BC(Ll), 3-1555, Bodnar et al., 5 figs., 1 tab. THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 258, No. 24, Issue of December 25, pp. 15206-15213,1983 Printed in U.S.A. Effect of Nucleotide Analogs on the Cleavage Restriction Enzymes AM, DdeI, HinfI, RsaI, of DNA by the and TaqP (Received for publication, June 6,1983) John W. Bodnarz, William Zempsky, Daryl Warder, ClareBergson, and David C. Ward From the Departments of Human Genetics and Molecular Biophysics-Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510 The cleavage of specific DNA sequences by the re- striction endonucleases AluI, DdeI, HinfI, RsuI, and TuqI has been studied by monitoring the effect of var- ious nucleotide modifications on the rate of DNA diges- tion. Bacteriophage fd DNA was completely substi- tuted in one strand with a single nucleotide analog, using an in vitro primed DNA synthesis reaction on a single-stranded viral DNA template. Twelve deoxy- nucleotide analogs were incorporated into these DNA substrates: 2-aminopurine, 2,6-diaminopurine, de- oxytubercidin, deoxyuridine, 5-bromodeoxyuridlne, 5-allylamine deoxyuridine, 5 - biotinyl deoxyuridine, deoxypseudouridine, deoxyinosine, 8-azadeoxyguano- sine, 5-iododeoxycytidine,and 5-bromodeoxycytidine. The restriction enzymes tested varied considerably in their ability to digest hemi-substituted DNAs contain- ing these modified nucleotides. Structural alterations in the base pairs immediately adjacent to thephospho- diester bonds cleaved by the enzyme reduced the rate of enzyme activity most dramatically, and in most cases more than a single determinant on each base pair al- tered activity. Interactions with nucleotides outside the recognition site seem to have little importance in the binding or catalytic activity of these enzymes. Restriction endonucleases have been used as model systems to study DNA-protein interactions because of their high spec- ificity for DNA sequences and thesimplicity of the DNA site recognized (for review see Modrich, 1979, 1982; Smith, 1979). One method of probing the DNA determinants required for restriction enzyme recognition has been to study the effect of modified nucleotides on the rate of DNA cleavage (Stahl and Chamberlain, 1978; Marchionni and Roufa, 1978; Berkner and Folk, 1977, 1979, 1983; Smith, 1979; Streeck, 1980; Back- man, 1980; Hofer and Koster, 1981; Gruenbaum et al., 1981; Huang et al., 1982). Although this approach has suggested sites of DNA-enzyme interaction, the exact points of inter- action have been only inferredsince a limited number of suitably modified DNA substrates have been available for analysis. To furtherdefine the effects of modified nucleotides on the rate of DNA cleavage by restriction enzymes we used a panel of 12 nucleotide analogs (see Fig. 1) that can be incorporated into DNA in uitro. We chose to study the restriction enzymes AluI (recognition site, AGCT), DdeI (CTNAG), HinfI * This research was supported by Grant GM-20124 from the Na- tional Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by Damon Runyon-Walter Winchell Postdoctoral Fel- lowship DRG-464-F. (GANTC), RsuI (GTAC), and TuqI (TCGA) for the following reasons. 1) They contain each nucleotide only once in their recognition sequence; thus there should be no ambiguity as to which modification might cause a given effect. 2) They con- tainbothATand GC base pairs so that the recognition determinants in both types of base pairs might be probed. 3) They cleave methylated DNA produced by dam+ strains of Escherichia coli, thus facilitating the preparation of appropri- ate substrate plasmids or bacteriophage DNA. Structural differences between AT and GC base pairs which could participate in restriction enzyme-DNA recognition are found at several points. For example, potential determinants could occur on the purine Cz, the purine C6, the pyrimidine C4, andthe pyrimidine Cs. (Foranindepthanalysis see Seeman et al., 1976 and Modrich, 1982.) We have altered the groups linked at these potential contact points to determine which of these sites may influence restriction enzyme activity. By analyzing the effect of these analogs on the rateof DNA cleavage several features of DNA-enzyme interaction were revealed. The results clearly indicated that different func- tional groups within thecleavage site influence the activity of the five enzymes differently. Structural alterationsin the base pairs immediately adjacent to the phosphodiester bond to be cleaved resulted in the most pronounced inhibitory effect. In contrast, the presence of modified nucleotides outside of the immediate recognition sequence had little effect on the rate of enzyme catalysis. MATERIALS AND METHODS Enzymes and Nucleotides-Restriction enzymes AluI, DdeI, HaeIII, HinfI, RsaI, and Tag1 were obtained from New England Biolabs as was E. coli polymerase I (large fragment). Deoxyuridine triphosphate and 5-iododeoxycytidine triphosphate were purchased from P-L Bio- chemicals, while 5-bromodeoxyuridine triphosphate was obtained from Sigma. 5-Allylamine deoxyuridine triphosphate and 5-biotinyl deoxyuridine triphosphate were synthesized as previously described (Langer et al., 1981). The deoxyribonucleotide triphosphates of 2- aminopurine, 8-azaguanine, and 2,6-diaminopurine were prepared as described by Cerami et al. (1967).Deoxyinosine triphosphate was synthesized as described by Kleinzeller (1942). Severaldeoxynucleotide triphosphate analogs were prepared by enzymatic reduction of their ribonucleotide triphosphates. Tubercidin triphosphate (Uretsky et al., 1968) and pseudouridine triphosphate (Ward etal., 1969) were converted to their deoxynucleotides with the ribonucleotide reductase from Lactobacillus leichmannii (Vitols and Blakley, 1965). This enzyme was the generous gift of Dr. R. L. Blakley, St. Jude Children’s Research Hospital, Memphis, TN. Each ribonu- cleotide triphosphate (2 mM) was incubated at 37 “C in the dark for 2 h in a reaction mix containing 0.2 M Tris succinate (pH 7.3). L. leichmannii ribonucleotide reductase (0.8 mg/ml), adenosylcobalamin (30 FM), EDTA (1 mM), and dithiothreitol(20 mM). The mixturewas then diluted 1:4 in H,O and applied to a column of diethylamino- ethylcellulose (Whatman DE52; 0.6 ml in 1-ml syringe, equilibrated in 10 mM Tris, 1 mM EDTA). After washing with 5 column volumes of 0.1 N triethylammonium bicarbonate the nucleotide was eluted in 15206 by guest on April 13, 2019 http://www.jbc.org/ Downloaded from
Transcript
BC(Ll), 3-1555, Bodnar et al., 5 figs., 1 tab.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 258, No. 24, Issue of December 25, pp. 15206-15213,1983 Printed in U.S.A.
Effect of Nucleotide Analogs on the Cleavage Restriction Enzymes A M , DdeI, HinfI, RsaI,
of DNA by the and TaqP
(Received for publication, June 6, 1983)
John W. Bodnarz, William Zempsky, Daryl Warder, Clare Bergson, and David C. Ward From the Departments of Human Genetics and Molecular Biophysics-Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510
The cleavage of specific DNA sequences by the re- striction endonucleases AluI, DdeI, HinfI, RsuI, and TuqI has been studied by monitoring the effect of var- ious nucleotide modifications on the rate of DNA diges- tion. Bacteriophage fd DNA was completely substi- tuted in one strand with a single nucleotide analog, using an in vitro primed DNA synthesis reaction on a single-stranded viral DNA template. Twelve deoxy- nucleotide analogs were incorporated into these DNA substrates: 2-aminopurine, 2,6-diaminopurine, de- oxytubercidin, deoxyuridine, 5-bromodeoxyuridlne, 5-allylamine deoxyuridine, 5 - biotinyl deoxyuridine, deoxypseudouridine, deoxyinosine, 8-azadeoxyguano- sine, 5-iododeoxycytidine, and 5-bromodeoxycytidine. The restriction enzymes tested varied considerably in their ability to digest hemi-substituted DNAs contain- ing these modified nucleotides. Structural alterations in the base pairs immediately adjacent to the phospho- diester bonds cleaved by the enzyme reduced the rate of enzyme activity most dramatically, and in most cases more than a single determinant on each base pair al- tered activity. Interactions with nucleotides outside the recognition site seem to have little importance in the binding or catalytic activity of these enzymes.
Restriction endonucleases have been used as model systems to study DNA-protein interactions because of their high spec- ificity for DNA sequences and the simplicity of the DNA site recognized (for review see Modrich, 1979, 1982; Smith, 1979). One method of probing the DNA determinants required for restriction enzyme recognition has been to study the effect of modified nucleotides on the rate of DNA cleavage (Stahl and Chamberlain, 1978; Marchionni and Roufa, 1978; Berkner and Folk, 1977, 1979, 1983; Smith, 1979; Streeck, 1980; Back- man, 1980; Hofer and Koster, 1981; Gruenbaum et al., 1981; Huang et al., 1982). Although this approach has suggested sites of DNA-enzyme interaction, the exact points of inter- action have been only inferred since a limited number of suitably modified DNA substrates have been available for analysis.
To further define the effects of modified nucleotides on the rate of DNA cleavage by restriction enzymes we used a panel of 12 nucleotide analogs (see Fig. 1) that can be incorporated into DNA in uitro. We chose to study the restriction enzymes AluI (recognition site, AGCT), DdeI (CTNAG), HinfI
* This research was supported by Grant GM-20124 from the Na- tional Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Supported by Damon Runyon-Walter Winchell Postdoctoral Fel- lowship DRG-464-F.
(GANTC), RsuI (GTAC), and TuqI (TCGA) for the following reasons. 1) They contain each nucleotide only once in their recognition sequence; thus there should be no ambiguity as to which modification might cause a given effect. 2) They con- tain both AT and GC base pairs so that the recognition determinants in both types of base pairs might be probed. 3) They cleave methylated DNA produced by dam+ strains of Escherichia coli, thus facilitating the preparation of appropri- ate substrate plasmids or bacteriophage DNA.
Structural differences between AT and GC base pairs which could participate in restriction enzyme-DNA recognition are found at several points. For example, potential determinants could occur on the purine Cz , the purine C6, the pyrimidine C4, and the pyrimidine Cs. (For an in depth analysis see Seeman et al., 1976 and Modrich, 1982.) We have altered the groups linked at these potential contact points to determine which of these sites may influence restriction enzyme activity.
By analyzing the effect of these analogs on the rate of DNA cleavage several features of DNA-enzyme interaction were revealed. The results clearly indicated that different func- tional groups within the cleavage site influence the activity of the five enzymes differently. Structural alterations in the base pairs immediately adjacent to the phosphodiester bond to be cleaved resulted in the most pronounced inhibitory effect. In contrast, the presence of modified nucleotides outside of the immediate recognition sequence had little effect on the rate of enzyme catalysis.
MATERIALS AND METHODS
Enzymes and Nucleotides-Restriction enzymes AluI, DdeI, HaeIII, HinfI, RsaI, and Tag1 were obtained from New England Biolabs as was E. coli polymerase I (large fragment). Deoxyuridine triphosphate and 5-iododeoxycytidine triphosphate were purchased from P-L Bio- chemicals, while 5-bromodeoxyuridine triphosphate was obtained from Sigma. 5-Allylamine deoxyuridine triphosphate and 5-biotinyl deoxyuridine triphosphate were synthesized as previously described (Langer et al., 1981). The deoxyribonucleotide triphosphates of 2- aminopurine, 8-azaguanine, and 2,6-diaminopurine were prepared as described by Cerami et al. (1967). Deoxyinosine triphosphate was synthesized as described by Kleinzeller (1942).
Several deoxynucleotide triphosphate analogs were prepared by enzymatic reduction of their ribonucleotide triphosphates. Tubercidin triphosphate (Uretsky et al., 1968) and pseudouridine triphosphate (Ward et al., 1969) were converted to their deoxynucleotides with the ribonucleotide reductase from Lactobacillus leichmannii (Vitols and Blakley, 1965). This enzyme was the generous gift of Dr. R. L. Blakley, St. Jude Children’s Research Hospital, Memphis, TN. Each ribonu- cleotide triphosphate (2 mM) was incubated at 37 “C in the dark for 2 h in a reaction mix containing 0.2 M Tris succinate (pH 7.3). L. leichmannii ribonucleotide reductase (0.8 mg/ml), adenosylcobalamin (30 FM), EDTA (1 mM), and dithiothreitol(20 mM). The mixture was then diluted 1:4 in H,O and applied to a column of diethylamino- ethylcellulose (Whatman DE52; 0.6 ml in 1-ml syringe, equilibrated in 10 mM Tris, 1 mM EDTA). After washing with 5 column volumes of 0.1 N triethylammonium bicarbonate the nucleotide was eluted in
Effect of Nucleotide Analogs on the Cleavage of D N A 15207
0.6 N triethylammonium bicarbonate, lyophilized, and resuspended in 10 mM Tris, 1 mM EDTA (pH 7.5) at 3-4 mM. The deoxynucleotide triphosphates were then used without further purification to remove unreacted ribonucleotide triphosphates.
Preparation of Modified DNA Substrates-Bacteriophage fd and the E. coli strain 71-18 were gifts of Eleanor Spicer (Yale University). The preparation of fd RF' DNA and single-stranded fd phage DNA have been described by Heidecker et al., (1980). Primers for in vitro DNA synthesis reactions were HaeIII fragments of fd RF DNA purified on 2% agarose gels. The DNA bands visualized by ethidium bromide fluorescence were cut out, electroeluted (125 V, overnight), phenol extracted, and ethanol precipitated twice.
In uitro synthesis of fd DNA was done using a modification of the procedures of Fiddes and Godson (1978) and Bourguignon et al., (1976). The primers, Hue111 fragments of fd RF DNA (either fragment 7 or 10 or a mixture of fragments 8 and 9), were used at a 10-fold molar excess over the fd viral DNA. Each of the primers used (all less than 160 base pairs) were located within a small segment of the fd genome (between nucleotides 5082 and 5829 base pairs; Beck et al. (1978)). Primers (2 pg/ml) and fd phage DNA (25 pg/ml) were boiled for 3 min in 80 ~1 of a solution containing 85 mM NaC1, 17 mM Tris- HC1 (pH 7.4), and 8 mM MgCl,. The primer-template complex was then formed by reannealing a t 67 "C for 15 min. The reaction mix was divided in half and each half adjusted to a 100-p1 final volume of reaction mixture containing 50 mM Tris-HC1 (pH 7.5), 10 mM MgCL, 1 mM P-mercaptoethanol, and the appropriate deoxynucleotide tri- phosphates. When dCTP analogs were studied, the control reactions contained dCTP, dTTP, and dGTP at 0.4 mM each, 0.15 p~ dATP and 50 pCi of [w3*P]dATP (4000 Ci/mmol, Amersham Cop.); the analog reactions contained 0.4 mM of the modified dCTP derivative rather than dCTP. For all other analogs studied, the control aliquot contained 0.4 mM dATP, dTTP, and dGTP, 15 p~ dCTP, and 50 pCi of [a-"*P]dCTP; in the analog reactions the appropriate normal nucleotide was replaced with its modified derivative at a concentra- tion of 0.4 mM. Since in each analog reaction the corresponding parent nucleotide was totally absent, enzymatic synthesis and 32P- label incorporation resulted in the production of polynucleotide chains fully substituted with the analog of interest. (This result could be confirmed by monitoring the extend of incorporation in control reactions that contained only the three normal nucleoside triphos- phates.) E. coli polymerase I (large fragment) was then added (0.6 unit), and both control and analog-containing reaction mixtures were incubated a t 37 "C. The rate of nucleotide incorporation was then monitored by acid precipitation. Aliquots of 2 pl were removed at various times and 10 ml of 5% trichloroacetic acid added after standing on ice, the samples were filtered through Whatman GF/A filters, the filters dried and counted by Cerenkov radiation. Although reactions were routinely incubated for 1 h, mixtures that contained analogs with low incorporation rates (see Table I) were often incu- bated for up to 5 h. The in uitro RF DNA was purified on P-60 columns (0.5 ml in 1-ml syringes) and eluted in 10 mM Tris, 1 mM EDTA (pH 7.4).
Assay for Restriction Enzyme Cleavage of Modified DNA Sub- strates-Restriction enzyme digestions were performed in the buffers recommended by the manufacturer of each enzyme. Aliquots of 20 pl, each containing 2000-8000 cpm of a control or analog-substituted fd RF DNA and 0.5 to 2 pg of carrier unlabeled fd RF DNA, were incubated for 1 h a t 37 "C with various enzyme dilutions. Starting dilutions contained 2 units/aliquot of Ddel, HinfI, RsaI, and TaqI or 8 units/aliquot for AluI; parallel samples contained 1:4, 1:16, or 1:64 dilutions of these initial concentrations. The reactions were stopped by addition of 4 p1 of 0.2 M EDTA, and the resultant DNA fragments were separated on 2% agarose gels. The gels were then stained with ethidium bromide and photographed. This control was used to dem- onstrate that the unmodified carrier DNA in control and modified nucleotide-containing samples were digested to the same extent in each reaction. Autoradiography of the dried gels was used to compare the rate of cleavage of the control and analog-containing DNA. The relative rates of cleavage could be estimated by comparing the cleav- age patterns of modified DNA with the patterns for control DNA as a function of the concentration of the restriction enzyme used.
I n Vitro Synthesis of fd RF D N A Containing One Strand Completely Substituted with Modified Nucleotides
Because of the technical difficulty of preparing DNA with nucleotide analogs in defined positions of both DNA strands we used hemi-substituted DNA substrates, prepared by i n uitro primed synthesis on a single-stranded fd DNA template. Although the analogs (see Fig. 1) are incorporated into only one strand of the duplex, previous studies have demonstrated that hemi-substituted DNAs are quite useful for evaluating the contribution of nucleotide components of individual strands in restriction enzyme-DNA interactions (Streeck, 1980; Gruenbaum et al., 1981) and repressor protein-operator DNA binding (Fisher and Caruthers, 1979). Nevertheless, we cannot exclude the possibility that the effect of analog sub- stitution would be different if the DNAs contained analogs in both strands. In spite of this caveat, the results presented below clearly delineate structural features of bases within the recognition sequence that, if altered, markedly inhibit the overall enzyme reaction ( i e . the cleavage of both normal and analog-substituted strands).
Double-stranded fd bacteriophage DNA circles with one strand containing a modified nucleotide were synthesized with E. coli DNA polymerase I (large fragment) on a template of single-stranded circular fd viral DNA that had been annealed to a gel-purified primer fragment of fd RF DNA (see under "Materials and Methods"). The extent of polymerization re- sulting from primer extension was monitored kinetically by following the appearance (in control reactions) of the various labeled fragments of known size and map position after sub- sequent restriction enzyme cleavage. Since the primers were
G-C BASE PAIR
G ANALOGS
0
B-AZAGUANOSINE
I N O S I N E
C ANALOGS
R = - I 5-~odocvt~dine
A-T BASE PAlR
A ANALOGS
2-AMINOPURINE 2,6-DIAMINOPURINE
TUBERCIDIN
T ANALOGS
0 HND 0 X - N \ 0
Pseudo- R = -H uridme uridine
-Br 5-bromocytidine -Br 5-bromourldlne - C H = C H - C H ~ - N H Z 5-allylamine
0
P H N X~~ -cH=CH-CH2-NH-c-(cH2)4-tSj
5-blotlnyl urtdtne
FIG. 1. Nucleotide analogs used in the experiments reported here.
15208 Effect of Nucleotide Analogs on the Cleavage of D N A
not labeled, any fragments cleaved by the restriction enzymes that were measurably shorter than full length linear DNA had to be due to cleavage at a restriction site containing a nucleotide analog.
Each reaction was run in duplicate with one reaction mix- ture containing the normal dNTPs, and the second containing a modified dNTP analog. The kinetics of the incorporation of the analog substrates was determined in comparison with the control reaction. The relative incorporation rate of the modified nucleotides is presented in Table I . Several nucleo- tides which showed slow kinetics of incorporation also ap- peared to cause premature chain termination. The adenine analog 2-aminopurine usually showed labeled restriction frag- ments extending only 1 to 2 kilobase pairs from the primer, and often the bands furthest from the primer were under- represented in digestions of in vitro RF made with AA-dU or 2,6-DAP.
Assay for the Effect of Modified Nucleotides on Restriction Enzyme Cleavage of D N A
Each double-stranded fd RF DNA sample made i n vitro with modified nucleotides was then assayed for its suscepti- bility to cleavage by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI. The "'P-labeled RF DNA was mixed with an excess of unlabeled carrier fd RF DNA which then served as an internal control. The fd RF DNA cleavage products gen- erated by the restriction enzymes were assessed by gel elec- trophoresis after the DNA samples had been incubated for a fixed period of time with various dilutions of each enzyme. In this way we could estimate the kinetics of cleavage of the modified substrates at various enzyme concentrations relative to the analog-free control samples. This approach gave similar results to that obtained by analyzing the effect of a single
concentration of enzyme as a function of time but required the analysis of fewer samples to assess the effect of the analog on enzyme activity. Typical results of such analysis are shown in Figs. 2 and 3, which illustrate data obtained with analog DNAs substituted with deoxypseudouridine and deoxytuber- cidin, respectively. As seen in Fig. 2 4 , ethidium bromide staining of the gel demonstrated that the digestion of carrier DNA in both control and analog DNA samples occurred to the same extent; this indicates that the restriction enzyme was as active in the presence of the modified DNA substrate as it was in the presence of unmodified (control) DNA. After drying, the gel was autoradiographed to allow side-by-side comparison of the rate of cleavage of the control and pseu- douridine-modified DNA substrates (Fig. 2B). The relative rate of cleavage could then be estimated by comparison of the digestion patterns. This assay technique does not permit one to determine if the analog effect is on the initial recognition of the site sequence, the binding of the site per se, or the catalysis of phosphodiester bond cleavage after specific bind- ing. Even though the primary process affected by the analog base cannot be precisely defined, the method provides a rapid and sensitive means of identifying structural determinants that are important for the overall cleavage reaction.
Fig. 2B shows that DdeI and TaqI cleave the GdU-substi- tuted DNA at rates similar to that seen with control DNA; however, the GdU-DNA substrate is totally resistant to AluI and RsaI, even though the carrier DNA in the same reaction mix is fully digested. HinfI cleaves the $dU-modified DNA; however, the rate is much slower than that observed with the control DNA. The enzymes also exhibit a different spectrum of activity when tested on the DNA substrate substituted with the adenosine analog deoxytubercidin (Fig. 3 ) . Both RsaI and TaqI cleave this DNA as rapidly as they do control DNA; DdeI and AluI are essentially inactive on this substrate, while
TABLE I Effect of modified nucleotides on cleavage by restriction enzymes and polymerization with E. coli polymerase I (large
fragment) Polymerization Rate of cleavage"
Analog (AGCT)
AluI (CTNAG)
DdeI (GANTC)
Hinfl (GTAC)
RsaI (TCGA) T d
Adenine 2-Aminopurine 2,6-Diaminopu-
Deoxytuberci- rine
din Thymine
dU WJ 5-BrdU 5-Allyamine dU 5-Biotinyl dU
dI 8-AzadGd
Guanine
Cytosine 5-IdC' 5-BrdC 5-BrdC + dU
Minor Groove 2.6-DAP + dI
+ + 0
+ 0 + 0 0
+ + 0 0 0
-
+ -
0
+ + + 0 0
+ + 0 0 0
-
- + +
+ + + + + + + + + + +
rate with E. coli
polymerase I (large
fragment)b
5 25
N D
72 ND 108 34 50
98 71
40 109 ND
41
a Rate: + = cuts >25% of control; - = cuts <25% of control; 0 = no cleavage detectable. Rate is per cent of radioactivity incorporated with the modified nucleotide relative to that of the unmodified
Effect of Nucleotide Analogs on the Cleavage of DNA 15209
A A l u I Dde I Hinf I Alu I Dde I Hinf I
Rsa I T a q I H h a I
B Alu I D d e I Hinf I nnn nnn nnn
1 2 3 1 2 3 1 2 3 A 0 A 0 A 0 10 A 0 A 0 AB A B A R - "" -_
Rsa I T a q I H h a I
FIG. 2. Assay for restriction enzyme cleavage of pseudour- idine-substituted DNA. Double-stranded '"P-fd DNA substrates substituted in one strand with deoxypseudouridine were synthesized
FIG. 3. Assay for restriction enzyme cleavage of deoxytu- bercidin-substituted DNA. Autoradiograph of 2% agarose gel of control ( A ) and deoxytubercidin-substituted ( E ) fd DNA assayed as described in the legend to Fig. 2.
H i n f f cuts at a much reduced rate. Enzymes that do not contain an AT base pair in their recognition sequence, e.g. HinfI and HhaI, restrict DNAs substituted with either deox- ypseudouridine or deoxytubercidin as efficiently as control DNAs (Fig. 2 B and Fig. 3).
DNA substrates containing each of the 12 nucleotide ana- logs were analyzed in a similar fashion for their ability to be restricted by AluI, DdeI, H i d , RsaI, TaqI. The results of these studies are summarized in Table I, and the significance of these results with respect to enzyme-DNA interactions is discussed below.
Effect of Modifications at the C-5 Position of the Pyrimidine Ring
Structural determinants which could be used to discrimi- nate between AT and GC base pairs in the standard B-DNA configuration occur in both the major and minor grooves. The most readily probed of the possible major groove determinants are structural differences at the 5-C position of the pyrimidine
as described under "Materials and Methods" and mixed with an excess of unlabeled fd RF DNA. Each sample was cut with a dilution of restriction enzyme and run on a 2% agarose gel. A, ethidium bromide-stained gels show that each enzyme retains full activity on the control DNA when assayed in the presence of modified DNA substrates; E , autoradiograph of dried gels. Lane A, control (unmod- ified) substrate; Lane B, deoxypseudouridine-substituted substrate. Sample I , each reaction contains 2 units of restriction enzyme except AluI assays which contain 8 units; sample 2, each reaction contains one-fourth the enzyme used in sample 1; sample 3, each reaction contains one-sixteenth the enzyme used in sample 1.
15210 Effect of Nucleotide Analogs on the Cleavage of DNA
ring. This position is normally methylated in AT base pairs but protonated in GC base pairs. We modified this segment of the thymidine base by removing the methyl group (i.e. using deoxyuridine) and by replacing the methyl group with a bromine atom or other more bulky substituents, such as allylamine and allylaminobiotin. In addition the C-5 carbon atom itself was substituted with a nonprotonated ring nitro- gen by incorporating deoxypseudouridine in place of thymi- dine. As shown in Table I, these alterations affected the enzymatic activity of each enzyme somewhat differently. AluI cleaved DNAs containing the dU or 5-BrdU analogs at rates near that observed with control DNA, but it was totally unable to restrict DNA containing the +dU, 5-AA-dU, or the 5-Bio- dU analogs. RsaI exhibited a similar pattern of activity, al- though it restricted dU-substituted DNA more slowly than AluI. DdeI rapidly cleaved dU-, +dU-, and 5-BrdU-substituted DNAs but it was also inactive on the DNAs containing the bulkier substituents. HinfI cleaved dU-, 5-BrdU-, and 5-AA- dU-substituted DNAs with high efficiency, +dU-substituted DNA slowly, but it was unable to restrict DNA containing the biotinylated nucleotide analog. In contrast, TaqI rapidly cleaved all of the analog-substituted DNAs. These results indicate that the C-5 domain of the thymidine ring is impor- tant for the activity of each enzyme tested except TaqI. We suggest that this observation reflects an enzyme-DNA inter- action, most likely hydrophobic in nature, although the methyl group per se is not an absolute requirement as each of the enzymes will digest DNA containing dU or 5-BrdU ana- logs. Since neither AluI nor RsaI will cleave +dU-substituted DNA, where the C-5 carbon atom is replaced with a more hydrophilic ring nitrogen, the hydrophobic interaction with this site may be stronger or more important for these two enzymes than for DdeI or HinfI. The observation that none of these four enzymes will cleave DNA with the bulky bio- tinylated substituent in the C-5 position is also consistent with an enzyme-DNA interaction at this site.
Methylation of cytidine residues at C-5 is a modification used in many restriction/modification systems to protect cel- lular DNA from cleavage (Mann and Smith, 1977; Streeck, 1980; Gruenbaum et al., 1981). We, therefore, expected to find that the activity of some of these enzymes would be reduced or abolished by introducing halogen atoms at this position. Addition of iodine or bromine atoms to the C-5 carbon of the cytidine ring completely stops cleavage by AluI and DdeI, slows the rate of cleavage by HinfI and RsaI, but has virtually no effect on the enzymatic activity of TaqI. These results support and extend the earlier work of van der Ploeg and Flavell (1980) who reported that AluI would not cleave DNA substituted with &methyl cytosine whereas HinfI and TaqI would. While it is apparent that the enzymatic activity of both AluI and DdeI is abolished by the introduction of small substituents on the cytosine C-5, studies with the thymidine analogs indicated that substituents in the thymidine C-5 position were also important for enzymatic activity. I t is interesting to note that in both cases the thymidine C-5 methyl group is in the same relative position in the DNA helix as the C-5 proton of cytosine but on the adjacent base pair (see Fig. 5, A and B ) . This raises the possibility that the recognition determinant could simply be the absence (-) of a C-5 C modification in combination with the presence (+) of a C-5 T substituent (e.g. CH, or Br) on the neighboring base. In the case of AluI, the C and T bases of the recognition sequence (AGCT) would thus be normally sensed as -,+; 5- BrC- or 5-MeC-substituted DNA (i.e. +,+) would thus not be cleaved. If the presence or absence of a C-5 group on these base pairs was the only factor that determined the ability of
the enzyme to discriminate between AT and GC base pairs, one would predict that DNA doubly substituted with 5-BrdC and dU (+,-I would be resistant to cleavage at the normal sites but would instead be cleaved at modified AGTC se- quences (i.e. AGdUSBrdC, -,+). Since DNA totally substi- tuted in one strand with both 5-BrdC and dU is completely resistant to digestion (with no new cleavage fragments appar- ent), the discrimination of T from C bases requires more than a simple means of detecting the coordinate presence and absence of methyl groups at the C-5 positions.
Minor Groove Modifications A second possible mode of discrimination between AT and
GC base pairs is the recognition of the presence of a C p amino group on guanosine or its absence on adenosine. This differ- ence is seen in the minor groove of the DNA helix and is essentially the only significant difference between AT and GC base pairs in the minor groove. We investigated the possible role of minor groove alterations in enzyme recogni- tion by using the G analog, dI, and by using the A analog, 2,6- DAP. By adding a Cp amino group the 2,6-DAP-T base pair mimics a GC base pair in the minor groove, and by removing the Cs amino group of G a dI-dC base pair mimics an AT base pair in the minor groove. If only minor groove determinants were important for recognition, incorporation of one or both of these analogs should change the restriction map. As sum- marized in Table I, DNAs containing either 2,6-DAP or dI as the sole analog were cleaved by all five enzymes, although in some cases a t somewhat slower rates than control DNA, and all fragments produced by digestion were similar to those seen in control DNA samples. Furthermore, all enzymes but HinfI cleaved DNA substituted with both analogs simultaneously. Changes in minor groove substituents, therefore, appear not to have an appreciable effect on the activity of these enzymes. Thus, with the possible exception of Hinfl, none of the enzymes seem to have specific recognition determinants in the minor groove of DNA. Studies with EcoRI (Modrich and Rubin, 1977; Lu et al., 1981) have shown that this enzyme also does not interact with determinants in the minor groove. The reduction in the rate at which the modified DNAs are cleaved may simply reflect subtle changes in secondary struc- ture induced by the altered pattern of hydrogen bonding expected with the 2,6-DAP and dI analogs.
Major Groove Modification of Purines
The other major difference between AT and GC base pairs is the orientation of the carboxyl and amino groups at the purine C, and pyrimidine C,. This is a difficult region of the DNA to probe with analogs since most modifications will affect the base pairing potential of the nucleotide and inhibit enzymatic incorporation of the nucleotide into DNA. We were able, however, to probe the effect of the Ns position on adenosine by the use of 2-AP and 2,6-DAP. 2-AP has a Cz amino group in the minor groove like 2,6-DAP, but its C, amino group is removed. Thus these analogs can coordinately probe the effects of each part of the adenine nucleotide; changes due to the addition of the Cs amino group will be seen for both analogs while changes due to removal of the CS amino group will be seen only in the 2-AP-substituted DNA. AluI and RsaI are insensitive to both changes and cleave these analog-substituted DNAs at rates similar to control DNA. Although the activity of HinfI and DdeI is reduced somewhat by the minor groove addition, they also restrict DNA substi- tuted with 2-AP. The removal of the c6 amino group markedly reduces cleavage by TaqI, suggesting that the CS amino group is part of the TaqI restriction enzyme-DNA interaction site.
Effect of Nucleotide Analogs on the Cleavage of DNA 15211
This is consistent with the fact that modification with an adenosine C,; methyl group stops Tag1 cleavage (Sato et al., 1980; Backman, 1980).
We also probed for interactions with two regions of DNA that appear to be similar in AT and GC base pairs but may be important for restriction enzyme activity nonetheless. In the tubercidin analog of adenosine the N, nitrogen is replaced by a carbon atom; we have found that this replacement slows cleavage by HinfI and RsaI and completely stops cleavage by AluI and DdeI. Thus hydrogen bonding to the adenosine N: may be important in binding these enzymes to the DNA. In contrast, replacement of the CR carbon and its associated proton with a nitrogen atom (i.e. using 8-azaguanosine) has shown no effect on any restriction enzymes tested including AluI, BamHI, ClaI, DdeI, EcoRI, HaeIII, HhaI, HinfI, RsaI, or TaqI.
Effect of Adjacent Sequences on Restriction Enzyme Cleavage Several investigators have noted that different restriction
enzyme sites on the same molecule are often cleaved a t different rates (Thomas and Davis, 1975; Forsblom et al., 1976; Halford et al., 1980; Armstrong and Bauer, 1982; Ber- kner and Folk, 1983). This suggests that factors other than the primary sequence of the restriction site may be involved in determining the rate of enzymatic cleavage. This could be due to either interaction of the restriction enzyme with a larger region of the DNA than the restriction site itself or due to changes in DNA conformation caused by the adjacent sequences. However, when synthetic oligonucleotides are used as substrates for EcoRI (Greene et al., 1975; Goppelt et al., 1980) and HpaII or MnoI (Baumstark et al., 1979) cleavage is observed with oligonucleotide duplexes that are only 2 base pairs longer than the restriction enzyme recognition site. These observations suggest that alternate DNA forms (such as cruciforms) or neighboring sequences are not involved in restriction enzyme binding or cleavage.
It has also been reported that DNA binding drugs affect the rate of cleavage of different enzymes in a manner that is dependent on the sequences neighboring the site (Malcolm and Moffatt, 1981; Nilsson et al., 1982). Actinomycin D, which binds at GC dinucleotides, inhibits cleavage at restriction sites which are flanked by GC base pairs while neotropsin slows cleavages a t sites flanked by its preferential binding site (AAA). Both of these drugs have been shown to distort the DNA double helix several base pairs from their binding site (Sobell, 1973; Pate1 and Canuel, 1977; Helene and Maurizot, 1981). Therefore, the mode of inhibition of restriction enzyme cleavage by these drugs is likely due to distortions in second- ary structure which sterically inhibit enzyme binding.
With this data in mind we have examined the pattern of cleavage of each analog-substituted DNA by AluI, DdeI, HinfI, RsaI, TaqI, HaeIII, and HhaI. We would expect that if any site were particularly affected by a modification at a neigh- boring base pair that certain partial digestion bands would be particularly prominent a t one or more of enzyme dilutions used. Such partial digestion products would be evident in side- by-side analysis with the partial and total digestion patterns seen for the control DNA. We examined the patterns of restriction fragments generated from all the modified DNAs and found that neighboring effects were detectable only in HaeIII digests of AA-dU- and Bio-dU-substituted DNA. Fig. 4 shows the digestion patterns for unsubstituted (lane A ) and Bio-dU substituted fd DNA (lane B) with HaeIII. (The Bio- dU-substituted DNA migrates more slowly in agarose gels due to the additional molecular weight of the added biotinyl side chains.) Fragment C is under-represented in the Bio-dU DNA
fd H a e m DIGEST
B- -B
-C/F
C -, -C
A =CONTROL B= Bio-dU SUBSTITUTED
C/F - - T C A A ’ G G ~ A A T C - JUNCTION - - AGBBCCGGBBAG-
FIG. 4. Cleavage of 5-biotinyl deoxyuridine-substituted DNA with HaeIII. Double-stranded fd DNA substituted with 5- biotinyl deoxyuridine was synthesized as described under “Materials and Methods,” cut with HaeII1, and run on a 2 4 agarose gel. Lane A, control (unsubstituted) fd DNA; Lune R, 5-biotinyl deoxyuridine- substituted fd DNA. (Fragments in this sample run slower than in the control DNA because their molecular weight is increased by the biotinyl side chains.)
and a new band is seen which has the size expected of a C/F fusion fragment. This is the same site that is cut very slowly in AA-dU-substituted DNA. The sequence of the viral strand at this HaeIII site (2245 base pairs; Beck et al., 1978) is -TCAAGGCCAATC. Thus the RF DNA synthesized in vitro in the presence of Bio-dU or AA-dU contains substituted thymidine nucleotides adjacent to the HaeIII site (GGCC) on both sides. Even with these extremely bulky modifications the restriction enzyme slowly cleaves the DNA. In every other case examined we have found that if the restriction enzyme cuts the modified DNA at all, both the pattern of partial digestion products and rate of their subsequence cleavage with increasing enzyme concentration were the same as for the unmodified DNA. Furthermore with DdeI (CTNAG) and HinfI (GANTC), where a fraction of the cut sites are also modified at the unspecified middle base pair, there was no evidence of different cleavage rates due to modification on that base pair. There was also no evidence that any other A or T analog significantly inhibited cleavage by HaeIII (GGCC) or HhaI (GCGC) at any other site. The only modified A or T analog which even marginally reduced the overall cleavage rate of either enzyme was 2-AP, which was cleaved slowly by HaeIII. In general the nucleotides which might be expected to distort the DNA secondary structure by changing the normal pattern of hydrogen bonding in base pairs (2-AP, 2,6- DAP, dI, and 2,6-DAP/dI together) tended to slow down the
15212 Effect of Nucleotide Analogs on the Cleavage of D N A
enzymes. These observations are entirely consistent with the oligonucleotide cleavage and drug inhibition studies and imply that the nearest neighbor effects of sequences outside the restriction sites are due mainly to distortions of secondary structure at the recognition site. All the restriction enzymes examined to date, therefore, appear to bind specifically to a very localized region of the DNA.
Determinants within the Restriction Enzyme Recognition Sequence
Specific structural alterations in the nucleotide residues that comprise one strand of a recognition sequence have been shown to inhibit the overall cleavage reaction of each enzyme studied. The spatial distribution of modifications within the recognition sequence that reduced the enzymatic activity of AluI, DdeI, HinfI, and RsaI are illustrated in Fig. 5 . In each case, alteration of functional groups on the base pairs imme- diately adjacent to the phosphodiester bonds to be cleaved exhibited that most pronounced inhibitory effect. Interest- ingly, even though our assay method measured only the ki- netics of the overall enzyme reaction, the results are similar to those one would expect from direct "footprint" analysis of enzyme-DNA binding site interactions. This observation sug- gests that the specific interaction between the enzyme and its recognition sequence per se is the major contributing factor to the overall kinetic process and that the analogs are, there- fore, indirect probes of determinants that are important in enzyme-DNA recognition. Our results can thus be interpreted to indicate the following features of restriction enzyme-DNA interactions.
AluI (Fig. 5A)"This enzyme appears to recognize cytidine by the lack of methyl on the cytidine Cs. It recognizes thy- midine by a hydrophobic interaction near the C, methyl; however, this is not the only determinant required for AT base pair recognition as dU-substituted DNA can be cut by AluI. Hydrogen bonding to the N7 of adenine may also be
A l u I ( A G C T )
FIG. 5. Location of functional group modifications (DNA determi- nants) that inhibit the activity of restriction enzymes AluI, DdeI, HinfI, and RsaI. 0, strong inhibition; 0, weak inhibition, b, location of phos- phodiester bonds cleaved by the restric- i n f I ( G A N T c tion enzymes. A , AluI; B, DdeI; C, Hinff ; D, RsaI.
important in binding to the DNA. It is interesting to note the Cs interaction sites on thymidine and cytidine and the phos- phodiester bond that is cleaved are adjacent on the DNA.
DdeI (Fig. 5B)"The interaction sites for DdeI are very similar to those of AluI, although the interaction at the thymidine Cs does not appear to be as strong, and there i s more effect caused by the minor groove addition in 2,6-DAP. Once again the Cs interaction sites on thymidine and cytidine are clustered adjacent to the cleaved phosphodiester bond.
HinfI (Fig. 5C)"This enzyme shows a complex pattern of interaction with DNA. Many modifications affected the rate of cleavage including those at thymidine and cytidine Cs, minor groove changes at adenosine Cz, and change of adeno- sine N7 to C7, but no single determinant seems important by itself for recognition. I t may be that several weak interactions in both major and minor groove are required for sequence recognition or that HinfI is very susceptible to minor pertur- bations in secondary structure that may be induced by these modifications.
RsaI (Fig. 5D)"Changes at both cytidine and thymidine Cs inhibited cleavage by RsaI, but in this case recognition of the Cs methyl of thymidine seems very important for recog- nition while absence of the Cs methyl on cytidine is less crucial. Inhibition of cleavage by dI and dTu suggests possible weak interactions at the minor groove guanosine Cz amino group and adenosine N7. Once again the strongest interaction is directly adjacent to the phosphodiester bond that is cleaved.
Taql-This enzyme is in a class by itself. Of all the modi- fications we incorporated into DNA only one (2-AP) even slowed this enzyme. This effect is consistent with data that the modification system in Thermus aquaticus methylates the Ns of adenosine (Sato et al., 1980; Backman, 1980). Huang et al. (1982) have also reported that TaqI is insensitive to many modifications at the pyrimidine C5. We found that even addition of the long and bulky biotinyl side chain at the thymidine Cg directly adjacent to the cutting site had no effect
Effect of Nucleotide Analogs on the Cleavage of DNA 15213
on the rate of enzyme cleavage. Since TuqI is isolated from a thermophilic bacterial strain and is assayed at 65 "C, one would expect that its mode of interaction with DNA might be different from other restriction enzymes. Determinants re- quired for recognition of DNA by Tug1 are still unclear.
In summary, the study described here has defined structural elements within the recognition sequence that effect. the en- zymatic activity of AluI, DdeI, HinfI, and RsuI. Most modifi- cations that reduced activity involved changes in major group substituents; alterations in minor groove components had little effect on any of the five enzymes tested. The predomi- nance of major groove substitutents in enzyme-DNA inter- actions had been observed previously with EcoRI (Lu et al., 1981). However, the differential response of each of the five enzymes to the same set of functional group modifications indicates that the precise mode of enzyme-DNA recognition will be almost as varied as the recognition sequences for the individual enzymes. Further characterization of the specific determinants essential for DNA binding and phosphodiester bond cleavage will require additional studies with new analog- substituted oligonucleotide or DNA substrates. Such studies are in progress.
Acknowledgments-We thank Paula Northrop for excellent tech- nical assistance and Dr. James Summerton and Dr. Maryellen Pol- vino-Bodnar for helpful discussion. Ribonucleotide reductase was provided by Dr. Robert Blakley, and bacterial strain 71-18 and fd bacteriophage were provided by Dr. Eleanor Spicer.
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