BspRI methyltransferase as a marker for electron microscopic physical mapping

Micron and Micro.scopica Acta, Vol. 22, No. 3, pp. 213—22!, 991. Printed in Great Bntain.

0739—6260/91 S3.00+0.00 Pergamon Press plc

BspRI METHYLTRANSFERASE AS A MARKER FOR ELECTRON MICROSCOPIC PHYSICAL MAPPING A. V.

KURAKIN,

L. S. ZARITSKAYA, A. Z. METLISKAYA, A. A. D. 1. CHERNY*

VOLODIN

and

Institute of Molecular Genetics, U.S.S.R. Academy of Sciences, Kurchatov Sq., 123182, Moscow, U.S.S.R. (Received 13 March 1991, revised 22 June 1991)

Abstract—An approach is described in this paper for direct physical mapping of DNA by electron microscopy. It implies visualization of specific DNA—methyltransferase complexes followed by computer analysis ofelectron micrographs. The BspRI methylase (recognition site GGCC) was used as a marker owing to the large difference (at least three orders ofmagnitude) between its specific and non-specific interation with DNA, as revealed by the gel retardation technique. For electron microscopic mapping the optimum conditions were established in order to produce the maps practically without non-specific noise. The approach was tested with well-characterized plasmid DNAs—pAO3, pUCI9 and pBR322 carrying 4, 11 and 22 GGCC sites respectively. The results were analyzed and the applications ofthe method are discussed. Index key words: Electron microscopy, DNA mapping, BspRI methylase, DNA binding, gel retardation.

INTRODUCTION Particular interest was shown in the investigation of protein—nucleic acid interactions a few years ago, and subsequently accelerated the development of preparative methods for the visualization of DNA—protein complexes in the electron microscope. The sites at which proteins bind to DNA can be determined over a large area by means of electron microscopy. However, the majority of studies performed were concerned with the so-called ‘functional mapping’ (for a review of this work see Brack, 1981), and only few attempts have been made to obtain data about the physical structure of genetic material (Miwa et al., 1979; Walter and Klotz, 1986). At the present time it is of special interest, especially for medical studies, to examine various genes or parts of genes important in terms of polymorphism, in order to make an effort to show that electron microscopy will be capable of application and of valuable help. In the present study we used DNA (cytosine-5) methyltransferase as a specific ligand for physical mapping of DNA molecules without respect to the structure of DNA—protein complexes or the manner of their formation. It is crucial in this case to have sufficient differences between specific and non-specific equilibrium binding constants of the ligand used and the possibility for artificial discrimination between specific and nonspecific binding. Because of these properties demonstrated by gel electrophoretic and Author to whom correspondence should be addressed. Abbreviations: SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; DTT, dithiothreitol; BSA, bovine serum albumin. *

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electron microscopical techniques. the enzyme should prove to he useful for the purposes of mapping. MATERIALS AND METHODS BspRl methylase was isolated from E. coil HBIOI strain with the pNTl2 plasmid carrying the gene of the enzyme under the Pr promoter of the lambda phage. The enzyme was purified by sequential chromatorgraphy on DEAE-sepharose. Cibacron F3Ga-sepharose and heparin-sepharose, yielding an electrophoretically homogeneous state (Sagitov and Alexandrov. 1988). The protein concentration was determined by spectrophotometry at 280 nm. The extinction coefficient, based on the amino acid composition (Posfai et a!., 1983), was adopted as E290=0.85 mlmg’ cm1. The enzyme activity was tested by the protection of lambda phage DNA from BspRl endonuclease action, by preincubation with BspRl methylase and S-adenosvlmethionine (Koncz et a!., 1978). Plasmid DNAs were prepared according to standard protocols and followed by cesium chloride density gradient centrifugation (Maniatis eta!.. 1982). We used pAO3. pUCl9 and pBR322 plasmids linearized by EcoRl endonuclease and the pLJCI9 plasmid fragmented by MspI endonuclease. DNA concentration was determined spectrophotometrically using an extinction coefficient E 2~,()of 20 ml mg cm BspRl methylase binding to plasmid DNA or plasmid DNA fragments was performed in buffer A (20 mM Tris -HCI. pH 7.5, 50 mM NaCI, l0% glycerol. 0.1 mg/mI bovine serum albumin (BSA), 1 mM DTT. 1 mM Na3 EDTA) with the addition of 50 1dM SAH at 37 C. The enzyme -DNA complexes were analyzed by gel electrophoresis in 40 mM Tris acetate buffer. pH 7.5, in l2% polyacrylamide. Complexes for electron microscopy were prepared by preincubation of DNA (lQ-20 fig/mI) and methylase (10 -20 j.ig/ml) in buffer A. excluding glycerol and bovine serum albumin normally in the course of 40 mm at 37C. Following complex formation, heparin was added to a final concentration 10 ~tg/ml, incubation was performed in the course of 2 mm and finally 20 volumes of 10mM Tris HCI. pH 7.5, 50 mM NaCI were added. The complexes were adsorbed onto carbon films activated by glow discharge in pentylamine, according to Duhochet ci a!.. (1971). The samples were then stained with a I % aqueous solution of uranyl acetate, then shadowed with Pt/C (95/5). The micrographs of DNA.. methylase complexes were digitized and analyzed with the aid of an HP9825B computer. The programs were written by F. I. Golovanov and Yu. A. Kalambet (Institute of Molecular Genetics, Moscow). RESULTS AND DISCUSSION Specificity of interaction u/the BspRI !nethyiase and DNA

The recognition of specific DNA sequences by DNA binding proteins is realized through a set of binding and dissociation steps with non-specific DNA sequences (for review see von Hippel. 1979). Therefore, the proteins that are capable of interaction with specific DNA sequences also show some degree of affinity to random nucleotidc sequences. To examine the specific interaction when pUCl9 plasmid DNA is hydrolyzed by Mspl endonuclease, the material was incubated with methylase under optimal condition for the enzyme’s catalytic function (buffer A) in the presence of a non-hydrolyzed substrate analog S-adenosylhomocysteine (SAH). The methylase is unable to methylate DNA in the presence of SAH (Modrich, 1982), and the reaction stops at an intermediate stage. Among the 13 fragments of the pUCl9/Mspl DNA. some contain no specific recognition sites of BspRI methylase (GGCC), some contain one site only, and others contain two or four sites (Yanish-Perron eta!.. 1985) (Fig. I When we used the above conditions and a protein/DNA weight ratio of less than l,the

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F4[21 F5[1 I F6[1] FJ[O] F8 9 [01 F10[1] F13[0] Fig. I - Ek’ctrophoresis oF comple~e’.fisrmed h~BspRI rncthslase and pL ‘(.10 MspI DNA (l2% PAAG, 10 v/cm, 4 h). The comple’scs acre formed in butler A by incubation ui 37 C for 30 mm. 0.3 jig of pUCl9/Mspl DNA; 0,0.026. (1.1)5, 0.1. (1.2 jig of methylase in tracks 15 respectively. The DNA restriction fragments with lengths 501, 489. 404, 331, 242, 190, 147, Ill, ItO. 67 and 34 are designated by the letter ‘F’ with indexes 1 11 respectively. The number of GGCC sites on a particular fragment is indicated within the brackets.

only fragments retarded were those containing the specific recognition site(s). Increasing amounts of BspRl methylase caused gradual attenuation of the bands corresponding to the fragments with the recognition sites(s), and also the simultaneous appearance of bands at the start (Fig. I, tracks 2-5). These data emphasize the specific character of the enzyme--DNA interaction under the chosen conditions. Twodimensional gel electrophoresis supported this suggestion (data not shown). For a fairly high degree of DNA saturation by enzyme molecules (Fig. I), fragments containing at least one GGCC site (e.g. F4, F3, F10) were completely retarded, while no retardation of the fragments without GGCC sites (F7, F8, F9, F11)was observed. These data demonstrate that complete saturation of the GGCC sites in these conditions is accompanied by rather low degree of occupation of non-specific sites by BspRl methylase (less than one molecule of the enzyme per 147 b.p.). Hence, in this situation, the efficiency of the methylase binding to the GGCC sites exceeds the interaction with non-specific DNA sequences by at least three orders of magnitude. The electron microscopic data confirmed the relatively low level of non-specific methylase binding under these conditions. Approximately one non-specific bound methylase molecules per whole pAO3 DNA molecule was observed when the saturation of specific binding sites was close to I. Electron microscopy

For our study we needed a DNA substrate of low molecular weight for which sequence data were available. The DNAs of the plasmids pAO3. pUCl9 and pBR322, which have 1683, 2686, 4363 b.p. and contain 4, II, 22 GGCC sites, respectively, were ideally suited for our purposes. BspRI molecules were relatively easy to locate on these

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DNAs by electron microscopy (Fig. 2). Linear plasmid DNAs were prepared by digestion with EcoRI endonuclease. Recognition complexes were formed by incubation of these DNAs with BspRI under conditions of specific enzyme—DNA interaction (buffer A without glycerol and BSA). During our first experiments we fixed the complexes with formaldehyde, glutaraldehyde or both at concentrations of 0.2% and 0.01%, respectively (in these cases the Tris buffer was substituted for triethanolamine buffer of the same concentration). It was found that fixation led to a substantial decrease in specific binding and an increase in non-specific binding compared to electrophoretic data. Omitting the fixation step resulted in a specific/non-specific relationship close to that expected from the electrophoretic data. Under the conditions employed the methylase molecules bound at most GGCC sites of the DNA molecules, though some non-specific binding was also observed. To diminish the non-specific methylase—DNA interaction we applied the polyanion heparin. The best results were obtained when heparin was added to the incubation mixture at the end of the course for 2 mm at a concentration of 10 fig/mI. However, this treatment also resulted in a slight decrease in specific binding to a value of 60—70% of untreated, while non-specific binding became practically undetectable. Thus, only the peaks representing the regions of the GGCC sites were observed in the binding maps (Fig. 3). Analysis of histogram

The histogram of binding is the result of computer assisted mutual orientation of DNA molecules (generally 50—100) carrying bound enzyme molecules, and it reveals the most probable distribution of bound enzyme along the DNA molecule under investigation. Table I represents results of mapping for linear pAO3, PUCI9 and pBR322 DNAs. in each case two independent experiments were performed at different molar enzyme/DNA ratios. It can be seen from the table that the positions of the binding sites for the same peaks coincide for all the peaks within experimental error (difference is not significant with the level of validity of 1%, t test). It clearly indicates that under the conditions chosen BspRl methylase may be used as a marker, and the binding sites or groups of them can be determined unambiguously by electron microscopy. Comparison of the binding maps for pBR322 and pUCI9, the nucleotide sequences of which overlap within about 2000 b.p., also gives a visual notion of the reproducibility of the method (Fig. 4). The resolution in the histograms of binding is proportional to the length of the DNA molecule analyzed and is approximately 50 h.p. for pAO3 and 100 b.p. for pBR322 DNAs. Therefore, the sites located at this distance or closer thus form a group of sites yield in one peak in the histogram, but higher and wider than for an individual site. The evaluation of the number of distinct sites in these groups depends on a particular group as well as a mean value of bound enzyme molecules per one site. For example, in our study we were able to determine four distinct binding sites out of three peaks for pAO3 DNA (four GGCC sites), 11 out of eight peaks in the case of pUCl9 DNA (11 GGCC sites) and no less than 21 distinct binding sites out of 13 peaks for PBR322 DNA (22 GGCC sites). The positions of centers of peaks on the histograms can be determined with an accuracy of 515 b.p. due to random DNA length fluctuations and measurement errors. But it is impossible to localize the real binding sites (GGCC in our case) with an accuracy exceeding the characteristic dimensions of the marker used (i.e. I 5—20 b.p.) as in the case of these experiments. A study of the number of bound enzyme molecules per individual binding site (for pBR322 we assumed 22 binding sites) suggests that the difference in binding affinity for different binding sites is no more than one order of magnitude.

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Table I. Positions of BspRI methylase binding sites, base pairs GGCC positions (from sequence data*)

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~ The short free ends of the DNA molecules were not seen, thus the binding was assumed to be at the very end of the DNA molecules. The results were obtained from the following experiments: pAO3 DNA: 55 DNA molecules/239 bound enzyme molecules (row I) and 113/226, respectively (row 2); pUCI9 DNA: 51/235 (row 1), 113/192 (row 2); pBR322 DNA: 64/442 (row I), 44/858 (row 2).

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Fig. 4. The comparison of the binding maps of methylase BspRI for pUCI9/EcoRI arid pBR322.EcoRI performed on the same scale. Solid line indicates the region of homology.

CONCLUSIONS 1. Conditions have been determined for direct electron microscopical mapping of GGCC sites of short DNA fragments using BspRI methyltransferase as a marker. Conditions were explored to maximize the ratio of specific to non-specific btnding and thus the ‘signal to noise’ ratio on the binding maps. 2. The method outlined above allows the construction of DNA physical maps in one or two electron microscopic experiments. 3. The short amount of time needed (several days), the high level of reproducibility. and also the minimum of material used, make the method suitable for obtaining primary information about the physical structure of short DNA fragments. 4. Presently the method is in the process of being expanded by testing the whole set of type II methyltransferases as possible markers for electron microscopic mapping.

.4cktiiiir/edcjents’nts--—-Wc wish to thank L. Ashornko for technical assistance in the work with bacterial cultures and Dr. V. Sagitov for the gift of the bacterial strain posscssirtg the pNTI2 plasmid. we also thank Dr. A. Alexandra.’ for useful discussions.

REFER ENC ES Brack. C’.. 1981. DNA electron microscopy. CRC (‘cit. Rs’r. Bloc/tern.. ID: 113 69. Duhochet, J.. Ducommun, M.. Zollinger. M. and Kellenherger. F., 1971. A new preparatiot] ntcthod for dark-held electron microscopy of hiomacromolecules. .1. Ultrastrtict. Re.’ .35: 147 167. son Hippel. P. H.. 1979. On the molecular bases of the specificity of interaction of transcriptiotsal proteins with genome DNA. I is: Biological Reguiat iiii said Dt’rt’lopnteitt , Gold herger. R - I. led. ), lien urn Press. New York. 1979, Vol. I. pp. 297 347. Koncz. C’.. Kiss. A. and Venetianer. P.. 1978. Biochemical characteriza(ion of the restriction—mochthictition system of Basil/us .sphaericu.s. Eur. J. Bloc/tent.. 89: 523 529. Maniatis. T. . Fritch, E. F. and Sambrook. J ., I 982. Moleculctr Cloning: ‘1 Laharatort Manual. (old Spring Harbor Laboratory. Cold Spring Harbor. New York. Miwa, T.. Takanami, M. and Yamagishi. H., 1979. Electron microscopic visualiiatton of rcsirtctiot] sites ot DNA molecules. Gent’, 6: 319 330. Modrich. P.. 1982. Studies on sequence recognition by type II resirictiots and modification cn/’sts]cs, F (‘cit. Rec. Bioc/tent.. 13: 287 323. Posfai . G . . Kiss, A.. Frdct, S.. Posfai . J . and Vcnettancr, P.. I 983. Struct urc at the Bitii/lts spltai’riiis’s R modification methylase gene ‘Sb/cc. Bial.. 170: 597 6 1(1. Sagitov. V. R. and Alexandros. A. A.. 1988. DokI. ‘lcisd. Vsitsk ( .5/SR.. 298: 266 1265.

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Walter, B. and Klotz, G., 1986. Mapping ofrestriction endonuclease binding sites by electron microscopy. J. Electron. Microsc., 5, Suppl. 3: 24313—2414. Yanish-Perron, C., Vieira, J. and Messing, J., 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the MI3mpl8 and pUCI9 vectors. Gene, 33: 103—119.