©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinct Regions of Cu(I)ACE1 Contact Two Spatially Resolved DNA Major Groove Sites(*)

Albert Dobi (1)(§), Charles T. Dameron (2)(¶), Stella Hu (1), Dean Hamer (1), Dennis R. Winge (2)(**)

From the (1) Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892 and the (2) University of Utah Health Science Center, Salt Lake City, Utah 84132

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The interaction between the Cu(I)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 CuACE1DNA complex. The NH-terminal segment of ACE1 is shown to contact the 5`-most distal major groove site.


INTRODUCTION

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-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) .

The Cu(I)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) .

The NH-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) .

CuACE1 is known to preferentially bind the left half-site as opposed to the right half-site of the UASpalindrome 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 UAShalf-site (23) .

In the present work we assessed the base-specific interactions between CuACE1 and a chemically modified CuACE1 with the left half-site of the UASsequence. 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 CuACE1DNA complex. The chemically modified CuACE1 was found to be incapable of binding to the distal major groove site of the UAShalf-site, whereas the minor and proximal major groove interactions remained unchanged.


EXPERIMENTAL PROCEDURES

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 10cpm) 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 NHOAc, 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 HO, lyophilized again, and then resuspended in HO 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 10cpm) 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 10cpm) 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(dIdC), 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 UASleft 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 UASleft arm. The open circle indicates the position of the DNA-protected protein core. B, protection by right left truncated UASleft arm probes.




RESULTS

CuACE1 Binds Specifically to the Minor Groove of the UAS Left Half-site between Two Adjacent Major Groove Contact Sites

In the binding of CuACE1 to the UASleft 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 UASwe used methylation interference experiments to test CuACE1 binding to guanine Nand adenine Nmethylated nucleotides. Methylation of guanines at the Nposition interferes with major groove contacts, whereas methylation of adenine at Nposition 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 UASbottom strand at position Gand 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 Gand Ginterfered with CuACE1 binding (Fig. 2 A; see Fig. 8).


Figure 2: Methylation interference footprint of nonmodified and modified CuACE1UASc left arm complexes. The UASleft 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 Nmethylation 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 Nposition. Inosine Nmethylation 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 Nmethylations interfered with the CuACE1 binding at position 129, 134, 135, 136, or 137 (Fig. 2 B). Interferences were enhanced at the distal Gand Gtop strand positions, while there were notable decreases at the proximal G, G, and Gpositions 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 Gand G(Fig. 2 B).


Figure 3: Mobility shift assay of CuACE1 with UASc left arm and mutant probes. P-Labeled UASleft 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 UASleft 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 Cyswere significantly less modified (<25-30%), and no modification was detectable at Cysand Cys. We could not examine Cysand Cyswhereas modification of Cysseemed to be ambiguous.

Incubations of modified CuACE1 with the CUP1 UASleft half-site revealed impaired binding by more than an order of magnitude compared with unmodified CuACE1 (see Fig. 5 A). In the bound modified CuACE1DNA complex the distal major groove interactions at positions Gand Gwere 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 UASLeft Arm

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) .

In initial studies with unmodified CuACE1 the top strand proximal region and the A-rich middle site showed continuous interferences between Gand Aand weak but detectable interferences at positions Aand A. The distal binding region from Gto Twas separated from the proximal region by two bases (Tand 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 TAor TApairs (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.

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 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).

In contrast, the ability of the modified CuACE1 to bind to DNA was unchanged by left 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 proteinDNA complex or a change in conformation of proteinDNA 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.

Right 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 proteinDNA complex formation at truncation positions 128 and 129 (Fig. 5 B). The 124 to 127 region may have a limited role in CuACE1 binding.

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 125, 126, and 127 is consistent with sugar-phosphate backbone interactions (Fig. 4 B).

Studies of the modified CuACE1 and right 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.

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 UASoligonucleotides. 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).

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 UASleft 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 Left Arm Distal Region Probes

The interaction between CuACE1 and DNA was further probed by changing the recognition sites through multiple substitution or insertions in UAS. 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: UASleft arm probe (UASleft arm); mutant distal binding site ( W); mutant middle minor groove binding site ( M); mutant proximal binding site ( A); completely mutated UASleft 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 UASleft arm.




DISCUSSION

The interaction of CuACE1 and CUP1 UASsequences 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 UASregion, and a C-terminal domain is responsible for the stronger binding of the middle and proximal regions.

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, 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 UASleft arm did not interfere with the protein binding. Thus, the detected A Nmethylation interference must derive from a minor grove interaction in the middle region.

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 UASDNA. 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 UASsites that lacked the distal region contacts with the same affinity as the unmodified protein. However, modified CuACE1 was bound to the complete UASleft 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 UASDNA binding.

Our biochemical experiments are supported by several previous results. The bases within the UASDNA-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 Cysresulted 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.

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-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 CuZn 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Developmental Neurobiology, NICHD, NIH, Bethesda, Maryland 20892. Tel.: 301-402-3148; Fax: 301-402-3149.

Current address: National Research Centre for Environmental Toxicology, University of Queensland, Brisbane 4072, Australia.

**
Recipient of National Institutes of Health Grant ES 03817 from the Institute of Environmental Health Sciences.

The abbreviations used are: UAS, upstream activator sequence; CuACE1, Cu(I)ACE1 complex.

R. Farrell and D. R. Winge, unpublished observation.


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