated salmon sperm DNA in a total
volume of 20 µl. The reaction mixture was incubated at 37 °C
for 15 min and then quenched by ethanol precipitation twice. The
pellets were washed with 95% ethanol, dried, and resuspended in 30
µl of binding buffer. For the C + T reaction, 30 µl of 95%
hydrazine was added to 2 µl (1.5 pmol) of probe (3.5
10
cpm) in the presence of 4 µg of sonicated salmon
sperm DNA in a total volume of 50 µl. The mixture was incubated at
23 °C for 7 min, and then the reaction was quenched by adding 240
µl of hydrazine stop solution (DuPont), precipitated twice, washed
as described above, dried, and dissolved in 30 µl of binding
buffer. The binding reaction, separation of the bound and free probes,
cleavage and fragment separation on a sequencing gel, and
autoradiography were carried out as described in the methylation
interference experiment.
Mobility Shift Assay
For binding reactions 6 ng of
CuACE1 protein or 6 ng of modified CuACE1 protein was mixed with 30
fmol of 5`-end-labeled probe in the presence of 0.2 mg/ml
poly(dIdC) in binding buffer in a total volume of 10 µl. The
reaction mixture was incubated at 0 °C for 10 min and then loaded
onto an 8% polyacrylamide gel (acrylamide/bisacrylamide, 40:1) in 0.5
TBE buffer, and the complex was separated from the free probe
by electrophoresis at room temperature and 4 watts constant power for 3
h. The gel was fixed in 10% acetic acid, transferred to 3 M
Whatman paper, dried, and exposed to Kodak X-Omat AR film. The bands
were also analyzed on the Radioanalytic Imaging System from AMBIS. The
probes are listed in Fig. 1.
Reverse Footprinting
S-Labeled
ACE1(1-122) was produced by in vitro translation in a
wheat germ system (Promega). Proteolysis assays contained 10
mM HEPES, pH 8, 5% glycerol, 1 mg/ml carrier tRNA, 0.1 mg/ml
poly(dI
dC), and 1 µl of wheat germ extract in a total volume
of 10 µl
(11) . Then 46 µM Cu(I)-acetonitrile
and 10 pmol of truncated probes (see Fig. 6) were added, followed
by a 10-min incubation at 0 °C. The protection was tested by adding
1 µl of 0.5% trypsin, and the reaction mixture was incubated for 15
min at room temperature. The protected peptide then was analyzed on a
20% SDS gel.
Figure 6:
Reverse footprinting. CuACE1 was labeled
with S-cysteine by in vitro translation. The
protein extracts were incubated with left
right and right
left truncated probes of the UAS
left arm in the absence or
presence of 46 µM Cu(I)-acetonitrile followed by
incubation with trypsin and finally SDS-polyacrylamide gel
electrophoresis. A, protection by left
right truncated
probes of the UAS
left arm. The open circle indicates the position of the DNA-protected protein core.
B, protection by right
left truncated UAS
left arm probes.
CuACE1 Binds Specifically to the Minor Groove of the
UAS
In the binding of CuACE1 to the UAS Left Half-site between Two Adjacent Major Groove
Contact Sites
left half-site, it was previously shown that the region contacted
spans 20 base pairs from nucleotide 125 to 144
(23, 24) . To map specific DNA contacts within the left
half of UAS
we used methylation interference experiments to
test CuACE1 binding to guanine N
and adenine N
methylated nucleotides. Methylation of guanines at the N
position interferes with major groove contacts, whereas
methylation of adenine at N
position alters minor groove
interactions
(28) . Potential minor groove interferences were
observed on the bottom strand at positions A
,
A
, A
, A
,
and A
(Fig. 2 A; summarized in
Fig. 8
). In agreement with the findings of others
(2, 23) strong major groove interferences were observed within the
proximal portion of the UAS
bottom strand at position
G
and on the top strand at position
G
, and detectable interferences were observed at
position G
. On the distal major groove surface of
the top strand, methylation at G
and
G
interfered with CuACE1 binding
(Fig. 2 A; see Fig. 8).
Figure 2:
Methylation interference footprint of
nonmodified and modified CuACE1UASc left arm complexes. The
UAS
left arm was
P-labeled on either the top
or bottom strand and methylated with dimethyl sulfate. The probes were
incubated with CuACE1, and the bound and free fractions were separated
on a mobility shift gel, eluted, cleaved with piperidine, and analyzed
on a sequencing gel. G, G sequencing ladder; B, bound
fraction; F, free fraction. Panel A, CuACE1
with wild-type probe. The positions of the major groove interactions
are shown on the top strand at positions
128,
131,
140, and
142 and on the bottom strand at positions
129 and
130. The interferences between
134 and
137 indicate minor groove interactions. Panel B, CuACE1 with the CI-substituted probe. The methylation
interferences between positions
134 and
137 on the bottom
strand are no longer evident. Panel C, the alkylated
CuACE1 with the wild-type probe. The top strand interferences at
128 and
131 and the bottom strand interferences at
134 to
137 are still evident. However, the proximal top
strand interferences at
140 and
142 have
disappeared.
Figure 8:
Summary
of the specific interactions between UASc left arm and CuACE1 and the
proposed arrangement of the cysteine residues in the protein. The
solid line shows the missing contact footprint of
CuACE1. The dashed line shows the missing contact
footprint of the modified CuACE1. Filled triangles indicate the sites of major groove interactions. Empty triangles indicate interactions at the middle minor
groove area. Continuous and dashed arrows indicate the borders of the minimal binding site deduced from the
mobility shift and protease protection assays, respectively. The
open circle shows the position of the dyad symmetry
center of UAS. The bottom part of the
diagram shows the proposed arrangement of cysteine residues within the
CuACE1.
To assess the importance
of the central AT-rich region, we replaced the TA pairs with CG base
pairs at positions 129,
134,
135,
136, and
137, thus changing both surfaces of the helix
(29) .
CuACE1 failed to bind the CG-substituted probe, suggesting the
importance of the contacts within the candidate site of minor grove
interactions (Fig. 3). The adenine N
methylation
interference can also be interpreted as an effect of methylation on the
local deviation potential of the DNA helix
(30, 31) . To
confirm the minor groove interactions and to exclude the possibility of
major groove contacts, we substituted the critical TA pairs with CI
base pairs
(32) . In a mobility shift assay CuACE1 bound to
CI-substituted probe, but the extent of binding appeared to be
decreased (Fig. 3). The inosine-containing probe was used in
methylation interference studies, since inosine can be methylated by
dimethyl sulfate at the N
position. Inosine N
methylation results in a methyl group sitting on the major groove
surface of the helix
(33) and therefore would be expected to
alter major groove contacts. None of the inosine N
methylations interfered with the CuACE1 binding at position
129,
134,
135,
136, or
137
(Fig. 2 B). Interferences were enhanced at the distal
G
and G
top strand positions,
while there were notable decreases at the proximal
G
, G
, and G
positions if compared with the nonmodified CuACE1 (Fig. 2,
A and B). The four consecutive CI base pairs might
slightly distort the DNA helix, resulting in a decreased interaction
with the tightly connected DNA-binding structures of CuACE1.
Interestingly, there were no such decreases in the distal major groove
interferences at positions G
and G
(Fig. 2 B).
Figure 3:
Mobility shift assay of CuACE1 with UASc
left arm and mutant probes. P-Labeled UAS
left
arm and mutant left arm probes were incubated with CuACE1 and analyzed
on an 8% polyacrylamide gel. The bound fraction ( I) and free
( Free) fractions are indicated. The first lane ( T/A) shows mobility shift with wild-type probe. The
second lane ( C/I) shows a probe in which TA
base pairs were substituted with CI at positions
134,
135,
136, and
137. The third lane ( C/G) shows a probe in which the same positions were
substituted by CG base pairs. The last lane ( M) shows the mobility shift of the UAS
left
arm minimal binding site probe.
Modified CuACE1 Does Not Bind to the Distal Major Groove
of UAS
Since CuACE1 contacts DNA at two spatially
separated major groove regions, the question arose of whether limited
modification of cysteinyl residues in CuACE1 would selectively disrupt
interactions in one site. CuACE1 was modified with iodoacetate to
alkylate chemically accessible cysteines. Edman sequencing of modified
CuACE1 revealed greater than 90% carboxymethylation of the three most
amino-terminal cysteinyl residues within the ACE1, namely
Cys, Cys
, and Cys
. As expected,
Cys
, which lies outside of the functional DNA-binding
domain
(11) , was also extensively modified. By contrast,
Cys
, Cys
, and Cys
were
significantly less modified (<25-30%), and no modification was
detectable at Cys
and Cys
. We could not
examine Cys
and Cys
whereas modification of
Cys
seemed to be ambiguous.
left half-site revealed
impaired binding by more than an order of magnitude compared with
unmodified CuACE1 (see Fig. 5 A). In the bound modified
CuACE1
DNA complex the distal major groove interactions at
positions G
and G
were
nondetectable by methylation interference (Fig. 2 C). The
interferences within the A-rich minor groove and the proximal major
groove regions appeared to be similar to those with unmodified CuACE1.
A slight increase in interference was found toward the dyad symmetry
center of UASc at position
124 (compare Fig. 2, A and C). Surprisingly, the selective modification of
several cysteine residues resulted in similar footprint as reported
earlier for the mutant ace1-1 protein containing a C11Y
substitution
(23) .
Figure 5:
Mobility shift assay of truncated UASc
left arm probes with CuACE1 and alkylated CuACE1. Left right and
right
left truncated UASc probes were
P-labeled and
incubated with either modified or unmodified CuACE1, and the complexes
were analyzed on an 8% polyacrylamide gel. I, the complex
formed with modified ( mod.) protein; II, the complex
formed with unmodified protein. A, mobility shift assay of the
left
right (from distal to proximal direction) truncated probes.
The positions of the truncations are indicated by the most 5`
nucleotide of the probe. B, mobility shift assay of the right
left (from proximal to distal direction) truncated probes. The
positions of the truncations are indicated by the most 3` nucleotide of
the probe. The change in mobility of the free probes is due to the
different lengths of oligonucleotides used.
Modified CuACE1 Contacts Only the Proximal and Middle
Binding Region of UAS
To corroborate the
altered footprint of the modified CuACE1, a missing contact
interference experiment was carried out. The missing contact
interference method can reveal both the structural and functional roles
played by the individual bases
(27) .
Left Arm
and
A
and weak but detectable interferences at
positions A
and A
. The distal
binding region from G
to T
was separated from the proximal region by two bases
(T
and C
) that were not
involved in the contact (Fig. 4 A). The four most
prominent missing contact interferences were found on the bottom strand
at positions A
, A
,
A
, and A
(Fig. 4 B). These are the four consecutive A
residues found to be involved in minor groove contacts as determined by
methylation interference mentioned above. Interestingly, there are
notable differences between the T and complementary A missing contact
interferences at the
136 and
137 positions, in which the
presence of the bottom strand adenines appeared to play a more
important role than the thymines at the top strand in the CuACE1
binding. No such A versus T selection could be seen at the
TA
or TA
pairs (Fig. 4,
A and B).
Figure 4:
Comparison of the missing contact
interference patterns of CuACE1 and alkylated CuACE1. The UASleft arm probe was
P-labeled on either the top or
bottom strand, and the probes were premodified for A + T and C
+ T reactions. The probes were incubated with CuACE1, and the
bound ( B) and free ( F) fractions were separated by
electrophoresis on an 8% polyacrylamide gel, eluted, cleaved with
piperidine, and subsequently analyzed on a sequencing gel. S indicates A + G and C + T sequencing ladders,
respectively. A, continuous interference through the top
strand proximal region and the A-rich site from position
137 to
128 relative to the transcription initiation site is indicated.
Missing contact interference with modified protein cannot be detected
at top strand positions
140,
142,
141, and
143, whereas interferences can be seen at these positions with
unmodified CuACE1. B, the reduced recognition site of modified
CuACE1 is indicated in the
125 to
135 region on the
bottom strand. In the proximal region, the complete loss of
interference is indicated at the
140,
141, and
142
positions.
The results for the modified CuACE1
protein were strikingly different from those for the intact protein.
The missing contact experiment showed that the modified CuACE1 failed
to contact the distal binding region of the UASleft arm
probe on both strands. The recognition site was reduced to the
128,
137 region of the top strand and to the
125,
135 region of the bottom strand (Fig. 4, A and
B). This observation coincides with the methylation
interference results.
128
and
136 sites that include the middle and proximal binding
regions if compared with nonmodified CuACE1 (Fig. 4 A).
The decrease may arise from a background enhancement derived from the
``forced binding'' or from decreased binding specificity of
the modified protein arising from a conformational alteration.
Minimal Binding Site for CuACE1
The gel shift
assay was used to test a series of UASleft arm synthetic
oligonucleotides truncated either from left to right or from right to
left in order to precisely define the minimal binding site for the
modified and unmodified forms of CuACE1. Left
right truncations
of the probe past the
142 position led to a major decrease in
CuACE1 binding. Once the truncations proceeded past the A-rich minor
groove binding site at position
137, all binding activity was
lost (Fig. 5 A). This experiment indicated that the
142 position was the distal extreme of the CuACE1 binding site
(see Fig. 8).
right DNA deletions of
nucleotides
142 to
139 (Fig. 5 A).
Furthermore, these complexes showed decreased mobility and intensity in
the mobility shift assay. The decreased mobility may arise from either
a reduced net negative charge of the protein
DNA complex or a
change in conformation of protein
DNA complexes. Neither the
mobility nor the binding was different past the
140 truncation.
This experiment corroborated the lack of distal DNA contacts with the
modified CuACE1 complex.
left truncations of the probe
past the
124 position, the dyad symmetry center of UASc,
resulted in mobility changes and greatly diminished CuACE1 binding
(Fig. 5 B). Approaching the proximal major groove binding
site from right to left resulted in loss of protein
DNA complex
formation at truncation positions
128 and
129
(Fig. 5 B). The
124 to
127 region may have
a limited role in CuACE1 binding.
125,
126, and
127 is consistent with sugar-phosphate backbone interactions
(Fig. 4 B).
left probe truncations revealed that the
124 base is the
last nucleotide required for modified CuACE1 binding. Further probe
shortening resulted in a complete loss of binding by modified CuACE1,
indicating that the modified ACE1 had an increased requirement for the
dyad symmetry center. This is consistent with the results of the
methylation interference experiment.
Reverse Footprinting
Standard DNA-protein
footprint assays map the sites in DNA that are protected by a protein.
However, it is unclear whether the observed contacts are due to
base-specific or sugar-phosphate backbone interactions. To get around
this problem, we developed a footprinting technique, called reverse
footprinting, that measures the ability of a DNA fragment to protect a
protein sequence.
oligonucleotides. As expected, the DNA-dependent
protection against trypsin became weaker as the left
right
truncation approached the
142 position (Fig. 6 A).
Past
142 the DNA-protected core was not detectable. This result
substantiated the
142 position as the left side of the contact
site. Using right
left truncated probes in the same
DNA-dependent protein protection assay, the
128 position was
found to be the last that still protected the protein against trypsin
degradation, suggesting that the right-hand boundary of the ACE1
binding site is closer to the
128 position than to the dyad
symmetry center (Fig. 6 B; see Fig. 8).
left arm must be 5`-GCGTCTTTTCCGCTGA-3`
(see Fig. 8). To directly test the ability of this sequence alone
to bind to CuACE1, we used the corresponding oligonucleotide in a
mobility shift assay. Fig. 3shows that this 16-base pair
oligonucleotide was capable of binding to CuACE1.
CuACE1 Exhibits Reduced Binding to Mutant UAS
The interaction between CuACE1
and DNA was further probed by changing the recognition sites through
multiple substitution or insertions in UAS Left Arm Distal Region Probes
.
Fig. 7
shows that mutation of the distal major groove binding site
severely reduced binding to CuACE1 but had little or no effect on the
already low level of binding to modified CuACE1 (Fig. 7). The
insertion of an increasing number of bases between the distal and
middle sites resulted in a progressive loss of binding by the intact
protein until it reached a low level similar to that observed for the
modified protein. These reductions indicate that there is limited
flexibility between the distal major groove and middle binding units of
CuACE1.
Figure 7:
Mobility shift assay of transversion and
insertion mutant UASc left arm probes tested with modified and
unmodified CuACE1. Probes were P-labeled and incubated
with either modified or unmodified CuACE1 and analyzed on an 8%
polyacrylamide gel. The probes were as follows: UAS
left
arm probe (UAS
left arm); mutant distal binding site
( W); mutant middle minor groove binding site ( M);
mutant proximal binding site ( A); completely mutated UAS
left arm ( Lmut.); insertions of +1, +2,
+3, +5, and +11 base pairs were introduced between
138 and
139 positions ( X). The positions of the
modified ( I) and unmodified ( II) CuACE1 complexes are
indicated. A, each comparison shows a single lane with the unmodified protein followed by two parallel lanes with modified proteins. B, diagram of the
positions of the mutant regions on the UAS
left
arm.
sequences is marked by three features summarized in Fig. 8.
First, the DNA binding site is comprised of a internal minor groove
site flanked on both sides by major groove sites. Second, the minor
groove and proximal major groove sites are essential for high affinity
binding of CuACE1, whereas the distal site contributes less to the
energy of stabilization of the complex. Third, we propose that distinct
segments of CuACE1 are responsible for the interactions; an
NH
-terminal domain appears important for the relatively
weak interactions with the distal UAS
region, and a
C-terminal domain is responsible for the stronger binding of the middle
and proximal regions.
,
G
, G
, G
,
and G
. As is well known, methylation interference
experiments cannot prove minor groove interactions. Therefore, we used
the approach of replacing AT base pairs with IC pairs at the middle of
the binding site
(32) . The experiment showed that methylation
on the major groove surface (I N
) in the middle of the
UAS
left arm did not interfere with the protein binding.
Thus, the detected A N
methylation interference must derive
from a minor grove interaction in the middle region.
DNA. The
evidence for this hypothesis was derived from the analysis of a
modified CuACE1 in which the three most NH
-terminal
cysteines were preferentially alkylated. The modified CuACE1 bound to
mutant UAS
sites that lacked the distal region contacts
with the same affinity as the unmodified protein. However, modified
CuACE1 was bound to the complete UAS
left arm with lower
affinity than unmodified CuACE1. This indicates that the DNA-binding
residues that are organized through metal binding to the three
NH
-terminal cysteines are important for the distal
UAS
DNA binding.
DNA-binding site that we defined as critical by footprint
analysis are almost identical to the bases demonstrated to be essential
for in vivo function
(2) . A C11Y mutation in the
ace1-1 protein that inactivated the distal binding domain still
had limited binding activity
(23) . Furthermore, mutation of the
cysteine residues at positions Cys
, Cys
,
Cys
, Cys
, Cys
, Cys
,
Cys
, and Cys
resulted in a complete loss of
DNA binding and in vivo function
(11) . This result can
also be predicted because these mutations inactivated the proximal
binding domain of CuACE1.
-terminal half of the ACE1 polypeptide contains four
Cu(I) ions in a tetracopper center and a single Zn(II) ion
(9, 19) . The ACE1 homologue in C. glabrata,
AMT1, is also isolated as a Cu
Zn
AMT1
complex, and the AMT1-bound Zn(II) is not displaced by exogenous Cu(I)
ions, suggesting that Zn(II) may be physiologically relevant
(19) . We have preliminary evidence that the Zn(II) in ACE1 can
be displaced by exogenous Cu(I) without altering DNA binding
activity.
(
)
The fact that Zn(II) depletion of
AMT1 does not impair DNA binding affinity is consistent with the most
NH
-terminal cysteinyl residues serving as Zn(II) ligands
(19) . The prediction is that ACE1 and AMT1 will consist of two
metal centers, a Zn(II) site organizing the NH
-terminal
segment, which binds to the distal major groove contacts in a UAS, and
a tetracopper site, which stabilizes a domain responsible for proximal
DNA major groove contacts. Additional structural studies are in
progress to test this prediction.
ACE1 complex.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.