Eur. J. Biochem. 67, 367-371 (1976)

Use of a Sequence-Specific DNA-Binding Ligand to Probe the Environments of EcoRI Restriction Endonuclease Cleavage Sites Jurgen KANIA and Thomas G. FANNING Institut fur Genetik. Universitat zu Koln (Received February 13/May 20, 1976)

The DNAs of bacteriophage lambda and adenovirus were incubated with the sequence-specific DNA-binding ligand 6,4’-diamidino-2-phenylindole. Digestion of the Iigand . DNA complexes with EcoRI nuclease and subsequent agarose gel electrophoresis demonstrated that the ligand inhibited nuclease activity at some sites, but not at others. The results suggest that diamidino-2-phenylindole can be used to probe the immediate environments of the EcoRI cleavage sites. Many regulatory proteins recognize and bind to specific sequences of double-stranded DNA. Studies on a number of such DNA sites (e.g. promoters [l], bacteriophage lambda operators [2]) have shown that the recognition sequences for a particular protein are similar in different areas of the chromosome, but not necessarily identical. Thus, although certain base pairs must be present at specific positions to assure maximal protein-DNA interaction, other base pairs in the immediate vicinity of the binding site are apparently nonessential. Because of this sequence heterogeneity, one might predict that sequence-specific DNA-binding ligands [3,4] would modify the binding characteristics of a particular protein at some DNAbinding sites, but not at others. We have tested this prediction by observing the action of the EcoRI restriction enzyme on the DNAs of bacteriophage lambda and adenovirus 2 (Fig.1) in the presence of the DNA-binding ligand 6,4’-diamidino-2-phenylindole (Fig. 2). Diamidino-2-phenylindolebinds strongly to ( A . T)-rich sequences in DNA and exhibits a preference for several continuous A . T base pairs [5] (and W. Miiller, personal communication). Our results suggest that sequence-specific DNAbinding ligands will prove to be useful tools for probing the binding sites on the DNA molecule of regulatory proteins and nucleases. MATERIALS AND METHODS Source of DNA

DNA from bacteriophage lambda and its derivatives were prepared by urea/EDTA treatment of the Enzyme. EcoRI restriction endonuclease (EC 3.1.4.32).

1

A U

2 3 L 5

i i F i nD i EnI C n

U

Adenovirus 2

0

0.2 0.L 0.6 0.8 Fractional length of chromosome

1.0

Fig. 1. EcoRI restriction cleavage mups of hacteriophage lambda and adenovirus 2 DNAs [13,14]. The cleavage products are lettered A-F according to the size of the fragment, i.e. migration distance in agarose gels. The open boxes represent the (A.F)-rich regions of the lambda [20] and adenovirus 2 114,211 genomes. The closed boxes above the lambda map represent the two (G.C)-rich regions in the right arm of the chromosome [20]. The circle on the lambda map represents the lambda attachment site

Fig. 2. The structure of6,4‘-diamidino-2-phenylindok

phage [6]. Adenovirus DNA was prepared by dodecylsulfate/pronase/phenol treatment of the virus. Virus and/or DNA samples were obtained from the following sources: lambda and lambda imm 434 bz (R. W. Davies and P. Schreier), lambda pgal 8 (J. Besemer), lambda h80 (B. Gronenborn), lambda pluc 5 (J. Shapiro), adenovirus serotype 2 (K. Baczko) and adenovirus serotype 12 (E. Fanning).

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DNA-Binding Ligand

Environments of EcoRI Cleavage Sites

1

2x10~)

6,4'-Diamidino-2-phenylindolewas synthesized by Dr Otto Dann, Universitat Erlangen [7], and was generously donated by D r W. Miiller, Universitat Bielefeld. Solutions of diamidino-2-phenylindole were made up in dimethyl sulfoxide and were stored frozen at 4 "C. (Diamidino-2-phenylindole has recently been made available commercially by Serva, Heidelberg.) Preparation of EcoRI Nuclease

EcoRI nuclease was isolated using the lac repressorllac operator filter binding assay as described previously [8]. Incubation with the Ligand and EcoRI Digestion Conditions

DNA samples were dialyzed against solution A (0.1 mM Tris-HC1, 0.1 mM EDTA, 1 mM NaC1, pH 8-9). The samples were then incubated with the ligand (normally one diamidino-2-phenylindole molecule/base pair at a concentration of 1- I0 pM diamidino-2-phenylindole) for 5 min at room temperature. Following incubation, the solutions were dialyzed against solution A for l h to remove dimethyl sulfoxide (failure to remove dimethyl sulfoxide results in a less pronounced effect on EcoRI nuclease activity, presumably due to a lowered affinity of the ligand for DNA in solutions containing dimethyl sulfoxide). The samples were adjusted to 36 mM Tris-HC1 (pH 8), 36 mM NaCl and 10 mM magnesium acetate prior to the addition of EcoRI nuclease. Incubation with the enzyme was conducted at 37 "C. The reactions were stopped by the addition of excess EDTA and the DNA concentrated by ethanol precipitation using yeast RNA as carrier. Agarose Gel Electrophoresis

Agarose gel electrophoresis was performed in 0.8 "/, agarose slab gels by a modification of standard procedures [9]. After electrophoresis the gels were stained with ethidium bromide [lo]. Under the conditions used the relationship of log (DNA molecular weight) vs distance migrated is linear below molecular weight values of 5 x lo6. Although nonlinear above this, the shape of the curve allows reasonably accurate molecular weight estimates up to lo7 (Fig. 3). RESULTS AND DISCUSSION Cleavage of Phage Lambda D N A in the Presence of Diamidino-2-phenylindole

In studying the interaction of sequence-specific DNA-binding ligands with DNA and DNA-binding

lo6 20

LO

60

80

100

Distance migrated I rnrn 1 Fig. 3. Calihration of'agavose gels with EcoRI cleavage products of lambda and adenovirus 2 DNAs. 3 kg of lambda DNA were mixed with 3 pg of adenovirus 2 DNA and digested with EcoRI nuclease as described in Materials and Methods. The digestion products were loaded on a 0.8% agarose slab gel and electrophoresed for approximately 18 h at 175 mA. After staining in ethidium bromide, the positions of the various fragments were plotted against their molecular weights [13,14]. (0)EcoRI fragments of lambda DNA. (0)EcoRI fragments of adenovirus 2 DNA

proteins we have chosen the EcoRI nuclease system for the following reason : the EcoRl nuclease recognizes a sequence of six nucleotide base pairs G-A-A-T-T-C . . . . . . , but outside this region no sequence

3-.L-L-v-v-9 homologies have been detected in those cases examined [ll]. Because of its large size (molecular weight of 80000 [12]>,however, it is expected that the protein will come into contact with regions of the chromosome outside of the recognition sequence. Thus, any differential effects on EcoRI activity seen in the presence of diamidino-2-phenylindole must be due to the interaction of the ligand with sequences near, but not within, the conserved recognition sequence. EcoRI cleavage of the ligand-lambda DNA complex resulted in the appearance of two predominant partial digestion products (Fig. 4). Based upon the staining intensity of the cleavage products, as well as the molecular weight of the partial cleavage products (determined from curves similar to that of Fig. 3), the two modified cleavage sites were determined to be sites 2 and 3 (see Fig. 1>.Time course experiments in which lambda [32P]DNAwas used and the bands were cut from the gel and counted for radioactivity indicated that site 3 in the presence of one diamidino-2-phenylindole molecule/base pair was cleaved at less than 1/20 the rate of the same site in the absence of the ligand. Site 2 was cleaved at an exceptionally slow rate in the presence of one diamidino-2-phenylindole molecule/base pair, at less than Ill50 the rate in the absence of the ligand

369

J . Kania and T. G. Fanning

2 0

A

15

1

2

3

L

Time I h )

Fig. 5. Rate of cleavage o f t h e hacteriophage lambda EcoRI nuclease site 2 in the presence of diamidino-2-phenylindole. The complexes between ligand and lambda ["PIDNA were formed and incubated with EcoRI nuclease as described in Materials and Methods. At various times the reactions were stopped with excess EDTA and the samples subjected to agarose gel electrophoresis. After staining with ethidium bromide, the bands corresponding to the E-D partial cleavage product were cut from the gel, boiled for 10 min in the presence of 1 ml of water to dissolve the agarose, and counted in a liquid scintillation counter

2

1

B

+a

+-b

1

2

3

L

5

6

Fig. 4. EcoRI cleavage of bacteriophage lambda and adenovirus 2 DNAs in the presence and absence of diamidino-2-phmylindole. The ligand concentration in both cases was approximately one diamidino-2-phenylindolemolecule/base pair. (A) Bacteriophage lambda DNA without ligand (track 1) and with ligand (track 2). Digestion with EcoRI nuclease was for 20 min. (a) represents a partial cleavage product containing the E, D and B fragments. (b) represents a partial cleavage product containing the E and D fragments. (B) Adenovirus 2 DNA without ligand (tracks 1, 2 and 3) and with ligand (tracks 4, 5 and 6). Digestion with EcoRI nuclease was for 5 min (tracks 1 and 41, 10min (tracks 2 and 5) and 15min (tracks 3 and 6). (a) Represents the presumed partial cleavage product containing the F, D, E and C fragments. (b) Represents a partial cleavage product containing the E and C fragments

(Fig.5). The other three EcoRI cleavage sites on the lambda chromosome were virtually unaffected by the presence of the ligand.

Diamidino-2-phenylindole Action on Phage Lambda DNA is Site-Specific Cleavage of the lambda genome by the EcoRI nuclease exhibits a gradient phenomenon, in which sites near the middle of the molecule are cleaved somewhat more slowly than sites near the end of the molecule [13]. To show that the inhibitory action of diamidino-2-phenylindole on EcoRI nuclease activity at site 2 is specific for that site and not for its position in the DNA molecule, we examined the cleavage patterns of lambda pgal8 and lambda imm 434 bz in the presence and absence of the ligand. Lambda pgal8 is a transducing phage for the E. coli galactose operon (the section of lambda DNA containing sites 1 and 2 has been deleted and replaced by E. coli DNA) and contains an EcoRI cleavage site of bacterial origin at approximately the same position as the lambda site 2. Lambda imm 434 bz contains a deletion from 45 % to 57 % on the lambda map (see Fig.1). Thus, in lambda imm 434 bz site 2 is deleted and site 1 is situated almost directly in the middle of the molecule (i.e. in the same position as site 2 in the wild-type DNA molecule). In neither case did the presence of diamidino-2-phenylindole have an inhibitory effect on the EcoRI cleavage at these two sites. Thus, the inhibitory action of the ligand on the cleavage of the lambda site 2 is apparently

370

due to the presence of ligand-binding sequences directly adjacent to the cleavage site and not to the position of the site in the DNA molecule. The effect of diamidino-2-phenylindole on EcoRI nuclease cleavage also appears independent of the overall base composition in the area of the EcoRI cleavage site. This is apparent when the macro-environments of lambda sites 2 and 3 are examined. Site 2 is in or near an ( A . T)-rich region of the genome, whereas site 3 is in or near a (G . C)-rich region (Fig. 1). Cleuvuge of Adenovirus 2 D N A in the Presence of Diumidino-2-phenylindole

In order to generalize the results obtained with diamidino-2-phenylindole and phage lambda DNA, we have investigated the action of the ligand at the EcoRl nuclease cleavage sites of another virus DNA. Adenovirus 2 DNA contains five EcoRI cleavage sites [14] (Fig.1). Site 1 appeared to be unaffected at all concentrations of diamidino-2-phenylindole tested. At one diamidino-2-phenylindole molecule/ base pair, however, site 5 was cut approximately ten times slower than in the absence of the ligand (Fig. 4). Also, early in the cleavage reaction in the presence of the ligand, a large partial cleavage product could be detected (Fig. 4). Based upon molecular weight this partial cleavage product probably contains the F-D-E-C region of the chromosome. The presumed F-D-E-C product disappeared approximately five times slower in the presence of the ligand than in its absence. Based upon the staining intensity of the DNA bands in the gel, the possibility also exists that site 3 is cleaved much more slowly in the presence of diamidino-2-phenylindole. Since, however, the F-D partial product co-migrates with the B cleavage product, we have not analyzed this further. As in the case with lambqa DNA the effect of diamidino-2-phenylindoleon EcoRI nuclease cleavage of adenovirus 2 DNA appears to be independent of the overall base composition in the area of the cleavage site. Adenovirus 2 site 1 (no effect with the ligand) and site 5 (inhibitory effect with the ligand) are both in or near (A . T)-rich regions of the genome (Fig. 1). Cleavuge of Other DNAs in the Presence of Diumidino-2-phenylindole

In addition to those of phage lambda and adenovirus 2 we have examined the following EcoRI cleavage sites in the presence of diamidino-2-phenylindole : two sites of bacterial origin on the transducing phages lambda pgaI8 and lambda pluc 5, six sites on the adenovirus 12 genome, and three sites of phage 80 origin in a lambda h80 hybrid phage. No detectable effect of the ligand on the rate of EcoRI nuclease cleavage at any of these sites could be detected. A site

Environments of EcoRI Cleavage Sites

of phage 434 origin on the lambda imm 434 bz chromosome was, however, cleaved approximately five to ten times more slowly in the presence of the ligand. CONCLUSION The experiments reported here suggest that the sequence-specificDNA-binding ligand 6,4'-diamidino2-phenylindole can be used to probe the environments of DNA-binding proteins. An examination of Fig. 5 demonstrates that the ligand . DNA complex near the EcoRI site 2 on the lambda chromosome is very stable. Since the cleavage reaction catalyzed by the EcoRI nuclease occurs rapidly in relation to the rate of breakdown of the ligand . DNA complex near site 2, the reciprocal of the half-life of the complex is a good approximation of the rate of dissociation of the ligand molecule(s) bound at this site. Thus, we calculate a dissociation rate of 2 x s-' (note that this value is for a ligand . DNA complex which very likely contains more than a single ligand molecule). The association rate of diamidino-2-phenylindole with DNA is not known, but is probably similar to that for the outside binding of proflavine to T2 DNA which is 6 x lo7 M-' s-' at 0.2 M sodium ion and 15 "C [15]. These two values give a Kd for the diamidino-2phenylindole . DNA complex at the EcoRI site 2 on the phage lambda chromosome of approximately 3.3 x M. Even in the event that this value is an overestimate by several orders of magnitude (which is probably not the case) the inhibitory action on EcoRI nuclease cleavage (K, = 30 nM using Simian virus 40 DNA [16]) is easily understood. The & value of 3.3 x lo-" M calculated above is only 10-fold lower than the Kd of lac repressor/lac operator interaction [17]. How can such tight binding occur between DNA and a small molecule such as diamidino-2-phenylindole? The binding mechanism of diamidino-2-phenylindole to DNA is unknown ; however, the ligand binds more strongly to doublestranded DNA than the intercalating dye ethidium bromide [5] and also binds tightly to single-stranded DNA [5].Our working hypothesis is that diamidino-2phenylindole intercalates into the DNA molecule analogous to proflavine and ethidium bromide. However, while both proflavine [18] and ethidium bromide [191show no sequence specificity, diamidino-2-phenylindole recognizes the DNA sequence at the intercalation site. Given the correct sequence, the ligand then binds tightly. Based upon the known sequence specificity of the ligand [ 5 ] , these sites are probably ( A. T)-rich. In the case of the bacteriophage lambda EcoRI site 2, the extremely slow cleavage would seem to indicate that several ligand-binding sites are present (very likely on both sides of the cleavage site). A confirmation of this, however, will require sequence data. Work along this line is currently in progress.

J. Kania and T. G. Fanning We thank Gisela Heidecker for discussion and criticism and Dr W. Miiller for communicating unpublished results and for the generous gift of diamidino-2-phenylindole.This work was supported by the Deutsche Forschungsgen7rinscl7a~tthrough SFB 74 by grants to B. Miiller-Hill and R. W. Davies.

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J. Kania, Institut fur Genetik der Universitat zu Koln, Weyertal 121, D-5000 Koln-Lindenthal, Federal Republic of Germany T. G . Fanning, Department of Biological Chemistry, University of California School of Medicine, Davis, California, U.S.A. 95616