The EMBO Journal vol.13 no.1 pp.200-204, 1994

Identification of amino acid - base contacts in theMyc- DNA complex by site-specific bromouracilmediated photocrosslinking

Qianping Dong, Erich E.Blatter,Yon W.Ebright, Klaus Bister1 andRichard H.Ebright2

Department of Chemistry and Waksman Institute, Rutgers University,New Brunswick, NJ 08855, USA and lInstitute of Biochemistry,School of Medicine, University of K6ln, D50924 Koln, Germany

2Corresponding author

Communicated by H.Buc

Myc binds to a 6 bp 2-fold symmetric DNA site:5'-C_3A-2C jG+jT+2G+3-3'. Using site-specific 5-bromouracil mediated photocrosslinling, we show thatHis336 of Myc contacts, or is close to, the thymine5-methyl group at 2-fold symmetry-related positions -2and +2 of the DNA site in the Myc-DNA complex. Ourresults strongly suggest that homologous amino acids ofMyc and Max make equivalent contacts in the respectiveprotein-DNA complexes.Key words: amino acid -base contacts/DNA bin-ding/Myc/photocrosslinking/protein-DNA interaction

IntroductionThe oncogene product Myc is a sequence-specific DNAbinding protein (reviewed by Marcu et al., 1992). Mycmediates dimerization and DNA binding through an 80amino acid structural motif termed 'basic/helix-loop-helix/leucine zipper' (b/HLH/Z) (Murre and Baltimore,1989; Anthony-Cahill et al., 1992; Halazonetis and Kandil,1992; Murre et al., 1992; Vinson and Garcia, 1992). Mycbinds to a 6 bp 2-fold symmetric DNA site:5'-C-3A-2C 1G+ T+2G+3-3' (Blackwell et al., 1990;Halazonetis and Kandil, 1991; Kerkhoff et al., 1991;Prendergast and Ziff, 1991; Papoulas et al., 1992). Thecrystallographic structure of Myc in complex with DNA hasnot been determined; however, the crystallographic struc-ture of the homologous b/HLH/Z sequence-specific DNAbinding protein Max in complex with DNA has beendetermined to 2.9 A resolution (Ferre-D'Amar6 et al.,1993).

Site-specific 5-bromouracil mediated photocrosslinkingpermits identification of contacts between individual aminoacids of a sequence-specific DNA binding protein andindividual thymines of the DNA site in the protein-DNAcomplex in solution (Allen et al., 1991; Blatter et al., 1992).The method uses 5-bromouracil (BrU), an isosteric photo-reactive analog of thymine (Figure 1; Hutchinson, 1973;Hutchinson and K6hnlein, 1980; Saito and Sugiyama, 1990;Allen et al., 1991; Blatter et al., 1992). In its simplest form,the method has two parts. (i) A set ofDNA fragments, eachcontaining the DNA site for the protein under investigationand each having one thymine of the DNA site replaced byBrU is synthesized. For each DNA fragment, the protein-

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DNA complex is formed, irradiated by UV, and it is thendetermined whether BrU mediated protein-DNA cross-linking occurs. (ii) For each DNA fragment for which BrUmediated protein -DNA crosslinking occurs, theprotein -DNA complex is formed on a preparative scale,UV irradiated, the crosslinked protein-DNA complex isisolated, and the amino acid(s) at which crosslinking occursis determined.

In the work in this report, we have used site specific BrUmediated photocrosslinking to identify amino acid-basecontacts in the Myc-DNA complex in solution.

ResultsDNA fragments containing the DNA site for Myc andhaving BrU at positions - 2 or + 2 crosslink with Mycupon UV irradiationWe have used a 119 amino acid protein fragment, 'Mycpl6'corresponding to the b/HLH/Z motif of Myc [amino acids310-416 of avian myelocytomatosis virus MC29 v-Myc(amino acids numbered according to the numbering for avianc-Myc)] (Kerkhoff and Bister, 1991; Kerkhoff et al., 1991).Mycpl6 contains all determinants of Myc for dimerizationand for DNA binding (Kerkhoff and Bister, 1991; Kerkhoffet al., 1991). We also have used a set of three 20 bp DNAfragments containing the DNA site for Myc: DNA fragmentsMRE, MRE/-2'BrU and MRE/+2BrU (Figure 2). In DNAfragments MRE/-2'BrU and MRE/+2BrU, a single thymineof the DNA site is replaced by BrU: i.e. at position -2 orat 2-fold symmetry-related position +2. Electrophoreticmobility shift DNA binding experiments establish that DNAfragments MRE, MRE/-2'BrU and MRE/+2'BrU are fullyfunctional in specific Mycpl6-DNA complex formation(Kobsl-1x 1066M-)To determine whether DNA fragments MRE/-2'BrU and

MRE/+2BrU can crosslink with Mycpl6 in the Mycp16-DNA complex, for each DNA fragment we formed theMycp16-DNA complex, UV irradiated it and analyzed thereaction products by denaturing polyacrylamide gel electro-phoresis. In each case UV irradiation resulted in theformation of a crosslinked Mycpl6-DNA complex (Figure3). The efficiency of crosslinking was 2%. Controlexperiments established that crosslinking required UVirradiation, Mycpl6 and that the DNA fragment containBrU. Additional control experiments established thatcrosslinking required specific Mycpl6-DNA complexformation. Thus, crosslinking did not occur with a 19 bpBrU-containing DNA fragment lacking the DNA site forMyc [DNA fragment GRE/+3'BrU of Blatter et al. (1992)].Furthermore, crosslinking was inhibited by a 5-fold excessof DNA fragment MRE but was not inhibited by a 5-foldexcess of a 19 bp DNA fragment lacking the DNA site forMyc [DNA fragment GRE of Blatter et al. (1992)]. Weconclude that a BrU residue at position 2 of each DNAhalf site can crosslink with Mycpl6 in the specific

© Oxford University Press

Amino acid - base contacts in the Myc - DNA complex

0

Br

5-BROMOURACIL

BrNH

0

H3CNH

THYMINE

hv l

,----0- Be 0 N H

_ I _

Fig. 1. 5-Bromouracil (BrU). (A) Structures of 5-bromouracil and thymine. (B) Photochemistry of BrU (reviewed by Hutchinson, 1973; Hutchinsonand K6hnlein, 1980; Saito and Sugiyama, 1990). Upon UV irradiation (254 nm), Br-C bond homolysis occurs and bromine and uracil-5-yl radicalsare formed. If there is organic material, 'X', in direct van der Waals contact with the uracil-5-yl radical, covalent bond formation can occur. Analternative photochemistry involves formation of a triplet excited state, followed by hydrogen abstraction or single-electron transfer from 'X',followed by combination of the resulting radicals.

-3 -2 -1 +1 +2 +3

MRE

MRE/-2 'BRU

MRE/+2BRU

C G C C G A C C ACG T G C T C C A G CG C G G C T G G T GC A C G A G G T C G

IABRU

IBRUA

Fig. 2. DNA fragments containing the DNA site for Myc. In DNA fragments MRE/-2'BrU and MRE/+2BrU, a single thymine of the DNA site isreplaced by BrU.

A 1 2 3 45 6 7 B 1 2 3 4 5

--CROSSLINKEDMYCP1 6-DNA

--DNA

--CROSSLINKEDMYCP1 6-DNA

--DNA

Fig. 3. Formation of crosslinked Mycpl6-DNA complexes. (A) Data for DNA fragment MRE/-2'BrU. Lane 1, crosslinking reaction; lane 2,control reaction omitting UV irradiation; lane 3, control reaction omitting Mycpl6; lane 4, control reaction using DNA fragment MRE in place ofDNA fragment MRE/-2'BrU; lane 5, control reaction using a 19 bp BrU-containing DNA fragment lacking the DNA site for Myc in place of DNAfragment MRE/-2'BrU [DNA fragment GRE/+3'BrU of Blatter et al. (1992)]; lane 6, control reaction in the presence of a 5-fold excess of DNAfragment MRE; lane 7, control reaction in the presence of a 5-fold excess of a 19 bp DNA fragment lacking the DNA site for Myc [DNA fragmentGRE of Blatter et al. (1992)]. (B) Data for DNA fragment MRE/+2BrU. Lanes as in (A).

A NLDSEENDKRRT*VLEdB yLDSEENDKRR*VLqC MASMTGGOQMGRLKQISNNRKCSSPRFLDSEENDKRR#*VLEFORRNELKLRFFALRDQIPEVANNEKAPKVVILKKATEYVLSLQSDEHKLIAE KEOLRRRREOLKHNLEOLRNSRA

I ~~~I I

b HLH Z

Fig. 4. Identification of the amino acid at which crosslinking occurs. (A) Amino acid sequence of the crosslinked peptide in experiments with DNA

fragment MRE/-2'BrU. At the 13th position, no standard amino acid was identified ('X'). (B) Amino acid sequence of the crosslinked peptide in

experiments with DNA fragment MRE/+2BrU. At the 13th position, no standard amino acid was identified ('X'). (C) Amino acid sequence of

Mycpl6 (Alitalo et al., 1983; Reddy et al., 1983; Kerkhoff and Bister, 1991; Kerkhoff et al., 1991). The basic ('b') helix-loop-helix ('HLH'),and leucine zipper ('Z') regions of Mycpl6 are indicated.

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A

B

Q.Dong et al.

AA

Fig. 5. Crystallographic structure of the Max-DNA complex showing the position of the homolog of His336 of Myc (Ferrf-D'Amard et al., 1993).(A) Interaction between the b/HLH/Z motif of Max and DNA. The Ca atom and side chain of the homolog of His336 of Myc in each Max subunitis indicated in dark gray. The thymine 5-methyl group at position 2 in each DNA half site is indicated in black. (B) Interaction between the basicregion a-helix of the b/HLH/Z motif of Max and positions 1-5 of the DNA half site (view parallel to the basic region a-helix). Interatomicdistances are in A.

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Amino acid - base contacts in the Myc - DNA complex

Mycpl6-DNA complex in solution. We further concludethat the thymine 5-methyl group at position 2 of each DNAhalf site contacts, or is close to, Mycpl6 in the specificMycp16-DNA complex in solution. [The C5 and Br5atoms of BrU are the reactive atoms for BrU-mediatedphotocrosslinking (Figure 1; Hutchinson, 1973; Allen et al.,1991; Blatter et al., 1992). These atoms correspond to theC5 atom and 5-methyl group of thymine.]

Crosslinking occurs at His336 of MycTo identify the amino acid(s) of Mycpl6 at which cross-linking occurs with DNA fragments MRE/-2'BrU andMRE/+2BrU, for each DNA fragment, we prepared thecrosslinked Mycp16-DNA complex in pmol quantities,digested the crosslinked Mycpl6-DNA complex withtrypsin, isolated the resulting crosslinked peptide-DNAcomplex and determined the amino acid sequence of thecrosslinked peptide. For each DNA fragment, the resultswere identical (Figure 4). The sequence of the crosslinkedpeptide was Thr-Leu-Asp-Ser-Glu-Glu-Asn-Asp-Lys-Arg-Arg-Thr-X-Asn-Val-Leu-Glu-Arg, which corresponds toamino acids 324-341 of Myc. For each DNA fragment,no standard amino acid was identified at the 13th positionof the crosslinked peptide, which corresponds to His336of Myc. We conclude that crosslinking occurs at His336 ofMyc. The fact that sequencing was able to proceed throughamino acid 336 indicates that crosslinking does not occurat the backbone amide nitrogen atom, the backbone carbonylatom or the backbone carbonyl oxygen atom (cf. Blatteret al., 1992). We conclude that crosslinking occurs at thebackbone Ca atom or at the side-chain of His336 of Myc.

DiscussionOur results establish that the Ca atom or side chain of His336of Myc contacts, or is close to, the thymine 5-methyl groupat position 2 of the DNA half site in the Myc-DNA complexin solution. Contacts with the thymine 5-methyl group canbe an important determinant of specificity for thymine inprotein-DNA interaction (Fisher and Caruthers, 1979;Wharton and Ptashne, 1987; Gunasekera et al., 1992). Wepropose that a van der Waals contact between the Cai atomor side chain of His336 of Myc and the thymine 5-methylgroup at position 2 of the DNA half site is part of the basisof specificity for thymine at position 2 of the DNA half site.Consistent with this proposal, His336 is one of threeabsolutely conserved, absolutely critical, amino acids in thebasic regions of Myc and Myc-related b/HLH/Z sequencespecific DNA binding proteins (Fisher et al., 1993). Alsoconsistent with this proposal, removal of the thymine5-methyl group at position 2 of the DNA half site reducesaffinity in Myc-DNA complex formation (4-fold; 0.8 kcal/mol; unpublished data).The b/HLH/Z sequence-specific DNA binding protein

Max exhibits amino acid sequence homology to Myc,including His336 of Myc (Blackwood and Eisenman, 1991;Prendergast et al., 1991), and recognizes the same DNAsite as does Myc (Prendergast et al., 1991; Fisher et al.,1992; Wechsler and Dang, 1992). The crystallographicstructure of the Max-DNA complex at 2.9 A resolutionrecently has been reported (Ferre-D'Amare et al., 1993).In the crystallographic structure of the Max-DNA complex,the side chain Cy, C02, and Ne2 atoms of the homolog of

His336 make van der Waals interactions with the thymine5-methyl group at position 2 of the DNA half site (inter-atomic distances = 4.3 A, 4.1 A and 4.3 A, respectively)(Figure 5). It is striking that His336 of Myc contacts, oris close to, the thymine 5-methyl group at position 2 of theDNA half site in the Myc-DNA complex and that thehomologous amino acid of Max contacts the thymine5-methyl group at position 2 of the DNA half site in theMax-DNA complex. Our results strongly suggest that,despite reported differences in DNA bending (Wechsler andDang, 1992), homologous amino acids of Myc and Maxmake equivalent contacts in the respective protein-DNAcomplexes.

Materials and methodsMycp 16Mycpl6 consists of 12 non-native amino acids, Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg, followed by amino acids 310-416 of avianmyelocytomatosis virus MC29 v-Myc (amino acids numbered accordingto numbering convention for avian c-Myc) (Kerkhoff and Bister, 1991;Kerkhoff et al., 1991). Plasmid pEwtAB encodes Mycpl6 under controlof the bacteriophage T7 gene 10 promoter (Kerkhoff and Bister, 1991;Kerkhoffet al., 1991). Mycpl6 was produced in Escherichia coli to a levelof -60% of total cell protein. Inclusion bodies containing Mycpl6 wereisolated and washed according to the procedure of Lin and Cheng (1992)and were solubilized in 9 M urea, 10 mM Tris-HCI (pH 8.0), 1 mMEDTA, and 10 mM dithiothreitol (2 ml/i of induced bacterial culture).Solubilized Mycpl6 was renatured by dilution into 30 vol of 20 mMTris-HCI (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol and 200 jg/mlbovine serum albumin at 4°C, and was purified by DNA-cellulosechromatography. Mycpl6 was 295% homogeneous as assessed bySDS-PAGE and was -25% active in DNA binding as assessed in electro-phoretic mobility shift DNA binding experiments performed understoichiometric binding conditions.

DNA fragmentsFor each DNA fragment, oligodeoxyribonucleotides corresponding to thetop and bottom strands were synthesized using solid-phase 3-cyanoethyl-phosphoramidite chemistry on an AB380A automated synthesizer. BrU wasintroduced using 5-bromo-2'-deoxyuridine-,B-cyanoethylphosphoramidite(Glen Research, Inc.). Products were purified by trityl-on C18 reversed-phase HPLC, followed by detritylation and ethanol precipitation. The BrU-containing oligodeoxyribonucleotide was [32P]5'-end labelled as describedin Ebright et al. (1989), and the oligodeoxyribonucleotides correspondingto the top and bottom strands were annealed (Ebright et al., 1989). Alloperations with BrU-containing oligodeoxyribonucleotides and DNAfragments were performed in darkness or under safelamp illumination.

Electrophoretic mobility shift DNA binding experimentsElectrophoretic mobility shift DNA binding experiments were performedessentially as described (Fried and Crothers, 1981; Gamer and Revzin,1981). Reaction mixtures contained (10 1l): 0-20 1tM Mycpl6, 1 nM[32P]5'-end-labelled DNA fragment (10 Bq/fmol), 30 ig/ml shearedherring sperm DNA (Promega, Inc.), 10 mM MOPS-NaOH pH 7.2,50 mM NaCl and 5% (v/v) glycerol.

Formation of crosslinked Mycp 16-DNA complex, analytical scaleCrosslinking reaction mixtures contained (50 tll): 5 ztM Mycpl6,1 1M[32P]5'-end-labelled DNA fragment (200 Bq/pmol), 70 ug/ml shearedherring sperm DNA (Promega, Inc.), 10 mM MOPS-NaOH pH 7.3 and50 mM NaCl. Reaction mixtures were incubated in the dark for 60 miat 22'C. Reaction mixtures then were UV irradiated for 90 s at 22'C(254 nm; 1280 ergs/mm2/s) in a Rayonet RPR100 photochemical reactor(Southern New England Ultraviolet, Inc.). (Control experiments establishedthat under these conditions UV irradiation does not affect the specific activityof Mycpl6.) UV irradiated reaction mixtures were ethanol precipitated,dissolved in 10 IL 5 M urea, 50 mM NaOH, 0.5 mM EDTA, 0.025%bromophenol blue and 0.025% xylene cyanol, and were electrophoresedon 10% polyacrylamide-8 M urea slab gels. After electrophoresis, gelswere dried and were autoradiographed. Efficiencies of crosslinking weredetermined by excision of bands followed by determination of Cerenkovradiation.

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Formation of crosslinked Mycp 16- DNA complex, preparativescaleFor each DNA fragment, crosslinked Mycpl6-DNA complex was preparedas described above, except that the reaction volume was 50 ml (UV irradiatedin 25 ml aliquots) and the specific activity of the DNA fragment was10 Bq/pmol. Crosslinked Mycpl6-DNA complex was purified fromuncrosslinked DNA by extraction into phenol and was recovered from phenolby back extraction into water. The UV irradiated sample was adjusted to0.1% SDS, incubated for 5 min at 70°C and extracted with 25 ml Tris-saturated phenol. The resulting phenolic phase was diluted in 25 ml etherand then was back extracted with 6 ml water. The resulting aqueous phasewas washed twice with 25 ml ether and lyophilized.

Identification of the amino acid at which crosslinking occursFor each DNA fragment, crosslinked Mycpl6-DNA complex was dissolvedin 400 pl 400 mM ammonium bicarbonate pH 8.8 and 8 M urea. Afterincubation for 1 min at 70'C, 1.2 ml water and 0.5 pg sequencing grademodified trypsin (Promega, Inc.) were added, and the sample was incubatedfor 7 h at 37'C. The resulting crosslinked peptide-DNA complex waspurified from uncrosslinked peptides by denaturing polyacrylamide gelelectrophoresis, then purified from low molecular weight contaminants bygel-filtration chromatography and lyophilized. The sample was ethanolprecipitated twice, redissolved in 20 p1 6 M urea, 0.025% bromophenolblue and 0.025% xylene cyanol, and electrophoresed through a 10%polyacrylamide-2 M urea slab gel. Crosslinked peptide-DNA complexwas excised from the gel and eluted into 1 ml 10 mM ammonium bicarbonate(pH 8.8) by shaking for 2 h at 22'C. Crosslinked peptide-DNA complexwas lyophilized, redissolved in 100 pl 10 mM ammonium bicarbonatepH 8.8, applied to a 5 ml column of Bio-Gel P-6DG (Bio-Rad, Inc.), elutedin the same buffer and lyophilized. Recovery was - 100 pmol. The aminoacid sequence of the crosslinked peptide was determined by M.Crawford(Yale University, New Haven, CT) using an AB477A automated gas-phaseprotein sequencer.

Molecular modellingCoordinates for the crystallographic structure of the Max-DNA complexat 2.9 A resolution (Ferre-D'Amar6 et al., 1993) were obtained fromS.K.Burley (Rockefeller University, New York, NY). Interatomic distanceswere measured using the program INSIGHT (BIOSYM, Inc.). Diagramswere prepared using the program MOLSCRIPT (Kraulis, 1991).

AcknowledgementsWe thank S.K.Burley for the coordinates for the structure of the Max-DNAcomplex. We thank E.Kerkhoff and C.Vinson for discussion. This workwas supported by a Searle Scholar Award to R.H.E., a Merck PostdoctoralFellowship to E.E.B and a Busch Predoctoral Fellowship to Q.D.

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Received on August 30, 1993

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