Sequence-specific spin labeling of DNA

38

Biochimica et Biophysica Acta, 1048 (1990) 38-42 Elsevier

BBAEXP 92023

Sequence-specific spin labeling of DNA Ivana Weygand-Durasevic and Slavoljub Susic Department of Organic Chemistryand Biochemistry, Faculty of Science, Universityof Zagreb, Zagreb (Yugoslavia)

(Received 25 July 1989)

Key words: Spin labeling; DNA modification;Guanine modification

Several DNA fragments deriving from plasmid pBR322 were used to determine the modification sites caused by the reaction with alkylating spin-labeling probes. At a high spin-label concentration, all guanines became alkylated, causing the cleavage of the phosphodiester bonds upon the treatment with piperidine. The lengths of the breakage products of 5'-end labeled DNA treated with spin labels were compared with the length of DNA scission products generated by Maxam-Gilbert procedure for DNA sequence analysis. The distribution of the guanine modifications is dependent on the amount of the reagent used for the alkylation and the ionic conditions of the reaction. The frequency of alkylation by spin labels was greatly enhanced within continuous runs of guanines in DNA. The stabilization of the DNA structure by magnesium or spermine directs the spin-label binding specifically to the most exposed region of DNA fragment containing GGTGG sequence. The sequence-dependent interaction of spin labels with DNA enables the development of the method for the selective spin labeling of DNA molecule.

Introduction Numerous spectroscopic techniques have been used to study the relationships between the structure and function of nucleic acid molecules. Some of the significant structure-function problems in this field have been illuminated by spin-labeling studies [1]. However, compared to other biosystems, spin labeling of nucleic acids have received little attention, because they contain essentially only four types of monomer, which most of the time are either stacked or hydrogen-bonded to one another, thus making specific spin labeling of a particular site or base very difficult. Because of this, most specific labeling studies of nucleic acids have been performed with transfer RNAs in which some rare bases are selectively reactive toward certain spin label reagents [2-4]. There were also some attempts to label DNA bases by chemical modifications [5]. The synthesis of the spin label analog of an antitumor agent [6] and of carcinogenic aromatic amines [7] raised the possibility of using ESR technique as a diagnostic tool for detection of cancer. The analysis of double-helix motions has been achieved by measuring the ESR spectral parameters of a family of spin-labeled probes non-covalently bound to DNA [8]. The base-specific spin-labeling

Correspondence: I. Weygand-Durasevic,Zavod za organsku kemiju i biokemiju, Prirodoslovno-matematickifakultet, Strossmayerovtrg 14, 41000 Zagreb, Yugoslavia.

method based on enzymatic incorporation of the spinlabeled building blocks developed by Bobst and coworkers [9,10] enabled the study of mobility of DNA base pairs. Spin-labeled polynucleotides were also used in a sensitive ESR approach designed to allow direct quantitative determination of relative nucleic acid affinities of protein binding under physiological conditions [111. We wanted to develop a suitable method which would enable the study of the interaction between specifically spin-labeled D N A and other macromolecules. Sequence selective reactions with D N A have been observed in the case of several compounds [12-17]. The mechanism of the interaction of D N A with nitrogen mustards, which are in use as anticancer drugs, has been recently elucidated [18]. They alkylate DNA primarily at the N v position of guanine. Treatment of modified DNA with piperidine results in breakage of the N-glykosylic bond and scission of the phosphodiester bond 3' to the alkylated base [19]. Different alkylating nitroxides have been used in our search for the most favorable conditions for selective spin labeling of DNA.

Materials and Methods Preparation of 5"-end labeled DNA fragments The DNA fragments of known sequence used in this work were obtained from plasmid pBR322. The purified plasmid DNA was cleaved with the restriction endonucleases B a m H I and AvaI, followed by dephosphoryl-

0167-4781/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)

39 ation with calf intestine phosphatase. 5'-end 32p-labeling was done by incubation with [y-32p]ATP (Amersham; 4000 Ci/mmol) and polynucleotide kinase (New England Biolabs) as described by Maxam and Gilbert [20]. Subsequent cleavage of two labeled DNA fragments with PstI and SalI yielded four fragments carrying the label only at one end. The fragments were resolved by electrophoresis on agarose gel, eluted and then taken for spin labeling. The restriction enzymes and calf intestine phosphatase were purchased from Boehringer-Mannheim.

El

CI

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I

I

I

o

CH2 CH3 ~N /

C-- CH2Br I NH

CH2 CH2 ~N /

I

I

I

0

0

0

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II

III

Fig. 1. Structures of nitroxide spin labels used in this study.

Alkylation reactions The alkylation of terminally labeled DNA fragments with spin labeling reagents (spin labels I and II were obtained from the J. Stefan Institute, Ljubljana, Yugoslavia; spin label III was from SYVA, Palo Alto, CA, U.S.A.) was performed at room temperature during 60 min., either in 25 mM triethanolamine-HC1 (pH 7.2), 1 mM EDTA [18] or in 50 mM sodium cacodylate (pH 8.0), 10 mM MgC12, 1 mM EDTA [20]. 14.10 -3 M stock solutions of spin labels were prepared in 50% ethanol. The concentrations of spin labels in total incubation mixtures (60 /~1) were from 2.3.10 -5 M to 2.3.10 -3 M. In experiments with spermine, the polyamine was incubated with DNA at room temperature for 60 min prior to the addition of spin label. The concentration of spermine in the reaction mixture was from 3 • 10 -7 to 6 • 10 -4 M. After the incubation, DNA was precipitated by addition of equal volume of an ice-cold solution containing 0.6 M sodium acetate (pH 5.2), 20 mM EDTA and 100 /~g/ml tRNA and three volumes of ethanol. The pellets were resuspended in 0.3 M sodium acetate, 1 mM EDTA and DNA was ethanol precipitated and washed with cold ethanol prior to vacuum drying. Saltfree DNA was resuspended in freshly diluted 1 M piperidine and incubated at 90°C for 20 min [20]. DNA fragments were recovered by several repeated precipitations with cold ethanol, dried and analyzed by polyacrylamide gel electrophoresis. Sequencing necessary for localization of the breaks at the sites of N T-guanine alkylation was performed according to Maxam and Gilbert [20].

Polyacrylamide gel electrophoresis High resolution analysis of DNA products on polyacrylamide gels was done as described by Maxam and Gilbert [20]. Electrophoresis was performed at 1500 V and the gels were autoradiographed at - 2 0 ° C. Results

To investigate whether alkylating spin-label probes shown in Fig. 1 can be used for sequence-specific modification of DNA, we have chosen double-stranded DNA

substrates

of

defined

sequences:

276-base

pair

BamHI/SalI fragment labeled at BamHI site, 774-base pair AvaI/SalI and 2184-base pair AvaI/PstI fragments both labeled at AvaI site. DNA was isolated from reaction mixtures and the length of DNA products analyzed on high-resolution denaturing polyacrylamide gels after treatment with 1 M piperidine. To faciliate the identification of the nucleotide at the site of cleavage, the products generated by reaction of the same DNA sequence with dimethyl sulfate were run in a parallel slot of a slab gel (Fig. 2, lane a). Under the reaction conditions used, (25 mM triethanolamine-HC1 (pH 7.2), 1 mM EDTA) treatment of BamHI/SalI fragment DNA with spin labels I (Fig. 2, lane c) or II (Fig. 2, lane b) followed by piperidine hydrolysis results in breakage of DNA only at guanine residues. No alkylation has been observed by spin label III when used in concentrations comparable to those of I or II (Fig. 2, lane f). The extent of DNA labeling with nitroxide probe can be controlled by changing the concentration of spin label used for DNA treatment. We found that all possible guanine residues of the template were accessible to modification by spin labels I and II. However, the rate of reaction of individual guanines with probes I and II varies, as apparent from the variation in the relative intensity of the bands (Fig. 2, lanes b and c). The intensity of each band reflects the frequency of alkylation and cleavage at the corresponding site because each molecule is labeled with 32p only at the 5' terminus. The most favorable sites for alkylation of DNA are the guanines within the contiguous runs. The comparison of the lanes b and c of Fig. 2, made us conclude that two spin-labeling reagents have slightly different preferences in attacking contiguous G residues. In the run of three contiguous guanines, SLII preferentially attacks the middle nucleotide, while the modification with SLI takes place equally at second and third nucleotide. The most surprising result was obtained when BamHI/SalI DNA fragment was treated with very limited amounts of spin-labeling reagents. Under such conditions, the primary site of reaction with SLII is GGTGG sequence (Fig. 3, lane c). The most probable explanation for this finding is that treatment

40

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B

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5G(535-539)

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~'-~ 3G(5'I;-513)

3G(485- ?) 3o(z,ss-z, .3G(47t-473)

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. o(461-4 ) Fig. 2. Autoradiography of a polyacrylamide gel used for determination of D N A modification sites caused by the reaction of 276-base pair Bam Hl/Sall fragment with spin-labeling probes. 5'-end 32p_ labeled D N A fragment was treated with (a) dimethyl sulfate; (b) 2.3.10 -3 M SLII; (c) 2.3-10 - s M SLI; and (f) 2.3.10 - 3 M SLIII, followed by piperidine treatment as described in Material and Methods. Spin labeling reactions were performed in 25 m M triethanolamine. HC1 (pH 7.2), 1 m M EDTA. Lane d contains untreated DNA, lane e treated with piperidine without prior spin labeling. The regions of contiguous guanines are marked and the base positions in pBR322 are shown in parentheses.

with higher concentration of spin label masks the first site of attack, because the DNA fragment gets modified and subsequently cleaved at several other sites. Such concentration dependent specific modification of DNA has not been observed with AvaI/SalI (Fig. 4.) and AuaI/PstI (Fig. 5.) fragments, although the preferential modification of contiguous guanines is also obvious. In our search for even more specific spin labeling of DNA than preferential attack to contiguous guanines, we tried to stabilize DNA structure with spermine and magnesium. The results of sequential additions of spermine to the 5'-labeled DNA fragments prior to the incubation with spin labeling reagents are presented in

Fig. 3. The effect of spermine on DNA-SLII interaction. (A) 276-base pair BamHI/SalI fragment, 5'-end 32p-labeled at BamHI end, was incubated with SLII in 25 m M triethanolamine-HCl (pH 7.2), 1 m M EDTA, either without or in the presence of spermine. Modified D N A was subsequently treated with piperidine, cleavage products were purified from reaction mixtures by repeated ethanol precipitations and electrophoresed on 8% polyacrylamide-urea gel. Lane a 2 . 3 . 1 0 - 3 M SLII; lane b, 2.3.10 - 4 M SLII; lane c, 2.3.10 5 M SLII; lane d, 2.3-10 3 M SLII, 3-10 7 M spermine; lane e, 2.3.10 -4 M SLII, 3.10 -v M sperrnine; lane f, 2.3.10 -3 M SLII, 6.10 6 M spermine; lane g 2.3.10 4 M SLII, 6.10 -6 M spermine.(B) Comparison of the degradation products, of same D N A fragment, obtained by incubation of D N A with 2.3.10 3 SLII in the absence of spermine (lane a) and in the presence of 6.10 - 4 M spermine (lane b). The regions of contiguous guanines are marked and the base positions in pBR322 are shown in parentheses.

Figs. 3, 4 and 5. The comparison of the amount of cleavage products obtained by spin labeling without spermine and in the presence of increasing concentrations of polyamine shows that the total extent of alkylation by spin label II (2.3. ] 0 - 4 M) followed by

41

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2G(t 2G(t 2G03 2G(~3:

04 -I501)\ 3G(tZ, 30(1,;2729)--.

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Fig. 4. Induction of alkali-labile sites in 774-base pair 5'-end 32p. labeled AvaI/SalI fragment by spin labeling with SLI1. Lane a 2.3.10 -3 M SLII; lane b, D N A fragment without treatment; lane c, 2.3-10 -4 M SLII; lane d, 2.3.10 3 M SLII, 6.10 -7 M spermine; lane e, 2.3.10 - 4 M SLII, 6-10 -7 M spermine. After the modification all samples except that in lane b were heated for 20 rain at 90 o C in 1 M piperidine. The electrophoresis was performed on 8% polyacrylamide gel as described in Material and Methods. Cleavage sites at some guanines are marked. The numbers given in parentheses correspond to the positions of cytosines in complementary strand of pBR322.

creation of alkali-labile sites is dependent on the structure of DNA fragment (Fig. 3., lanes b, e and g; Fig. 4., lanes c and e; Fig. 5., lanes c, h and j). Binding of spermine to DNA protects the macromolecule from alkylation, having as a consequence lower amounts of breaks caused by binding of spin label and piperidine treatment. Moreover, in the case of BamHI/SalI fragment, spermine directs alkylation only to the most accessible site, e.g., G G T G G sequence (Fig. 3, lane g). Under condition used (2.3-10 - 4 M SLII; 6 . 1 0 - 6 M spermine) this is the only site of binding for the nitroxide probe within the 5'-labeled strand of 276-base pair BamHI/SalI fragment of pBR322. Fig. 3B shows the

Fig. 5. The cleavage of D N A induced by the binding of spin label to 2184-base pair AvaI/PstI fragment, 5'-end 32p-labeled at Aval site. D N A was reacted with SLII in 25 m M triethanolamine-HCl (pH 7.2), 1 m M E D T A either without or in the presence of spermine, followed by piperidine treatment in all cases except in lanes a and e. Lane a, D N A without treatment; lane b, 2.3.10 -3 M SLII; lane c 2.3.10 4 M SLII; lane d, 2.3-10 -5 M SLII; lane e, 2.3.10 -4 M SLII, no piperidine treatment; lane f, D N A treated with piperidine without prior spin labeling; lane g, 2.3.10 -3 M SLII, 6.10 7 M spermine; lane h, 2.3.10 - 4 M SLII, 6-10 -7 M spermine; lane i, 2.3.10 -3 M SLII, 6.10 5 M spermine; lane j, 2.3.10 4 M SLII, 6.10 -5 M spermine; lane k, D N A treated with dimethyl sulfate according to Ref. 20. The regions of contiguous guanines are marked and the base positions in pBR322 are shown in parentheses.

cleavage of the same DNA fragment by incubation with 2.3. 10 -3 M SLII, followed by high-temperature piperidine treatment without spermine (lane a) and in the presence of 6 . 1 0 - 4 M spermine (lane b). The same effect was observed when 10 mM magnesium was used instead of spermine in the spin-labeling experiment (not shown). Discussion

The work presented in this paper offers direct evidence that some alkylating spin-labeling probes can attack DNA in a sequence-specific manner which opens the possibility to use this method for obtaining selectively spin-labeled nucleic acid suitable for various investigations. The sequence specificity of the interaction between spin labels and DNA were determined by using a DNA sequencing technique [20] and 5'-end-labeled DNA fragments of a defined sequence as substrates. Alkylating spin labels I and II (structures are presented in Fig. 1) modify guanines in double-stranded

42 DNA, inducing alkali-labile sites. For relatively high concentrations of spin labels, cleavage products were the same as those generated by modification of D N A with dimethyl sulfate followed by alkali-treatment [20]. Mattes et al. [18] have recently observed that several nitrogen mustard analogs (bifunctional alkylating agents) produce alkali-labile lesions at positions of guanine, attacking preferentially those within the contiguous runs, which is also the case with alkylating spin labels used in our study. Crosslinks through bifunctional alkylation of guanine-N 7 positions in D N A helix can arise by reaction with two adjacent guanines in the same D N A strand. Since both mono (SLII) and bifunctional (SLI) alkylating spin labels show the preference for contiguous guanines (Fig. 2), the enhanced reactivity of such sequences can not be explained by crosslinks. Instead, another possibility of altered reactivity of the guanine N 7 position by neighboring bases should be considered [21]. Interestingly, according to Mattes et al. [18], the sequence specific alkylation of D N A by nitrogen mustards is not markedly dependent on the solvent condition of the reaction. On the contrary, we have observed that both the extent and in one case also the specificity of alkylation can be changed by including the magnesium or spermine into the reaction mixtures (Figs. 3, 4 and 5). We have previously shown the profound effect of polyamine on t R N A structure [22,23]. Formation of strong associations between spermine and D N A by electrostatic interactions could explain at least some effects of spermine on DNA, such as the protection against shearing, denaturation, radiation damage and intercalation of aromatic compounds [24]. The stabilization of D N A structure by polyamine lowers the extent of alkylation by spin labeling probes (Figs. 3, 4 and 5) and at the same time directs the attack of the spin label probably to the most accessible site in 276-base pair BamHI/SalI fragment of pBR322. The same site (517-521 G G T G G sequence) is alkylated by spin label II in the presence of 10 mM magnesium or even in the absence of divalent cations or polycations when the amount of spin label is limited (Fig. 3). This observation can be very useful in developing the method for selective spin labeling of D N A molecule. At the moment, we are not able to explain the reasons that make this particular region of D N A exposed to alkylating agents, although we have observed that some other factors such as prolongated piperidine treatment and nuclease $1 attack cause the cleavage of the BamHI/SalI fragment at the same site. On the other hand, the interactions of spermine and magnesium with two other examined pBR322 fragments are reflected only in overall stabilization of the polynucleotides giving rise to lower extent of spin labeling (Figs. 4 and 5). The site-specific binding of spinlabeling probe was not observed in those fragments

within the stretch of nucleotides that can be resolved on polyacrylamide gels. It would be interesting to examine in the same way, other D N A fragments having the same or similar stretch of nucleotides ( G G G T A T G G T G G C ) . The described method might also be suitable for tracing some control regions of the genes, which are expected to adopt less compact structure than the rest of the D N A molecule.

Acknowledgments The authors are indebted to Dr. Vesna Nothig-Laslo and Professor Zeljko Kucan for their continuous encouragement and valuable discussions. This work was in part supported by a grant from the National Institutes of Health (No. JFP 757).

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