Ethidium Binding Sites on Plasmid DNA Determined by Photoaffinity ...

. 259, No. 17, Issue of September 10, pp. 11090-11097,19&

THEJOURNAL OF BIOLOGICAL CHEMISTRY 1984 by The American Society of Biological Chemists, Inc

(c\

Prrnted in U.S.A.

Ethidium Binding Sites on Plasmid DNA Determined by Photoaffinity Labeling* (Received for publication, September 28, 1983)

J. Marie Hardwickt, R. Suzanne von SpreckenS, K. Lemone Yielding#, and Lerena W. YieldingST From the ZDeDartment of Biochemistry and the $DeDartment of Anatomy, University of South Aidama,College of Medicine, - . Mobile, Alabama 36688

Photoaffinitylabeling of pBR322with ethidium 5’-C-G-3’, in synthetic molecules has been demonstrated for monoazide (8-azido-3-amino-5-ethyl-6-phenylphenethidium bromide but at relatively high levels of drug (4-6). anthridinium chloride) was used to provide evidence Azido analogs of ethidium have been developed as photoaffor the sequence specifity of ethidium binding to native finity probes (7) to permitcovalent attachment of bound drug DNA. DNA-drug interactions were examined at con- and to facilitate the study of DNA-drug interactions with centrations of eight covalently bound ethidium drugs native DNA at the very low concentrations of drug which are per molecule of pBR322 (4363 base pairs). Restriction responsible forbiological effects. The photoaffinityprobe used enzyme cutting wasblocked by the covalentbinding of in this studyis an ethidium analog inwhich the aminogroup a drug molecule at (or near) the enzyme recognition in the 8-position has been replaced with anazido group. After sequence. This phenomenon was observed with all rethe drug has been allowed to intercalateDNA, the azido group striction enzymes tested and was not limited to specific is activated by exposure to visible light to form a covalent regions of the pBR322 molecule. Double-digestion exbond with DNA via a nitrene radical. In the dark, the monperiments indicated that a drug molecule may bind 2 to 3 base pairs outside the recognition sequence and oazido analog of ethidium binds to DNA noncovalently in a similar manner to its parent compound (8, 9), and also bestill block restriction enzyme digestion. Intact plasmid was treated with [3H]ethidiummono- haves like ethidium in the induction of petite mutations in azide and digestedwith restriction enzymes. The yeast (10) and in anti-trypanosoma1 activity (11).However, amount of covalently-linked ethidium analog was photoactivation of the analog in situ, enhances petite mutaquantitated for different restrictionfragments and the tion induction (10, E ) , and trypanocidal activity (11),and G-C content of each fragment was determined from the induces frameshift mutations inSalmonella (13-14) and DNA DNA sequence. In approximately half of thefragments repair synthesis in mammalian cells (15, 16). Thus, the biothe drug appeared to preferentiallybind at a G-C base logical activity of ethidium monoazide can be enhanced via pair. However, no preference for specific sequences adduct formation. such as 5’-C-G-3‘ was detected, as had been suggested We reported recently the blocking of HhaI restriction enby previous modeling studies with ethidium bromide. donuclease by ethidium monoazide binding to pBR322 (17). The other fragments were located in specific map re- We now demonstrate that this blocking of restriction enzymes gions of the plasmid and did not bind drug with a strict by ethidium monoazide is a general phenomenon. We also dependence on GC content suggesting that binding examine the bindingspecificity of ethidium monoazide for a specificity may depend on more than one structural native DNA molecule at very low drug concentrations. feature of the DNA. MATERIALS ANDMETHODS

Plasmid-One to eight 1-liter flasks, each containing 500 ml of M9 medium with 20 pg/ml of tetracycline or 100 pg/ml of ampicillin, The interactionsof small molecules with DNA are believed and 1 mg/ml of uridine (18) were inoculated with a 25-ml culture of to trigger various biological processes such as mutagenesis Escherichia coli strain RR1 containing pBR322 (obtained from G . V. and carcinogenesis, and provide the basis for some forms of Paddock, Medical University of South Carolina, Charleston SC). For cancer chemotherapy. However, the mechanisms involved in radiolabeled plasmid, 2 mCiof [3H]thymidine (80 Ci/mmol, ICN the productionof these biological effects are largely unknown. Pharmaceuticals, Inc.) or 250pCi [14C]thymidine (58 mCi/mmol, The reversibility of the DNA-drug interactions and thus the Amersham Corp.) were added per 500 ml. Chloramphenicol (150 pg/ ml) was added when the culture reached an Asso of 0.3 to 0.4 and necessity of using high concentrations of drug to achieve the incubation with rapid shakingwas continued overnight at 37 “C. The steady-state conditionsrequired for their detection have hin- cells were harvested and plasmid was purified by a large scale version dered the elucidation of these mechanisms. The reversibility of the alkalineextraction procedure of Birnboim and Doly (19). makes the isolationof the ligand-substrate complex difficult, Following alkaline extraction, the preparation was passed through cheesecloth to remove particulates and then precipitated by addition and increased drug levels used experimentally may produce of solid polyethylene glycol (8000) to 10% (w/v). The precipitate was additional nonspecific interactions which do not appear to be dissolved inTris-EDTA buffer (10 mM Tris-HCI, pH 8.0, 1 mM relevant to biological activity. I n vitro model studies have Na2EDTA) and was treated with 100 units of T1 ribonuclese for 1 h revealed a strong association between ethidium andDNA (1- at 25 “C. Following nuclease digestion, the preparation was extracted 3). A preference for pyrimidine-purine sequences, particularly twice with an equal volume of buffer-saturated chloroform-isoamyl alcohol (201), and theDNA was ethanol-precipitated. A Bio-Gel A* The costs of publication of this article were defrayed in part by 5m column (2 X 90 cm) was used to remove low molecular weight the payment of page charges. This article must therefore be hereby RNA, and DNA fractions were concentrated in a dialysis bag coated marked “advertisement” in accordance with 18 U.S.C. Section 1734 with polyethylene glycol and theDNA was ethanol-precipitated. DNA was resuspended in Tris-EDTA buffer with 100 pg/ml of Proteinase solely to indicate this fact. K for 1 h a t 37 ‘C. After the mixture was extracted twice with bufferfl Supported by National Institutes of Health Grant AI-17683.

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Ethidium Binding to Plasmid DNA

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Blocking of Restriction Enzymes-The pBR322 DNA with covalently linked drug was digested with restriction enzyme HhaI. Higher molecular weight bands a to g and i were observed consistently from restriction digests of DNA containing the covalently bound ethidium moiety (Fig. 1, Lane 2). These bandswere absent when DNA was not treated with drug (Lane I ) or when drug-treated samples were not photoactivated(not shown).Overdigestion with HhaI did not alter the intensitiesof these bands (Lune 3 ) . Size estimations of the drug-induced bands, determined from the nucleotide sequence (21), indicated that each new band resulted from the blocking of one HhaI recognition sequence (17). To confirm the origin of the drug-induced bands, pBR322 was digested with HaeII, which has a 6-base recognition sequence, d(Pur-G-C-G-C-Pyr), containing the 4-base recognition sequence of HhaI d(G-C-G-C). Therefore, HaeII recognizes a subset of the HhaI sites. The HaeII fragment A (the largest of the HueII fragments) includes some of the largest of the HhaI fragments as diagrammed in Fig. 1. A drug bound to HhaI site 30 would produce the largest of the drug-induced bands, the 669-bp' fragment, a. Likewise blocking of HhaI sites 31, 27, 28, 29, 26, and 25 would produce fragments of 538 ( b ) , 502 (c), 486 (dl, 430 (fi), 283 0'1, and 274 bp ( k ) , respectively. After covalent attachment of drug anddigestion with HhaI the isolated HaeII-A fragment produced the expected bands (a, b, c, d , j , and k ) which were lacking in the control not treated with drug (Fig. 1, Lanes 4 and 5 ) . The f z fragment was obscured in Lanes 5 and 6 by trailing of DNA which was due to ultraviolet damage during recovery of the HueII fragment from a preparative gel. Drug-induced HhaI fragments b and h span the uniqueEcoRI site. Digestion with EcoRI (Lane 6) resulted in the disappearance of fragment b. Also observed in the EcoRI digest was the appearance of the expected 237-bp digestion product of the 338-bp h fragment (which otherwise co-migrated with a normal digestion product). Fragmentx is a doublet resultingfrom the HhaI digestion of a HueII drug-induced fragment which was co-purified with the HueII-A fragment.Fromthe gel patternin Fig. 1 all detectable HhaI sites appeared to be blocked. Smaller druginduced fragments which were masked by the normal digestion products were identified by isolating the other HaeII fragments and treating with HhaI (not shown). Therefore, the blocking by ethidium monoazide does not appear to be specific for a subset of HhaI sites. Also, the drug does not preferentially bind to the HhaI restriction site d(G-C-G-C), since other restrictionenzymes were also blocked. The digestion patterns for HpaII, d(C-C-G-G), HaeIII, d(G-G-C-C), and Hinfl, d(G-A-N-T-C), are shown in Fig. 2. Again the expected fragmentscontaining blocked enzyme sites were observed. Blocked sites have also been observed with HaeII, RESULTS ThaI, d(C-G-C-G), HincII, d(G-T-Pyr-Pur-A-C),EcoRI, d(GEthidium monoazide (8-azido-3-amino-5-ethyl-6-phenyl-A-A-T-T-C),and AuuII, d(G-G-(A/T)-C-C)(notshown). phenanthridinium chloride)was added to pBR322 supercoiled Based on the locations of all the blocked restriction sites, DNA at a concentration of 17.4 molecules of drug/molecule there does not appear to be any region of the plasmid which Thedrug does not bind drug. of pBR322(drug:nucleotide ratioequalto 2 x was equilibrated with DNA in the dark for 10 min. The sample Quantitution of Drug-induced Bunds-If drug binding is a was then irradiated for 10 min with visible light to activate random event then only asmall portionof any specific restricthe azidogroup to form a nitrene radical which can then tion site would be expected to be blocked since there areonly covalently bind the drug to theDNA. The product consisted eight drug molecules/plasmid. To test this hypothesis, the of 8.3 f 0.7 covalently bound drugs per molecule of pBR322 amount of DNA found in the drug-induced bands was quanas determinedby quantitating bound and unbound 3H-labeled titated by slicing and countinggels containing [3H]thymidinedrug after photoactivation. The noncovalently bound drug as labeled DNA treated with ethidium monoazide and digested well as any free drug wasremoved effectively by ethanol with HhaI. The HhaI drug-induced bands were selected for precipitation as demonstratedby the loss of >99% of the [3H] quantitation since they were well separated from each other ethidium monoazide from drug-treated DNA samples that were held in the dark. The abbreviation used is: bp, base pair.

saturated, redistilled phenol, the DNA was ethanol-precipitated, and stored in Tris-EDTA buffer at 4 "C. Drugs-Ethidium bromide (Sigma) was recrystallized once from methanolprior to use. Ethidium monoazide (8-azido-3-amino-5ethyl-6-phenylphenanthridiniumchloride) was synthesized by the technique of Firth and coworkers. (20). Proton nuclearmagnetic resonance analysis, performed by Dr. Charles Watkins (Chemistry Department, University of Alabama in Birmingham) with a Nicolet 270 mHz Spectrometer, revealed that the ethidium monoazide preparation consisted entirely of the 8-azido isomer. Drug concentrations were determined spectrophotometrically a t pH 7.0 using extinction coefficients of 5680 M" cm" at 480 nm and 5220 M" cm" at 462 nm for ethidium bromide and the monoazide, respectively. Drug Treatment and Photouctiuation-Plasmid DNA, a t a nucleotide concentration of 2.5 X 10" M, was equilibrated for 10 min on ice in the dark with ethidium monoazide a t a concentration of 5 X M in Tris-EDTA buffer at pH 7.2. Samples were photoactivated by exposure to light from two General Electric Daylight #F15-T8D hulhs (total energy delivery at a rate of 80 J m-' s-') for 10 min on ice. After irradiation, DNA samples were digested with the appropriate endonuclease. Restriction Endonucleases-HhaI, EcoRI, HpaII, HaeII,HaeIII, Hinff, PstI, ThaI, T q I , and AuaII were purchased from Bethesda Research Laboratories. Reaction conditions were those suggested by the manufacturer, except that enzymes were used at 1.5 times the number of units recommended per pg of DNA. Electrophoresis-DNA samples were electrophoresed on 16- or 30cm 3.5% acrylamide gels (acry1amide:bisacrylamideratio = 201) or 2% agarose gels using a Hoefer slab gel apparatus. DNA samples were also analyzed on agarose gels (5 x 7.5 cm) in a horizontal system (Ann Arbor Plastics). The gels and running buffer consisted of 90 mM Tris-HC1, 90 mM borate, 2.9 mM EDTA, pH 8. DNA precipitates were dissolved routinely in gel buffer containing 3.0% (w/v) Ficoll (Sigma) and 0.0125% (w/v) bromphenol blue. All gels were electrophoresed at 100 V for 3 to 5 h. Bands were visualized by staining the gels with 0.1 Fg/ml of ethidium bromide in water and trans-illuminating with a short-waveUV light box (UV Products) for photography or by illuminating with ahand-held short wave UV source (UV Products) for band excision. Gels were photographed using a Polaroid MP-3 LandCamera with Polaroid Positive/Negative Land film, type 55. Preparation and Quantitation of Drug-treated DNA FragmentsDNA was recovered from gels by placing the DNA-containing gel slice in a pipette tip and electroeluting into a dialysis bag at 1 mA/ sample overnight, followed by ethanol precipitation of the DNA. For quantitation of radiolabeled drug or DNA, stained bands were excised or frozen gels were sliced into 0.9- or 1.8-mm fractions and gel slices were counted in Protosol/water/Econofluor (8.5:1.5:90%) after incubating overnight at 37 "C. DNA was also quantitated by scanning the photographic negatives with a Hoeffer GS300 scanning densitometer and an LKB 2210 recorder. Synthesis of r3H/Ethidium Monoazide-Ethidium bromide which was tritium labeled by aqueousexchange(Amersham Corp.) was immediately purified by cation-exchange chromatography. The monoazide (8-azido isomer) was synthesized and purified as described for the nonradiolaheled monoazide and had an absorption maximum of 462 nm (20). The specific activity was 3 Ci/mmol.


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1

2

3

4

5

6

abCd-

-b

FIG.1. HhaI restrictionenzyme sites blocked by ethidium monoazide. Untreated and drug-treated pRR322 was digested with Hhal, and electrophoresed on a 3.5% polyacrylamide gel (10 pglwell). Lane I, DNA not treated with drug; Lane 2, DNA treated with drug, then digested with HhaI; Lane 3, like Lane 2 except with a 2.7-fold overdigestion with HhaI; Lane 4 , untreated HaeII-A fragment digested with HhaI; Lane 5, like Lane 4 except pretreated with drug; Lane 6, like Lane 5 except digested with both HhaI and EcoRI. The expected HhaI drug-induced bands are shown in the diagram.

Hae II fragment A 2722

I !

236 I I

1 1

I

I

I

I

I I

I I

1 I

1

HhaIsite 3312029 #: 282726254

-k Drug-induced fragments:

3 4

-j

274 bp 283 bD c-502 - d

-2f -

bp 486 bp

1

2

4 3 0 bp 669 bp 5 3 8 bp

a

TABLE I Quantitation of drue-induced frwments Drug-induced fragmentso

% of Hhol sites blockedb

a

1.94 f 0.57 1.68 k 0.88 2.51 k 1.41 1.95 k 0.95 2.02 k 0.97

b C

d a-d

’HhaI drug-induced fragments as diagramed in Fig. 1. *The per cent of [3H]thymidine-labeled DNA found in the druginduced fragment compared to thatfound in the two normal digestion fragments from which each drug-induced fragment was generated. Data represent the mean and S.D. for five experiments.

Approximately 2% of the normal digestion products were foundinthedrug-inducedform,therefore 2% of plasmid FIG.2. Blocking of other restriction enzymes by ethidium molecules were blocked at any given restriction site. These monoazide. Untreated pBR322 was digested with HpaII (Lane I ) , experiments were repeated using [“]ethidium monoazide to HaeIII (Lane 3 ) , or HinfI (Lane 5) and electrophoresed on a 3.5% of drug bound to those fragments with polyacrylamide gel (10 pg/well). DNA pretreated with drug was quantitate the amount digested with HpaII (Lane 2). H a d 1 (Lane 4 ) . or Hinfl (Lane 6 ) . a blocked site. The results were essentially identical to those for[‘Hlthymidine andhad a mean of approximately 2% Expected drug-induced bands are marked. (1.97%).This indicated that drug was not preferentially bound and from the normal digestion products ongels. The amount to drug-induced fragments, but was bound throughout the suggested that each restricof [“]thymidine label found in drug-induced bands a, b, c, plasmid molecule. These data also tion site was blocked to a similar degree. and d was compared to that found in those normal fragments Restriction Enzyme Digestion Near the Drug-bindingSitefrom which the drug-induced bandswere generated (Table I). Eachdrug-inducedbandcontainedone blocked HhaI site If drug binding is random then the predicted blocking frebetween two adjacent fragments. The per cent froma of DNA found quency would also be dependentonthedistance restriction site that a drug could bind and still block digestion. in the drug-induced form should be equivalent to the per cent of plasmid molecules with that specific H h I site blocked. T o determine whether this is thecase, the distance between


11093

the drug-binding site and the restriction site was measured. on the second strand from the cutting site of the HincII an excess The HhaI-a fragment of 669 bp has one blocked HhaI site restriction enzyme. Evenafter digestionwith which in view of the binding stoichiometry is most likely due amount of HincII enzyme, densitometric analysis indicated to one covalently bound drug molecule (Fig. 1).Fragment a that no more than 69% of theHincIIsites were cleaved was isolated by electroelution from a polyacrylamide gel and yielding the expected 615- and 249-bp fragments (Lane 2). aliquots were redigested with ThaI, HpaII, or HincII, all of Therefore, approximately one-third of the bound drug was which have recognition sequences within 30 base pairs of the located such that it blocked both enzymes. Ifwe definea blocked site. Because of the low drug toDNA ratio, less than domainto be the area where a drugbindsand blocksa within that domain, then the 2% of the HhaI-a fragments would beexpected to have a restriction enzyme site centered second drug bound within 30 base pairs of the blocked HhaI HpaII and HincII domains must overlap by about one-third site. Therefore, any blocking of a second enzyme would be of their distances. We interpret this tomean that a drug may due to the same covalently bound drugthat blocked the HhaI bind anywhere within a domain of approximately nine base site. There are two HpaII sites in the HhaI-a fragment; one pairs andblock a restriction enzyme site within that domain. a t 26 bp from the blocked HhaI site toyield a fragment of 358 The asterisks in diagram B represent those positionswhere a drug could intercalate and block both sites. The domainsize bp (Fig. 3, diagram A ) and a second at a distant site (not of 9 basepairsis only anapproximation sincedifferent shown) to produce a 242- and a 69-bp fragment. The HhaI-a restriction enzymes may have blocking domains of different fragment was completely digested by HpaII (Fig. 3A, Lane 3). sizes. H i n d which cuts 19 bp from the HhaI enzyme site also Specificity of Drug Binding-Previous modeling studies digested HhaI-a completely toproduce a 351 and 318-bp have assignedaspecificityfor ethidiumbindingto DNA fragment (Lane 4 ) . The relativemobilities of these fragments correlate by size to the ThaI and HpaII digestion products of regionsrich in G-C content, particularly to 5'-C-G-3' seuntreated pBR322 in Lanes 1 and 6, respectively. Therefore, quences. However, such conclusions were based on studiesa t a drug must bind less than 19 base pairs from a restriction high levels of drug binding. This study of the distribution of to content provided site to causeblocking. ThaI shares the same cutting site withthe covalently bound drug in relation G-C an opportunity to look for binding specificity at a low level of HhaI on one strand and cuts two bases away on the other drug (one drug molecule/500+ base pairs). strand. No ThaI digestion products were observed (Fig. 3A, Plasmid DNA was photolabeled using [3H]ethidium monLane 2) and the ThaI-treated HhaI-a fragment co-migrated oazide and freed of noncovalentlybound drug by ethanol with the undigested fragment ( L a m 5 ) . A drug blocking the precipitation. The treated DNA was digested withTaqI,yieldHpaII site shown in diagram A produces an 864-bp HpaII ing fragments with a wide range of different G-C composifragment (Fig. 3B, Lanes 3 and 4). The blocked HpaII site is tions. The Tag1 DNA fragments were excised from a n acryllocated seven bases away on one strand and five bases away amide or an agarose gel and counted in a liquid scintillation system (Table 11). The variation in G-C content from 50% I 2 31 42 53 64 5 was computed for each fragment by dividing the per cent of G-C base pairs by 50% so that a fragment with 50% G-C A. would have a value of1.0. The amount of radioactive drug was expressed as counts/min/bp tocompensate for the varying fragmentlengths. The datawere then placed on a relative scale and the amount of radioactive drug bound to DNA of 50% G-C content was set equal to1.0. If all of the drugwere bound at a G-C base pair, then theradioactive material would HhaIgexhibit the same distributionas the G-C content. This is the case as described in Table I1 with the possible exception of 358 the 616-bp fragment. ["]Thymidine-labeled DNA was 242treatedwith nonradioactive ethidium monoazide, digested TABLEI1

TaqI digest of ethidium momazide-treatedpBR322 ['HjThymidine monoazide ['HIEthidium T h aH I haI

Hpa II

cpm Fragment

A.

cpm/bp

Relative radioactivity Theo- Experireticar mentalb

cpm c p m h

Relative radioactivity Thm- Experiretical mental

bP B.

FIG.3. Redigestion of isolated drug-induced fragments. For size standards, 0.5-pg pBR322 was digested with HpaII ( A , Lane 6; R, Lane 5 ) , T h I (A,Lune I ) , or Hinff ( B ,Lane I ) and electrophoresed on a 2% agarose gel. The drug-induced HhI-a fragmentwas electroeluted from a preparative gel and redigested with T h I (A, Lane 2). HpaII ( A , Lane 3 ) , or HincII (A, Lane 4). Undigested HhaI-a is shown in A, Lane 4. In a similar manner, the HpaII-a fragment was redigested with HincII (B,Lane 2) or was not redigested ( R , Lanes 3 and 4). The partial sequence for HhI-a and HpaII-a are shown in diagrams A and B, respectively.

1444 1307 616 368 315/312

6074 6148 2338 1274 2964

4.21 4.70 3.80 3.46 4.73

1.00 1.11 1.24 0.79 1.18

1.00 1.12 0.90 0.82 1.13

2819 2103 835 737 865

1.95 1.61 1.36 2.00 1.38

1.00 0.89 0.76 1.21 0.82

1.11 0.91 0.77 1.14 0.78

"Theoretical values are those expected for a perfect correlation between G-C content and ethidium monoazide binding. The values were obtained by dividing the per cent G-C content by 50%,thereby setting 50% equal to 1.0. * The experimental relative radioactivityis a ratio of the amount of radioactivity per base pair for each fragment to the amount of radioactivity at the 50% G-C point on the best fit line for the experimental data(see Fig. 4).

11094


with TaqI and counted in a similar manner. This control demonstrated that each fragmentwas recovered in equimolar ratios and that therewas a correlation between A-T content and the amountof incorporated thymidine label (Table 11). The relative amounts of covalently bound 3H-labeled drug were determined for eight experiments in which fragments were generated by digestion with TuqI or by double digestion with TuqI plus PstI which cuts the 1444-bp TaqI fragment to produce two fragments of 1038 and 406 bp. The mean and S.D. for the amount of bound [3H]ethidium monoazide per fragment were plotted uers’sus G-C content(Fig. 4A).All except for the 616-bp fragment (areaI) approximated the theoretical slope of 2.0 (solid line). The mean slope of eight individual experiments excluding the 616-bp fragment (and the 368-bp fragment, see Fig. 5) was 1.83 which closely approximated the theoretical slope of 2.0. The S.D. limits of the slopes from all experiments are indicated by the dashed lines in Fig. 4. The relative amounts of [3H]thymidine in TuqI-digested controls were plotted uersus A-T contentin a similar manner and had a mean slope of 2.05 with all fragments included (Fig. 4B). Although the range from 39 to 62% G-C content was small for statistical analysis, there appeared to be a close correlation between G-C content and the ethidium monoazide binding with theexception of the 616-bp fragment which consistently bound less drug thanexpected based on G-C content. The pBR322 was photolabeledwith [3H]ethidium monoazide and then digested with HinfI plus EcoRI to extend the analysis of base-specific drug binding. Only five fragments which represented about 70% of the pBR322 molecule (998, 633,517/506, and344 bp) showed a good correlation between drug binding and G-C content(Fig. 4C). The mean slope for these fragments was 1.80. The [3H]thymidine controls hada mean slope of 1.83 with all fragments included (Fig. 40). The fragments found in area 1 (298,221/220, and 154 bp) appeared to bind less drug than would be expected based on the G-C content. T o insure quantitative recovery, gel systems were selected to best suit the size range of DNA fragments, and the [3H]thymidine controls indicated equimolar recovery of all fragments. Also, adouble-labeled experimentin which [3H]ethidiummonoazide was bound to[l4C]thymidine-1abeled DNA produced the same results and the internal [14C]thymi-

dine control again indicatedequimolar recovery (not shown). The HinfIIEcoRI fragments in area 1 are allfound in the same region of the pBR322 molecule as the TaqI fragment of 616 bp (between positions 700 and 1500 base pairs). There was one remaining fragment(396 bp) which did not bind drug with a G-C correlation (area 2, Fig. 4C). When pBR322 was with [3H] predigested with Hinfl plus EcoRI and then treated ethidium monoazide, the same distribution of drug as shown in Fig. 4C was observed (not shown). This indicated that supercoilingwas not responsible for the variation in drug binding. The [3H]ethidiummonoazide data from severalenzyme EcoRI, HhaI, and digestsincluding TuqI k PstI, HinfI Hue11 were corrected for any variation inequimolar quantities by the thymidine recovery and plotted versus G-C content (Fig. 5). When all the points areincluded, the result is a slope of 0.27 with a poorcorrelation coefficient of r = 0.36. Most of thepointsin Fig. 5 were determined fromeight or more experiments, and the mean S.D. for all the points in Fig. 5 was only 0.08 relative radioactivity units (y axis). Since each point in Figs. 4 and 5 was reproducible, we concluded that different fragments consistently bind different amounts of drug. In this light, there are three interesting observations to bemade. The data in Fig. 5 are not randomly distributed; instead, about 50% of the fragments do closely approximate the theoretical slope for G-C correlation (solid line, Fig. 5 ) . The slope calculated from those points in Fig. 5 that are not labeled with a size designation is 1.56 (dashed line)and has a good correlation coefficient of r = 0.93. Second, those fragments which do not appear to exhibit G-C correlation fall into groups (designatedareas 1 to 4 ) based ontheir map positions on pBR322. Thus, the fragments within each area bind similar amounts of drug and are derived from overlapping map regions (Fig. 6). These data indicate that drug binding is not entirely random and that different regions of the plasmid consistently bind different amounts of drug. Third, the sizes of those fragments which appeared to exhibita G-C correlation extended over the full range of fragment sizes that were examined. However, all fragments longer than 650 base pairs demonstrated G-C specificity. This suggests that if G-C content isa determining factor, then thoseexceptions to therule

+

1,3

’H-Ethidium monoazide

,,yc

_ji

FIG. 4. Correlation of ethidium monoazide binding with G-C content. pBR322 was pretreated with 3H drug,digestedwith TaqI or TaqI plus PstI ( A ) and Hinfl plus EcoRI (C), electrophoresed on a 3.5% polyacrylamide or 2% agarose gels and the amount of radioactivity in each fragment determined by liquid scintillationcountingasdescribed under “Materials andMethods.” The mean and S.D. foreightexperiments was plottedon arelativescale versus the G-C content of each fragment (as described in the text). The dashed lines are the S.D. limits for the slopes of all experiments and the solid line has the expected slopefor perfect correlation between drug binding and G-C content. Theexperiments were repeatedusing [3H]thymidine-labeled DNA pretreated with unlabeled drug and digesting with TaqI ( B )or H i n f f plus EcoRI (Dl.

11.1 .o

.OQ

-

.08

-

.o7

,,’/

’ti-Thymidine

% G-C or

A-T

% G-C or A-T

D


11095

than the 998-bp fragment. A comparison of the amount of radioactive drug bound covalently to these fragmentsyielded a ratio of 1.29 which reflects more closely G-C content than the fragment size. The same was true for the other three examples. This again suggested thatethidium monoazide prefers to bind at a G-C base pair in these fragments. The ratio of bound [“]ethidiummonoazide for ‘the same two fragments was compared to the ratio of the frequency of the sequence 5’-C-G-3’ to determine if the drug preferred this sequence. In examples 1, 2, and 4, the ratio of the number of occurrences of the sequence 5’-C-G-3’ was not similar to the O.* ratio of radioactive drug, therefore, we can conclude that for 45 40 50 55 60 65 these fragments the ethidium monoazide did not preferen% G-C tially bind at the sequence 5°C-G-3’. Example 3 has a 5°CFIG. 5. Fragments which do not binddrug according to their G-3’ ratio which reflects the G-C content and since we have G-C content. The experimental approach and computation of results already established a correlation between the drug-binding are as described in the legend to Fig. 4 except that the experimental values were corrected for recovery based on the [3H]thymidine con- and G-C content, this example was not useful for examining trols. Those fragments which did not appear to hind drug with a G- the affinity of ethidium monoazide for thisparticularseC correlation are identified by size in base pairs. Theoretical slope quence. Examples 1 and 3 demonstrate that thedrug also did forperfectG-C correlation (solid line), experimentally determined not bind selectively at the sequence 5’-T-G-3’. Examples 1 slope for fragments without size designations (dashed line). 0, TaqI and 3 suggest a lack of affinity for 5’-Pyr-Pur-3’ sequences. PstI; W, H i n f f + EcoRI; 0, HhuI; HueII. Acomparison was also madewithonly those pyrimidinepurine sequences thatcontained aG or C (omittingthe sequence 5‘-T-A-3’);however, examples 3 and 4 indicate that EcoRl there was also no preference for these sequences. Other se4362 I quences andfragments were compared but no additional I correlations were apparent. EcoR I/Hinf & \ /yI

t

Hae IU-“.

DISCUSSION

Intheseexperiments,lightirradiation of pBR322 with ethidium monoazide after equilibration with 17 drug moleroo3 I 2 I I I cules/plasmid resulted in covalentattachment of the ethidium moiety to the extent of an average of eight molecules/plasmid. This covalentlabeling was associated withblockage of restriction enzyme cutting sites within or near the site of the drug lesion. It seems likely that the blockage results from the presence of the covalent drug adduct although it cannot be FIG. 6. Map of fragments lacking G-C correlation. Frag- determined whether attachment of the drug is accompanied are by additional damage. It is relevant, however, that noncovaments which bind less drug than expected based on G-C content designated by open burs, while fragments which bind more drug than lent drug binding high at levels of drug saturation can produce expected based on G-C content are designated by closed bars. Short lines represent restriction enzyme cleavage sites. The map positions blockage also suggesting that thepresence of the drug on the (in base pairs) are indicated on the inside circle. The areas in Fig. 5 DNA is responsible (22, 23). The utilizationof the photoaffinmay be localized on the map; 4000 to 100 bp (area 3),700 to 1500 bp ity labeling approach permitted exploration of the questionof (area I ) , 2400 to 2800 bp (area Z), and 3100 to 3300 bp (area 4 ) . specificity at very limiting drug concentration. Whatever the mechanism, these experiments have established two imporof the drug effect extends over aredetectable only onsmallerfragmentsandtendto be tant points. First, the domain averaged out over the domain of a large fragment. It was not about 9 bp of the plasmid fragment. Second, ethidium binding possible to analyze every fragment from an enzyme digest is not dictated only by G-C content of the DNA. The blocking of restriction enzyme sites by covalently since some are very small and others co-migrate with fragments froma distant location. Hence, there may be other bound drug resulted in new fragments each composed of two small regions of the plasmid which bind drug in a different consecutive restriction fragments. These drug-induced fragments were detectable onpolyacrylamide gels and were quanproportion but have escaped detection. Sequence Specificity of Drug Binding-Those fragments titated using [”]thymidine-labeled DNA. Approximately 2% that appeared to bind drug in proportion to the G-C contentof the plasmid molecules were blocked at any oneof the four were analyzed further to determine if there was a specific restriction sites examined. The probability of a drug binding sequence preferred for binding. The numberof occurrences of to a restriction enzyme site can be calculated if drug binding a specific sequence such as 5’-C-G-3’ wasdetermined for each is random asindicated by the finding that the restriction sites fragment, and the ratio of the number of such sequences of all enzymes examined could be blocked. The restriction between two given fragments was computed (Table 111). In enzyme site for HhaI is 4 bp and is a G-C rich sequence. With eight drugs bound per plasmid (4363 bp), there is a 0.73% addition, therelative amounts of covalently bound [“lethidium moiety were compared to the totalbase composition and chance of a drug binding within a specific 4-basepairsethe sequence frequency. In example 1 of Table I11 the two quence. The double-digestion experiments indicated that the Hinfl EcoRI fragments of 998 and 633 bp were compared. drug molecule may bind within a 9-base pairdomain and still domain thereby The ratioof the total number of base pairs for these fragments block a restriction site contained within that equals 1.58, but the ratio of the number of G-C base pairs doubling the chances of blockinga site to about1.6%. If equals 1.28 because the 633-bp fragment is more G-C rich ethidium monoazide bound specifically at a G-C base pair in Taq V P s t I

+

“n1-


11096

TABLE 111 Sequence specificity of ethidium monoazide Sequence“ Example

+ + + +

1 ( H i d EcoRI) 2 ( H i d EcoRI) 3 (Tuq I PstI) 4 (TuqI PstI)

998/633 bp 517 + 506/633 bp 1038/406 + 368 bp 1038/315 + 312 bp

1.58 1.62 1.34 1.66

1.291.28 f 0.03 0.98 1.391.47 1.28 1.33 1.37 f 0.18 0.95 1.52 1.38 1.30 1.51 1.52 f 0.13 1.54 1.25 1.30 1.14 1.40 1.43 f 0.07 1.03 1.25 1.39 1.39 The ratio of the number of occurrences of the indicated sequence found on both strands in the pBR322 fragments indicated. The ratio of the total number of base pairs in the pBR322 fragments indicated. The ratioof the total number of G-C base pairs in thepBR322 fragments indicated. The mean f S.D. (for three to eieht exwriments) of the ratios of cpms of [3H]ethidium monoazide that were covalently bound to each fragment. ”

this region of the plasmid, then theprobability of blocking a for theincreased drug bindingof the 206-bp fragment apparsite would depend on the G-C content of the restriction site ently was averaged into the larger 368-bp fragment. Since and surrounding sequence (approximately 2.2%). Neverthe- these two fragmentshave very similar G-C compositions less, this estimationroughly approximates the experimentally (39.8% and 39.7%), the amountof drug bound was not strictly determined blocking frequency of 2.02 k 0.97%. Our results regulated by base composition. It is interesting to note that it have indicated that a single covalently bound drug molecule is in this region of the plasmid that the promotors for the is responsible for blocking a restriction enzyme, since the ampicillin and tetracycline genes are located. Another very probability of a second drug binding independently to the A-T richregion is present inpBR322 between approximately same 9-base pair domain isso small that if blocking required 3100 and 3300 bp (Fig. 6), and restriction fragments from this two drug molecules, it would not have been detected by our region also bind more drugthan expectedbased onG-C analysis. Alternatively, if drugs were bound in a highly COOF- content (area 4 , Fig. 5). The indirect repeat of transposon erative manner such that the binding of the firstdrug directed Tn3 is also located in this region. The G-C rich region found the binding of a second drug nearby then the effective drug between 700 and 1500 bp on the plasmid appeared to bind concentration would be 4 drug pairs per plasmid molecule much less drug thanwould be expected based on G-C content rather than 8 single molecules. In this situation theexpected (area 1, Fig. 5). Although another region of the plasmid with blocking frequency would be approximately 1.1% (when the similar G-C composition appeared to exhibit G-Cspecificity, G-C content of the HhaI domains are taken into considera- several fragments from differentenzyme digests confirmthat tion), which is only 1 S.D. away from the experimentalvalue the G-C base composition does not predict drug binding in of 2.02%. Thus, this possibility cannot be completely overthis region of the plasmid. This section of pBR322 includes ruled. some of the coding sequences for the tetracycline gene. The Although ethidium monoazide blocked restriction enzyme segment between 2400 and 2800 base pairs on pBR322 (Fig. sites all around the plasmid molecule with a frequency roughly 6) also bound less drug than expected if the drug boundonly predicted for random binding, this did noteliminatethe to a G-C base pair (area 2, Fig. 5) and this area includes the possibility of any base or sequence specificity. Therefore, we origin of replication for pBR322. There are at least three explanationsby which those fragbound [3H]ethidium monoazide to pBR322 and analyzed the plasmid by using a variety of restriction enzymes to determine ments that do notshow a correlation for drug binding based where thedrug wasbound. The[3H]ethidium monoazide on G-C content could be explained. One explanation is that, appeared to bind different regions of the plasmidby different for some reason, those fragments were not present in equi[3H]thymidine-labeled criteria. For approximately half of the plasmid there appearedmolar quantities.Thedrug-treated specific- fragments from TaqI and HinfI plus EcoRI digests indicated to be a correlation between G-C content and binding ity of ethidium monoazide. It is unlikely that 50% of the that this was not the case. To confirm this conclusion [3H] fragmentsexamined would indicate a G-Ccorrelation by ethidium monoazide was bound to [14C]thymidine-labeled chance. That is, the data inFig. 5 are not a random distribu- plasmid DNA and the same resultswere obtained. Second, it tion of points. Therefore, G-C content probably does play a is possible that a structural effect related to the supercoiled role in drug specificity at least for some regions of pBR322 conformation of the plasmid was responsible since drug was under the conditionsused in this study. This correlationwas boundtoprimarily supercoiled DNA. However, when the observed for fragments which varied in size from 190 to 1444 plasmid was digested with HinfI plus EcoRI prior to drug bp and varied with G-C compositions from 44.3 to 62.3%. A treatment, again the same results were obtained. Third, it very A-T rich region around the unique EcoRI site (Fig. 6) seems likely that ethidium monoazide does not bind to all regions of the plasmid with the same affinity. We suggest that appeared to bind more drug than would have been expected if drug binding were strictly dependent onG-C content (area structural differences determined bya unique sequence of 3, of Fig. 5). The 206-bp HhaI fragment which contains the DNA affect the extentof drug binding. A comparison of those fragments which apparently bound EcoRI site, bound druglike a fragment of approximately 47% G-C even though only 39.8% of the base pairs are G-C. The drug preferentially at a G-C base pair (Table 111) indicated 368 bp-TaqI fragment overlaps about 60% of the HhaI 206- that there was not a specific dinucleotide sequence preferred bp fragment and extends further left of the EcoRI site. The for drug binding. This is in contrast to earlier reports with 5’368-bp TaqI fragment bound drug like a 43% G-C fragment ethidium bromide which indicated apreferencefora even though it only has 39.7% G-C. Therefore, the 368-bp pyrimidine-purine-3’ sequence, specifically 5’-C-G-3’ (4-6). as much as It is difficult to make a direct comparison since the earlier fragment bound more drug than expected but not the 206-bp HhaI fragment. Whatever factor was responsible reports used high levels of drug andwere often based on drug

EthidiumPlasmid Binding to

DNA

11097

To our knowledge, this is the first report of its kind on binding to dinucleotidesor to short synthetic fragments. This analysis does not prove the lack of sequence specificity drug-binding specificities on a native molecule using such low for drug binding, sincea localized specificity in a given region drug concentrations. However, this system is still removed of pBR322 would have escaped our detection. Also, if only from a n in uiuo model. In these studies, drug was bound to 10% of the drug demonstrated an affinity for a specific se- naked DNA and the effects of proteins which coat cellular known. quence while the remaining drug bound to random sequences, DNA, salts, and divalent cations are not we would not have detected it. However, if 25% of the drug would like to thank Drs. Gaubatz and Firth had bound to a specific sequence indicated in Table 111, it forAcknowledgments-We their criticisms and discussions. We would also like to thank most likely would have been detected since theexpected ratio Ginny Hollinger for technical assistance, and Mary Burnsfor typing would have been outside theS.D. of the experimental values. this manuscript. We have not searched for sequences longer than two base REFERENCES pairs which might be preferred binding sites. Furthermore, 1. Waring, M. J. (1965) J. Mol. Biol. 13, 269-282 the data suggest that there is no overall preference for 5’pyrimidine-purine-3’ sequences for those regions of the plas- 2. Waring, M. J. (1970) J. Mol. Biol. 54, 247-279 3. Bresloff, J. L., and Crothers, D. M. (1975) J . Mol. Biol. 9 5 , 103mid where drug appears to bind aatG-C base pair. 123 If ethidium monoazide doesindeedbindDNApriorto 4. Krugh, T. R., and Reinhardt, C. G. (1975) J . Mol. Biol. 97, 133photoactivation in the same manner as the parent compound, 162 5. Kastrup, R. V., Young, M. A., and Krugh, T. R. (1978) Biochemthenit may serveas a photoaffinityprobe for ethidium i s t 17,4855-4865 ~ bromide. Although the precise molecularinteractions between 6. Reinhardt, C.G., andKrugh, T. R. (1978) Biochemistry 17, covalently boundethidium monoazide and DNA have not 4845-4854 been identified, many other parameters have been used to 7. Yielding, K. L., and Yielding, L. W. (1980) Ann. N. Y. Acad. Sci. 346,368-378 show that the photoaffinity probe in question does behave 8. Garland, F., Graves, D. E., Yielding, L. W., and Cheung, H. C. similarly to ethidium bromide. (1980) Biochemistry 19, 3221-3226 Spectrophotometric analyses indicated that the noncova9. Graves, D. E., Watkins, C. L., and Yielding, L. W. (1981) Biolent and covalent interactions of ethidium monoazide are chemistry 20,1887-1892 essentially identical to those of the parent ethidium, therefore,10. Hixon, S. C., White, W. E., and Yielding, K. L. (1975) J. Mol. Biol. 92,319-329 no reorientation occurred when photoactivated (9). Analysis 11. Cox, B. A., Firth, W. J., Hickman, S., Klotz, F. B., Yielding, L. by steady-state and nanosecond fluorescence and stoppedW., and Yielding, K. L. (1981) J. Parasitol. 67, 410-416 flow kinetics indicated that the 8-azido analog used in this 12. Fukunaga, M., Yielding, L. W., Firth, W. J., 111, and Yielding, K. study bound nucleic acid similarly to ethidium bromide (8). L. (1980) Mutat. Res. 7 8 , 151-157 Recently, Laugaa andcoworkers (24) showed that the8-azido 13. Yielding, L. W., White, W. E., and Yielding, K. L. (1976) Mutat. isomer of ethidium monoazide is a photoactivatable derivative Res. 34, 351-358 whichbehavedexactlylike ethidium bromide in ‘H NMR 14. Yielding, L. W., and Firth, W. J., 111 (1980) Mutat. Res. 71, 161168 spectroscopy used to examine the drug binding to dinucleo15. Cantrell, C . E., and Yielding, K. L. (1977) Photochem. Photobiol. tides. Competition by ethidiumbromide for thecovalent 2 5 , 181-191 binding of ethidium monoazide in vivo was shown for yeast 16. Cantrell, C. E., and Yielding, K. L. (1980) Photochem. Photobiol. 32,613-619 mitochondrial DNA (25), Salmonella DNA (26), and chromatin of human lymphocytes (27). Ethidium monoazide has 17. Coffman, G. L., Gaubatz, J. W., Yielding, K. L., and Yielding, L. W. (1982) J. Biol. Chem. 257, 13205-13207 also been shown to elicit the same but enhancedresponse in 18. Norgard, M. V., Emigholz,K., andMonahan, J. J. (1979) J . assays for trypanocidal and mutagenic activities (10-12). It is Bacterial. 138, 270-272 also significant that competition was demonstrated between 19. Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7, 15131523 the azido probe and ethidium bromidefor petite mutagenesis in yeast (28). This evidence indicates that ethidium monoa- 20. Firth, W. J., Watkins, C. L., Graves, D. E., and Yielding, L. W. (1983) J . Heterocycl. Chem. 20, 759-765 zide is a photoaffinity probe for ethidium bromide. 21. Sutcliffe, J. G. (1978) Cold Spring Harbor Symp. Quant. Biol. 4 3 , The most probable event is that the ethidium monoazide 77-90 binds noncovalently like the parent compound and the gen- 22. Goppelt, M., Langowski, J., Pingoud, A., Haupt, W., Urbanke, C., Mayer, H., and Maass,G. (1981) Nucleic Acids Res. 9,6115eration of a short-lived nitrene leads to a covalent linkage. 6127 The possibility that a radical is generated which subsequently S. E., and Johnson, N. P. (1981) Biochem. J. 1 9 9 , 767binds to DNA in a manner different from the parent com- 23. Halford, 777 pound cannot be eliminated. Although the effects of bound 24. Laugaa, P., Delbarre, A., LePecq, J. B., and Roques, B. P. (1983) drug described here are most likely due to the drug molecule Eur. J. Biochem. 134,163-173 itself, it is also possible that drug-inducedDNA damage 25. Morita, T.,and Yielding, K. L. (1978) Mutat. Res. 5 4 , 27-32 occurred elsewhere due to a free radical cascade for example. 26. Yielding, L. W., Graves, D. E., Brown, B. R., and Yielding, K. L. (1979) Biochem. Biophvs. Res. Commun. 87. 424-432 However, since it is known that photoactivation enhances the 27. Cantrell, C. E., and Yielding, K. L. (1980) Biochem. Biophys. Acta same biological effects induced by noncovalently bounddrug, 609,173-179 we stand to learn much from the studyof photoaffinity probes. 28. Morita, T., and Yielding, K. L. (1977) Mutat. Res. 56, 21-30