tris(tetramethylphenanthroline)ruthenium(ii): a chiral probe that

5
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 1339-1343, March 1988 Biochemistry Tris(tetramethylphenanthroline)ruthenium(II): A chiral probe that cleaves A-DNA conformations HOUNG-YAU MEI AND JACQUELINE K. BARTON* Department of Chemistry, Columbia University, New York, NY 10027 Communicated by Nicholas J. Turro, October 9, 1987 ABSTRACT A-Tris(3,4,7,8-tetramethyl-1,10-phenanthro- line)ruthenium(ll) [A-Ru(TMP)2+] was found to be a distinc- tive molecular tool to examine the local variations in confor- mation along the strand. The metal complex binds coopera- tively to A-form helices of various base sequences under conditions where little or no binding was found to analogous B-form DNAs. Photoactivated DNA cleavage may be coupled to this conformation-specific binding by taking advantage of the photophysical properties of ruthenium(ll) complexes. A- Ru(TMP)2+ cleaves preferentially 3H-labeled A-form polynu- cleotides upon irradiation with visible light. The photoinduced DNA strand scission is likely to be mediated by singlet oxygen, which leads to a preferential cleavage of guanine residues. Comparative mapping of cleavage sites on a linear pBR322 fragment for tris(phenanthroline)ruthenium(II), which binds to B-DNA and cleaves also by sensitization of singlet oxygen, and for Ru(TMP)2 I shows the selective binding of A- Ru(TMP)2+ to conformationally distinct sites along the frag- ment. These sites correspond to 5- to 13-base-pair homopyri- midine stretches. It has become increasingly clear that a remarkable confor- mational heterogeneity may be present along the DNA strand. Local variations in DNA structure include bends, kinks, cruciform loops, and even left-handed Z-DNA (1-5). Segments of altered conformation may serve as recognition sites for the binding of regulatory proteins, and indeed the correlation of conformationally distinct sites with the bor- ders of gene coding regions has been observed (6-9). Small molecules that recognize and react at distinct sites along the DNA strand provide sensitive probes for the local variations in DNA structure. We have focused on chiral metal complexes in developing tools to map local DNA secondary structure (10). A-tris(4,7-diphenyl-1,10-phenan- throline)cobalt(III), for example, has been shown to cleave conformationally distinct sites, such as Z-DNA, and has been used to examine altered conformations along the simian virus 40 genome (6, 7, 11). We have determined that tris(3,4, 7,8-tetramethylphenanthroline)ruthenium(II) [Ru(TMP)" +] binds preferentially to A-form polynucleotides, displays chiral discrimination in its binding, favoring the A-isomer in binding to right-handed helices, and upon photoactivation promotes cleavage of the bound polymer (12). The complex associates with the polymer in a surface or groove-bound mode rather than through intercalation, the primary mode of binding for the parent chiral complex tris(phenanthroline)ru- thenium(II) [Ru(phen)2+] (13). We report here the applica- tion of Ru(TMP)2 + to synthetic polynucleotides and to probe local A-form sites through photoactivated cleavage. The structure of A-Ru(TMP)2+ is as follows: CH3 2+ CH3 CH3 )CCH3 CH3 CH CH3 CH3 CHH MATERIALS AND METHODS Materials. [Ru(TMP)3]C12 was synthesized and enantio- mers were separated as described (12, 14, 15). Labeled nucleotides were from New England Nuclear, and most enzymes were from Bethesda Research Laboratories. Instrumentation. Radioactivity was monitored by using a Beckman model LS-6800 liquid scintillation counter. Irradi- ations were performed by using a 1000 W Hg/Xe lamp (Oriel, Stamford, CT) narrowed to 442 + 6 nm with a monochrometer for photocleavage of tritiated polynucleo- tides or a He/Cd laser [Liconix (Sunnyvale, CA), model 4200NB, 442 nm, 40 mW] for fine mapping experiments. Densitometry was conducted by using a LKB 2202 ultrascan laser densitometer interfaced with a Nelson analytical box. Binding Experiments by Equilibrium Dialysis. All polynu- cleotides (Pharmacia) were dialyzed first exhaustively in buffer R (5 mM Tris.HCI/50 mM NaCl, pH 7.3) to remove small fragments. DNA (1 ml of 300 ,uM nucleotides) reten- tates were dialyzed against 3.2-ml dialysates containing racemic Ru(TMP)3 + concentrations ranging from 8 to 48 ,uM for at least 48 hr at 25°C, with shaking, after which time equilibration was achieved. Bound and free concentrations were then determined spectrophotometrically (A442) (12). Photocleavage of Synthetic Polynucleotides. The tritiated polynucleotides were synthesized according to methods adapted from Sigman and Pope (16). The A+T-containing polymers were labeled with [3H]dTTP (83 Ci/mmol; 1 Ci = 37 GBq) and G + C-containing polymers, with [3H]dGTP (33.9 Ci/mmol). In 20 ul of buffer R containing 1.2 mM histidine, the tritiated polymer was diluted with 200 uM unlabeled polymer and irradiated at 442 nm in the presence of 20 ,uM Ru(TMP)2+. Cleavage was monitored by the retention on filters of acid-precipitable radioactivity (12). Abbreviations: Ru(TMP)3 +, tris(3,4,7,8-tetramethylphenanthro- line)ruthenium(II); Ru(phen)3+, tris(phenanthroline)ruthenium(II). *To whom reprint requests should be addressed. 1339 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Page 1: Tris(tetramethylphenanthroline)ruthenium(II): A chiral probe that

Proc. Natl. Acad. Sci. USAVol. 85, pp. 1339-1343, March 1988Biochemistry

Tris(tetramethylphenanthroline)ruthenium(II): A chiral probe thatcleaves A-DNA conformationsHOUNG-YAU MEI AND JACQUELINE K. BARTON*Department of Chemistry, Columbia University, New York, NY 10027

Communicated by Nicholas J. Turro, October 9, 1987

ABSTRACT A-Tris(3,4,7,8-tetramethyl-1,10-phenanthro-line)ruthenium(ll) [A-Ru(TMP)2+] was found to be a distinc-tive molecular tool to examine the local variations in confor-mation along the strand. The metal complex binds coopera-tively to A-form helices of various base sequences underconditions where little or no binding was found to analogousB-form DNAs. Photoactivated DNA cleavage may be coupledto this conformation-specific binding by taking advantage ofthe photophysical properties of ruthenium(ll) complexes. A-Ru(TMP)2+ cleaves preferentially 3H-labeled A-form polynu-cleotides upon irradiation with visible light. The photoinducedDNA strand scission is likely to be mediated by singlet oxygen,which leads to a preferential cleavage of guanine residues.Comparative mapping of cleavage sites on a linear pBR322fragment for tris(phenanthroline)ruthenium(II), which bindsto B-DNA and cleaves also by sensitization of singlet oxygen,and for Ru(TMP)2 I shows the selective binding of A-Ru(TMP)2+ to conformationally distinct sites along the frag-ment. These sites correspond to 5- to 13-base-pair homopyri-midine stretches.

It has become increasingly clear that a remarkable confor-mational heterogeneity may be present along the DNAstrand. Local variations in DNA structure include bends,kinks, cruciform loops, and even left-handed Z-DNA (1-5).Segments of altered conformation may serve as recognitionsites for the binding of regulatory proteins, and indeed thecorrelation of conformationally distinct sites with the bor-ders of gene coding regions has been observed (6-9).

Small molecules that recognize and react at distinct sitesalong the DNA strand provide sensitive probes for the localvariations in DNA structure. We have focused on chiralmetal complexes in developing tools to map local DNAsecondary structure (10). A-tris(4,7-diphenyl-1,10-phenan-throline)cobalt(III), for example, has been shown to cleaveconformationally distinct sites, such as Z-DNA, and hasbeen used to examine altered conformations along the simianvirus 40 genome (6, 7, 11). We have determined that tris(3,4,7,8-tetramethylphenanthroline)ruthenium(II) [Ru(TMP)"+]binds preferentially to A-form polynucleotides, displayschiral discrimination in its binding, favoring the A-isomer inbinding to right-handed helices, and upon photoactivationpromotes cleavage of the bound polymer (12). The complexassociates with the polymer in a surface or groove-boundmode rather than through intercalation, the primary mode ofbinding for the parent chiral complex tris(phenanthroline)ru-thenium(II) [Ru(phen)2+] (13). We report here the applica-tion of Ru(TMP)2+ to synthetic polynucleotides and toprobe local A-form sites through photoactivated cleavage.The structure of A-Ru(TMP)2+ is as follows:

CH3 2+CH3

CH3)CCH3

CH3 CH CH3

CH3CHH

MATERIALS AND METHODSMaterials. [Ru(TMP)3]C12 was synthesized and enantio-

mers were separated as described (12, 14, 15). Labelednucleotides were from New England Nuclear, and mostenzymes were from Bethesda Research Laboratories.

Instrumentation. Radioactivity was monitored by using aBeckman model LS-6800 liquid scintillation counter. Irradi-ations were performed by using a 1000 W Hg/Xe lamp(Oriel, Stamford, CT) narrowed to 442 + 6 nm with amonochrometer for photocleavage of tritiated polynucleo-tides or a He/Cd laser [Liconix (Sunnyvale, CA), model4200NB, 442 nm, 40 mW] for fine mapping experiments.Densitometry was conducted by using a LKB 2202 ultrascanlaser densitometer interfaced with a Nelson analytical box.Binding Experiments by Equilibrium Dialysis. All polynu-

cleotides (Pharmacia) were dialyzed first exhaustively inbuffer R (5 mM Tris.HCI/50 mM NaCl, pH 7.3) to removesmall fragments. DNA (1 ml of 300 ,uM nucleotides) reten-tates were dialyzed against 3.2-ml dialysates containingracemic Ru(TMP)3 + concentrations ranging from 8 to 48 ,uMfor at least 48 hr at 25°C, with shaking, after which timeequilibration was achieved. Bound and free concentrationswere then determined spectrophotometrically (A442) (12).

Photocleavage of Synthetic Polynucleotides. The tritiatedpolynucleotides were synthesized according to methodsadapted from Sigman and Pope (16). The A+T-containingpolymers were labeled with [3H]dTTP (83 Ci/mmol; 1 Ci =

37 GBq) and G + C-containing polymers, with [3H]dGTP(33.9 Ci/mmol). In 20 ul of buffer R containing 1.2 mMhistidine, the tritiated polymer was diluted with 200 uMunlabeled polymer and irradiated at 442 nm in the presenceof 20 ,uM Ru(TMP)2+. Cleavage was monitored by theretention on filters of acid-precipitable radioactivity (12).

Abbreviations: Ru(TMP)3 +, tris(3,4,7,8-tetramethylphenanthro-line)ruthenium(II); Ru(phen)3+, tris(phenanthroline)ruthenium(II).*To whom reprint requests should be addressed.

1339

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Page 2: Tris(tetramethylphenanthroline)ruthenium(II): A chiral probe that

1340 Biochemistry: Mei and Barton

Fine-Mapping Experiments. 32P-end-labeled HindIII-Ssp IDNA fragments were prepared from the pBR322 plasmids byfirst linearizing with HindIII and then labeling either at the 5'end with T4 polynucleotide kinase with [,y-32P]ATP (4500Ci/mmol) or at the 3' end by a fill-in reaction with theKlenow fragment of DNA polymerase I with [a-32P]dATP(3000 Ci/mmol). Following secondary restriction enzyme

digestion with Ssp I (New England Biolabs), uniquely la-beled fragments (224 or 221 base pairs) were separated on a

nondenaturing polyacrylamide gel and eluted at 40C (17).The HindIII-Ssp I fragments were further purified by spin-column chromatography with a NACS 52 ion-exchange resin(Bethesda Research Laboratories). The purified 32P-end-labeled fragments were diluted in 20 Al of buffer R containingas carrier 100 AM sonicated calf thymus DNA and 1 mMhistidine. After irradiation for 20 min at 442 nm in theabsence or presence of 30 AM ruthenium, the fragmentswere precipitated with ethanol, treated with piperidine (10%vol/vol distilled piperidine in deionized water) at 90'C for 30min, and precipitated with ethanol again. DNA fragmentswere lyophilized for at least 1 hr and electrophoresed on 8 Murea denaturing 8% polyacrylamide gels. To determine theposition of the nicks generated by the ruthenium complexes,untreated DNA was sequenced by the method ofMaxam andGilbert (18) and coelectrophoresed in adjacent lanes. Forautoradiography, gels were exposed to Kodak XAR-5 film at- 60°C for 1-3 days, using Dupont Cronex Lightning Plusintensifying screens.

RESULTS AND DISCUSSIONBinding to Synthetic Polynucleotides. Binding isotherms

were determined by equilibrium dialysis of synthetic poly-nucleotides against racemic Ru(TMP)"+ at 25°C. Fig. 1shows plots of the ratio of bound ruthenium per nucleotide(rb) versus the ratio of formal added ruthenium per nucleo-tide (rf). In Fig. 1A are given the binding results for G + C-containing polymers, and in Fig. 1B, for A+T-containingpolynucleotides. Cooperative binding of the complex topolynucleotides adopting A-like conformations (19) underconditions where little binding is apparent to other nucleicacid forms is observed.

This preferential association to A-form helices is evidentclearly in Fig. 1A where binding to synthetic polymers ofidentical cytosine content is compared. The highest level ofbinding is seen to the double-stranded RNA, poly(rI)-poly(rC) and a moderate level of binding is observed withpoly(rG)-poly(dC). Under similar conditions, no binding isfound to the alternating B-DNA polymer poly[d(G-C)]. Weexamined also binding to Z-form poly[d(G-C)] in the pres-

ninni n inn .

o.

.tvAXA.0 0.0450.i0A

~.050

0.0-o 14

0.0 0.045 0.105

rr

.B

0.050

0.0 -r'+"- r

0.0 0.045 0.105

FIG. 1. Binding of Ru(TMP)"+ to synthetic double-strandedpolynucleotides. (A) A, Poly(rI)-poly(rC); *, poly(rG)-poly(dC); A,

poly(dG)poly(dC); o, B-form poly[d(G-C)]; and x, Z-formpolyld(G-C)]. (B) A, Poly(rA)-poly(rU); *, poly(rA)-poly(dT); o,polyld(A-T)I; and A, poly(dA)-poly(dT). rb, Ratio of bound ruthe-nium to nucleotide concentration; rf, formal added ratio of metal pernucleotide.

ence of cobalt hexammine (20). Although 40 ,uM Co(NH3)3 +reduces Ru(TMP)"+ binding also to poly(rI)-poly(rC) by afactor of two, no binding comparable to the A-form isapparent with Z-DNA.

Poly(dG)-poly(dC) has been seen to adopt the A-confor-mation in solution by NMR and in fibers through x-raydiffraction experiments (21-24). Ruthenium binding to thisdouble-stranded polymer at somewhat lower levels than tothat with poly(rG)-poly(dC) is seen consistent with a prefer-ential association to A-forms. We earlier (12) found littlebinding to calf thymus DNA, ostensibly in the B-form, undersimilar conditions. Fig. 1B shows binding results for A + T-containing polymers. For these polynucleotides the highestassociation is again to the double-stranded RNA, poly-(rA)-poly(rU), with a cooperative association also to theDNARNA hybrid poly(rA)-poly(dT). Possibly the some-what reduced binding to this DNARNA hybrid results fromits propensity to adopt B-like conformations as well as theA-form (25, 26). In the case of the A + T-containing DNAcopolymers, little binding is evident either to alternatingpoly[d(A-T)] or to the homopolymer duplex poly(dA)-poly-(dT), both of which likely adopt a B-conformation in solution(19, 27). The absence of binding to poly(dA)-poly(dT) shows,furthermore, that it is not simply a preference for a homo-purine-homopyrimidine sequence that is the determiningfactor in promoting binding. The binding results, for exampleat an rf of 0.075, indicate also a preference for the G + Cpolynucleotides over A+ T-containing duplexes of =2:1. Itshould be noted that for all polymers some precipitation maybe evident with time at high rf values (>0.1), given the poorsolubility of Ru(TMP) +, and this precipitation may accountfor some cooperativity seen at high formal ratios.For all dialysis experiments with racemic Ru(TMP)f ,

levels of chiral discrimination were determined throughmeasurement of optical enrichment in the dialysate in theless-favored enantiomer. In all instances, the preferentialbinding of A-Ru(TMP)2 + was observed. The level of enan-tiomeric preference varied from 56% to 92% depending uponrb, and lowest binding levels showed the highest associateddiscrimination. There was no variation observed in levels ofstereoselectivity with base composition or polynucleotideform.We examined also whether binding of the ruthenium

complex leads to the induction of conformational changes inthe polymer. Circular dichroism spectra in the ultravioletregion as a function of titration of each of the polynucleo-tides and conditions shown in Fig. 1 with racemicRu(TMP)2 + were conducted. Interestingly, these titrationsrevealed an increase in the positive band (280 nm) of thecircular dichroism for poly(rI)-poly(rC) with increasing ru-thenium bound, though the negative peak at 262 nm re-mained constant (28). This result may indicate the inductionof greater A-form character for this polymer with rutheniumbinding or may reflect an induced circular dichroism associ-ated with binding. No change in circular dichroism wasobserved for any B- or Z-form polymer despite the additionof high levels of racemic Ru(TMP)2 .

Photocleavage of Synthetic Polynucleotides. Visible irradi-ation of the intense metal to ligand charge transfer band inRu(phen)2 + and tris(bipyridyl)ruthenium(II) have beenshown (29, 30) to promote DNA cleavage in a reaction likelymediated by singlet oxygen (31). Analogous photochemistry(H.-Y.M. and G. Duveneck, unpublished results) may beapplied to Ru(TMP)2 + to convert conformation-specificbinding into conformation-specific cleaving.

3H-labeled synthetic polynucleotides were incubated withruthenium complex and irradiated at 442 nm to promotecleavage of the polymer. The level of polymer degradationwas then assayed by acid precipitation of the polynucleotide,and the amount of label solubilized was determined. As

Proc. Natl. Acad Sci. USA 85 (1988)

v.

Page 3: Tris(tetramethylphenanthroline)ruthenium(II): A chiral probe that

Proc. Natl. Acad. Sci. USA 85 (1988) 1341

reported (12), irradiation of Ru(TMP)" leads to cleavage ofpoly(rC) poly([3HldG) but not poly([3H]dG-dC) and an enan-tiomeric preference, favoring the A-isomer, is observed.Table 1 summarizes photocleavage results obtained forG + C- and A+ T-containing polynucleotides. The resultsresemble but do not parallel binding results given above.The A-form poly(rC)-poly(dG) and poly(dC)-poly(dG) bind

A-Ru(TMP)"+ and are cleaved by the ruthenium complex.The relative cleavage levels reflect to some extent theirrelative levels of binding. A similar trend is not apparent forA+ T-containing polynucleotides. Although poly(rA)-poly-(dT) binds the ruthenium complex, indeed to the same levelas poly(dC)-poly(dG), no similar degree of cleavage is ob-served. Poly[d(A-T)] also neither binds nor is cleaved byA-Ru(TMP) +. Consistent with the binding results, photo-cleavage by A-Ru(TMP)"+ of the polymers was found to beapproximately twice that with A-Ru(TMP) +.These results may be understood in terms of the singlet-

oxygen-mediated cleavage mechanism. The relative rates(32) of reaction of 102 with deoxynucleotides decrease in theorderdGMP>> dTMP> dCMP dAMP. The efficiency ofcleavage of guanine-containing polymers would, therefore,be expected to be greater (as much as two orders ofmagnitude) than that of thymine-containing polymers at thesame level of bound ruthenium. Also consistent with thesinglet-oxygen mediation of the reaction is the observation,apparent in Table 1, that a small level of cleavage ofpoly[d(G-C)] can be detected despite the fact that no bindingis apparent. The observed level of cleavage can be ac-counted for by the diffusion of singlet oxygen to the DNAstrand, and in particular to guanine-containing residues,after sensitization by free ruthenium in solution. The results,therefore, reflect a preferential cleavage of polynucleotidesphotoinduced by Ru(TMP)2+ based in part upon the char-acteristics of binding, that is favoring the A-conformation,and in part upon the efficiency of singlet-oxygen-mediatedcleavage reactions, favoring guanine residues.Mapping Cleavage Sites Along a Linear DNA Fragment.

The coupling of photochemistry to conformation-specificbinding by the metal probe permits the determination ofwhere locally along a DNA strand conformationally distinctsites recognized by the probe may occur. To locate discretesites bound and cleaved by the metal complex, a 32P-end-labeled double-stranded DNA fragment is incubated with thecomplex, irradiated to promote cleavage, and then electro-phoresed on a high-resolution denaturing polyacrylamidegel. From the length of the single-stranded end-labeledfragment, the position of cleavage may be deduced.

Fig. 2 shows the autoradiograph of a polyacrylamide gelcontaining linear fragments (HindIII-Ssp I) cleaved by race-mic Ru(phen)2 +, A-Ru(TMP)2 +, A-Ru(TMP)2 +, or aMaxam-Gilbert guanine-specific sequencing reaction. Con-sider first the pattern obtained after photocleavage withRu(phen)2 . The pattern observed resembles that of theguanine-sequencing reaction and is consistent with the pref-erential reactivity of singlet oxygen with guanine residues.

Table 1. Polynucleotide cleavage by A-Ru(TMP)3

Polymer rb % cleavagePoly(rC).poly([3H]dG)* 0.032 47.3Poly(dC)-poly([3H]dG) 0.02 18.9Poly([3H]dG-dC)-poly(dG-dC) 0.0 7.4Poly(rA)-poly([3H]dT) 0.02 0.0Poly(dA)-poly([3H]dT) 0.0 0.0

Photocleavage experiments were performed in buffer R contain-ing 1.2 mM histidine. Cleavage was monitored by the retention on

04

w -1

n--

ONW

v5'

W-4295

'e-4344

4.11-3.

kf. w 48

.: .:

I...n8

ii-4344RA

~~4bv-8

FIG. 2. Mapping of cleavage sites for Ru(phen)31 and A- and

A-Ru(TMP)I' along a linear fragment of pBR322, 31P-end-labeled at

3~~~~~~

its 3' terminus. Lane G contains the Maxam-Gilbert guanine se-

quencing reaction for the end-labeled fragment and thus shows the

positions of guanine residues. Singlet-oxygen-mediated photocleav-

age by the ruthenium complexes yields reaction at guanine residues.

A-Ru(TMP)"~binding sites, where preferential cleavage is appar-

3~~~~~~~~~~~~

ent, are indicated by the arrows.

The apparently selective guanine reaction obtained may

occur either as a result of uniform binding of Ru(phen)2 to

:~~~~~~~~~

all B-form sites coupled to a cleavage selectivity for gua-

nines, or the diffusion of singlet oxygen fromRu(phen)3 3free in bulk solution to all sites on the DNA strand and,

again, preferential reaction with guanines. A different pat-

tern of cleavage is observed after irradiation with

Ru(TMp)ar isomers. A- andA-Ru(TMp)2w also appear to

promote cleavage through photoreaction mediated by 102,and hence the same pattern of guanine-selective cleavage as

is seen for Ru(phen)sc is found. Yet in the case of A-

Ru(TMei , binding along the strand is nonuniform, andtherefore, superimposed over the guanine reaction might be

expected the preferential reaction of locally high concentra-tions of singlet oxygen generated at preferentially bound,conformationally distinct sites. Major sites of reaction with

A-Ru(TMP)32+ on the 5' strand are evident at positions4301-4308, 4316-4326, 4330-4341, 4349-4358, and 1-11.These sites are apparent also but to a lesser extent afterphotoreaction with A-Ru(TMP)32, consistent with the bind-ing stereoselectivity.A-Ru(TMP)2 + cleavage sites on the opposite strand can be

seen in Fig. 3. The 5'-32P-end-labeled double-stranded frag-ment was irradiated at 442 nm with A-Ru(TMP)32+ in buffersolution, in buffer solution containing 80%o (vol/vol) 2H20,or in buffer solution containing 1 mM sodium azide. In buffersolution, four major cleavage sites for A-Ru(TMP)2+ are

3

found, at positions 4293-4301, 4323-4333, 4337-4347, and

filters of acid-precipitable radioactivity, and given here is thepercentage of counts solubilized.*Diluted with unlabeled poly(rG)-poly(dC).

Biochemistry: Mei and Barton

Page 4: Tris(tetramethylphenanthroline)ruthenium(II): A chiral probe that

1342 Biochemistry: Mei and Barton

A B C1* a& is

4297-

4325----$#

4341- ,

$ L

I4358--.* 4FIG. 3. Map of A-Ru(TMP)"2

cleavage sites on the oppositestrand, labeled at the 5' terminus,where cleavage has been con-ducted in buffer (H20, lane A),80%o (vol/vol) 2H20 (lane B), or inthe presence of 1 mM sodiumazide (lane C).

4358-4369. Another feature of the cleavage pattern at theruthenium-bound sites, evident in Figs. 2 and 3, is the strongintensity at center residues with a decreasing distribution ofintensity outward from the center of these sites in the 5' and3' directions. This characteristic distribution, unlike thespecific reaction found with cobalt complexes, is consistentwith a diffusible species, singlet oxygen, emanating from thebound site. This cleavage pattern increases both in intensityand in the width [8 base pairs in 80% (vol/vol) 2H20 versus6 base pairs in H20 for the site centered at 4359] over whichthe cleavage is seen when irradiations are carried out in 80%o(vol/vol) 2H20 (Fig. 3). The lifetime of 102 is increased in2H20 relative to H20 (45). Consistent with that increasedlifetime is an increased diffusion distance along the DNAstrand and increased intensity of reaction. The longer-livedsinglet oxygen sensitized by bound ruthenium at a particularsite leads to no extra sites of cleavage but instead leads tomore intense cleavage and a wider distribution at each site.The binding site parameters versus the cleavage parametersmay also be probed through the addition of sodium azide tothe solution. Azide ion competes with the DNA polyanion informing complexes with Ru(TMP)2+ and actually promotesprecipitation of the ruthenium complex. In the presence ofazide, then, a reduction in intensity at the specifically boundsites is seen; the background singlet oxygen reaction fromfree ruthenium in solution remains, however. Lower levelsof bound Ru(TMP)2I lead not to fewer sites but to areduction in intensity at all sites, an observation that doesnot indicate cooperative binding locally along this fragment.A-Ru(TMP)"+ Cleavage Sites. Fig. 4 displays a histogram

that indicates A-Ru(TMP)O3 cleavage sites and intensitiesalong both strands of the double-stranded pBR322 fragment

1'- TAA

42"

(lower 111 residues). The intensities for each residue wereobtained after subtraction of light controls and Ru(phen)3+cleavage intensities, so as to remove the guanine biasassociated with the 102 reaction. What remains, then, shouldbe the cleavage pattern for specifically bound A-Ru(TMP)2+.The two strongest cleavage sites for A-Ru(TMP)f3 are

seen at positions 4346-4358 on the 5' strand along thehomopyrimidine stretch CCCLTTlCGTCTTC and at posi-tions 4323-4333 on the 3' strand along another largelyhomopyrimidine stretch TATTTTTATCC. Indeed almost allcleavage sites that can be assigned for A-Ru(TMP)' + corre-spond to homopyrimidine stretches, and these are identifiedby the boxed regions in Fig. 4. The asterisk denotes onecleavage site of medium intensity where no obvious stretchof homopyrimidine is apparent. The stretches vary in lengthfrom 5 to 13 base pairs. There is no clear sequence homologyassociated with these binding sites, but there is a strikingpreference for homopyrimidine sequences.The asymmetry of cleavage pattern of the bound molecule

has frequently been used to determine features of bindingorientation (33). In contrast to intercalative binding for thechiral metal complexes, which appears to yield cleavagefrom the major groove (7), surface binding, the mode favoredfor A-Ru(TMP)"+, appears to be associated with the minorgroove (ref. 13; J. Rehmann and J.K.B., unpublishedresults). Moreover, the A-conformation, bound preferen-tially by A-Ru(TMP)'+, includes a wide and shallow minorgroove surface. Examination of the histogram shows noregular asymmetry in cleavage pattern to the 3' or 5' side. Infact only one strand of a given binding site, the homopyri-midine stretch, appears favored. Furthermore here, wheresinglet oxygen is presumably generated directly at the com-plex and subsequently diffuses outward, the greatest inten-sity of cleavage appears at the center of the homopyrimidinestretch, rather than at the edge (internal) with a matchingasymmetry shifted on the opposite strand, which is seen withmolecules bound with tethered Fe(EDTA) (33). If the com-plex were associated more toward one strand, then diffusionof 102 would give largely cleavage at the bound site andthen, by diffusion above and below, cleavage on the oppositestrand at flanking sites 5 or 6 base pairs on the 3' and 5'sides, with the intensity pattern like that which we see.Alternatively, the pattern of cleavage could be explained bythe binding of Ru(TMP)2+ to an opened groove surface atthe junctions of a homopyrimidine-homopurine segment.According to Calladine's rules (34), 5' Y-R 3' segments(where Y is a pyrimidine and R is a purine) would give riseto clashes in the minor groove and so widen the majorgroove surface. Hence for a 5' R-(Y),-R 3' stretch, onewould find a minor groove surface on the 5' side, a majorgroove surface on the 3' side, and thus a pattern of cleavage(arrows indicate cleavage) of high intensity in the central

I111111 Ill. III11I1TCI ll ll I I ll,,. 111111 1GIAACC~iTATT A nrAT GACATTlAACCT AT AAAAA iiG'Gkhlyi; iGAGG2rccr1rTclT1 tGr1 ic1 l rrtt

o--ttt_...... ................----- .lC -. ........... . ____......... .

I l I I

FIG. 4. Histogram indicating cleavage by A-Ru(TMP)2+ along the DNA fragment. The boxes indicate the homopyrimidine stretches (5-13base pairs) that may constitute ruthenium binding sites. A 12-residue cleavage site marked by asterisks does not show the homopyrimidinepattern.

Proc. Natl. Acad Sci. USA 85 (1988)

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Proc. Natl. Acad. Sci. USA 85 (1988) 1343

oligopyrimidine segment and on opposite strands both in the3' and 5' directions.

a14 45' R-(Y)n-R 3'3' Y-(R)n-Y 5'

Specific footprints of the ruthenium binding sites could notbe obtained using hydroxyl radical footprinting techniques(35). Therefore, we cannot distinguish between these twomodels. Actually the pattern is reminiscent of that observedalong homopurine-homopyrimidine tracts that are hypersen-sitive to cleavage by S1 nuclease. These tracts also show apattern of cleavage in the interior on the homopyrimidinestrand and cleavage by the nuclease on the opposite strandflanking in 3' and'5' directions (36-38).

It is interesting that one of the two strong sites (position4325) corresponds to the AAAAA tract that hydroxyl radicalfootprinting experiments have suggested might be bent (39).Bent sites could conceivably bind the ruthenium complex;Ru(TMP)2+ reacts with only one of the two long (6, 8)adenine tracts in the promoter region of simian virus 40 DNA(40), however. That the TTTTT tract on the pBR322 frag-ment is preferentially cleaved becomes interesting also toconsider when recalling that the synthetic polynucleotidepoly(dA)-poly(dT) showed no appreciable cleavage (Table1). In the context of the pBR322 fragment, a distinct confor-mation apparently is formed and recognized at this position.Unlike the pattern observed with hydroxyl radicals, how-ever, the intensity of cleavage does not diminish over thetract from the 5' to the 3' side and is only distinctly apparenton one strand. The pattern of cleavage is understandablebased on our second model and the crystal structure re-ported (46).What the sites seem to have in common is that, for the

most part, they constitute homopurine-homopyrimidine seg-ments. There have actually been several experimental indi-cations, from a variety of techniques, that homopurine-homopyrimidine segments may adopt distinct non-B-conformations, among them bent, tripled, and A-form (1, 24,39, 41). It is also interesting to consider that the Watson-Crick base-paired homopyrimidine-homopurine strands of atriple helix resemble in conformation the A-form and mayreflect the propensity of these sequences to adopt the alteredconformation. It has also been suggested that locally A-formsites may serve as recognition elements for protein binding,in particular the transcription factor binding sites alongsimian virus 40 DNA (42) and along the 5S RNA gene ofXenopus (43, 44).What does the probe reveal about the structure of the local

sites? What A-Ru(TMP)3 + is able to recognize is the shallowsurface feature, apparent with A-form segments and possiblyapparent with other conformations as well. The convexsurface feature of the Z-DNA major groove is not similarlybound however. Certainly the basis of recognition cannot behydrogen bonding interactions, since none are possible withthe metal complex. Instead what must at least be recognizedis a distinct shape and a distinct symmetry. Whether or notthe sites bound are A-like, they are surely distinctive in theirconformation.A-Ru(TMP)2 + becomes, then, another molecular tool to

examine the local variations in conformation along thestrand. As more DNA fragments are examined, and moresequences recognized, we may be able to understand betterrelationships among these sequences and the local second-ary structures they adopt as well as how they may be

recognized by our molecular probes and perhaps by proteinsin regulating the expression of the information containedalong the strand.

We thank N. J. Turro, C. V. Kumar, and D. Patel for helpfuldiscussions. This work was supported by grants from the NationalInstitutes of Health (GM33309) and the National Science Founda-tion (CHE85-17354, Alan T. Waterman Award to J.K.B.).

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Biochemistry: Mei and Barton