Generation of DNA fragments by enzymatic cleavage at sites sensitive to denaturation

Biochimica et Biophysica Acta, 299 (1973) 264-272 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97618

G E N E R A T I O N OF D N A F R A G M E N T S BY E N Z Y M A T I C CLEAVAGE AT SITES SENSITIVE TO D E N A T U R A T I O N A R T H U R L A N D Y , W I L M A ROSS and C A R L F O E L L E R

Division o f Biological and Medical Sciences, Section o f Microbiology and Molecular Biology, Brown University, Providence, R. 1. 02912 (U.S.A.) (Received October 30th, 1972)

SUMMARY

Fragments of bihelical bacteriophage @80 D N A have been generated by enzymatic cleavage at denaturation-sensitive sites. The D N A is exposed to subcritical denaturation conditions which are known to result in the generation of small singlestranded loops (Inman, R. B. (1967) J. Mol. Biol. 28, 103-116), presumably at loci enriched for A • T base pairs. The loops are "fixed" by reaction with formaldehyde. In rapid succession the unreacted formaldehyde is removed and the D N A is digested with the single strand-specific endonuclease from Neurospora crassa. Polyacrylamide agarose gel electrophoresis is used to monitor the reaction or isolate the DNA fragments. This procedure provides one approach to studying the chromosomal distribution of regions that are strongly biased toward A • T or G" C base pairs and also afford a flexible complement to the site-specific endonucleases in isolating and studying specific regions of the chromosome.

INTRODUCTION

The sensitivity of a D N A double helix to strand separation induced by alkali, heat or other denaturants is a function of its base composition 2. Early experiments on the denaturation-induced differential inactivation of genetic markers in transforming D N A 3, 4 and more recent experiments involving the analysis of denaturation induced hyperchromic shifts in the DNA from different Escherichia coli bacteriophage 5, 6 suggested that a D N A molecule should possess a characteristic"denaturation map". Such a map, which indicates the relative sensitivity to denaturation over the entire length of a D N A molecule, was realized for 2 DNA in the remarkable electron micrographs of Inman 1. In his procedure D N A is exposed to subcritical conditions of thermal or alkaline denaturation in the presence of high concentrations of formaldehyde. By reacting specifically with the non-hydrogen bonded bases the formaldehyde prevents reformation of a double stranded structure. The electron micrographs clearly show that by using formaldehyde in this way to "fix" small locally denatured regions it is possible to generate D N A molecules with a characteristic distribution of singlestranded regions.

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By altering the conditions of partial denaturation the distribution of singlestranded loops can be manipulated in a progression of sites from those which are most susceptible to denaturation to those which are most resistant. The ability to specifically cleave a molecule at these sites would be exceedingly u s e f u l - both analytically and preparatively - - in biochemical studies on the organization of genetic information and control sequences in DNA. We have been successful in defining a system in which enzymatic cleavage of DNA is entirely dependent upon the generation of locally denatured regions within the molecule. MATERIALS AND METHODS Preparation of DNA and N. crassa endonuclease Lysates of bacteriophage 480 were grown on E. coli CA275 in LP medium containing 32Pi at 5 pCi/ml (ref. 7). The phage were purified by differential centrifugation and banding on two successive CsC1 step gradients. The phage suspension in 10 mM Tris-HC1 buffer (7.4), 0.3 M NaCI, 10 mM EDTA, 1 M NaC104 and 0.2 ~o sodium dodecyl sulfate was extracted twice with water-saturated phenol. The DNA was dialyzed against 10 mM Tris-HC1 buffer (pH 7.4) and when necessary was concentrated by vacuum dialysis to a final concentration of 200 #g/ml. Specific activities were in the range of 2 • 104 cpm/#g. The single strand-specific endonuclease was isolated according to the procedure of Linn s from mycelia of N. crassa (ATC 9279) grown in Vogel's minimal medium9. One unit of enzyme is defined as that amount which produces 1 pmole of acid-soluble nucleotide in 30 min in the standard assay mixture, 0.1 M Tris-HCl buffer (pH 7.5), 10 mM MgClz, 10 #g/ml bovine serum albumin (Fraction V) 8. The final specific activity of the enzyme was 18.1 units/#g protein. No acid solubilization of native 480 or E. coli DNA was detectable when incubated with 5 times the quantity of enzyme necessary to render denatured DNA 40 ~ acid soluble. Controlled partial denaturation of DNA The conditions used for partial alkaline denaturation of the q~80 DNA were those described by Inman and Schn6s 1°. The following procedure was adopted for carrying out the denaturation. A set of standard stock solutions containing Na/COa, EDTA and an appropriate quantity of NaOH were constructed. These solutions were stable for several weeks when kept sealed from the atmosphere. Reagent grade formaldehyde (Mallinckrodt, 37 ~ ) was filtered through a 0.45-#m millipore filter and before use was heated in a boiling water bath for 10 min to eliminate polymeric forms 11. Immediately before each experiment the appropriate stock solution is mixed with 37 ~ formaldehyde to obtain a triple strength denaturation buffer: 6 mM NazCO3, 15 mM EDTA 30 ~ (v/v) formaldehyde. (This must be made up fresh for each experiment since at high pH formaldehyde is slowly converted to tormic acid.) The denaturation is carried out at the desired pH (in the range 10.90-11.40) by mixing 20 #1 of the appropriate 3 times denaturation buffer with 40 ktl of 32p-labeled 480 DNA (200/~g/ml) and incubating for 5 min in a 25 °C water bath. The reaction is terminated by chilling in ice and adding 5.6 #1 of 5.3 M Na2HPO4. Enzymatic digestion of partially denatured DNA Preparation of the sample for endonuclease digestion, involving removal of

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formaldehyde and buffer exchange, is accomplished on a column of Sephadex G-25 previously equilibrated with 0.2 M Tris-HC1 buffer (pH 8.7) in a spin-filter device fabricated in our laboratory for this purpose (in preparation). A 15-/fl aliquot of the eluate containing the partially denatured D N A in 0.2 M Tris-HC1 buffer (pH 8.7) is brought to 10 mM MgCI2, 0.5 mM CoC12 and 10/~g/ml bovine serum albumin in a total volume of 21/fl and incubated for 30 min at 37 °C with 1.2 • 1 0 - 3 unit of Neurospora endonuclease. The digestion is terminated by adding 0.10 vol. of 0.2 M E D T A or by precipitating with 10 ~o trichloroacetic acid. All of the steps from the initial preparation of denaturation buffer to final termination of the endonuclease digestion are carried out without any interruptions. It is possible to hold digested samples overnight in 20 mM E D T A without any observable effect on the gel profiles; however, in the experiments reported here samples were loaded directly onto the gels.

Electrophoresis of DNA fraoments on composite 9els Polyacrylamide agarose slab gels were formed according to the method of Peacock and Dingman in an ED Model 470 cell using an 8 place slot former 11. The final concentration of the composite gel was 2.5 ~o acrylamide (0.125 ~ methylenebisacrylamide), 0.1 ~ N,N,N',N'-tetramethylenediamine, 0.1 ~ ammonium persulfate and 0.5 ~ agarose. The gel buffer (both for preparing the gel and running) was that of Loening 13. Before loading on the gels each sample was brought to 10 ~ sucrose and 0.1 ~ bromophenol blue. Gels were run at 4 °C for 625 V • h or 975 V • h (70-90 mA). The gel slabs were cut into vertical strips corresponding to each slot and each strip was cut into fifty 3.3-mm slices. The gel slices were counted in a toluenebased solution of Omnifluor (New England Nuclear) in a liquid scintillation spectrometer. The distribution of radioactive D N A fragments in the gel slices is expressed as the percent of total counts in the gel strip. RESULTS AND DISCUSSION

The effect of formaldehyde on the Neurospora endonuclease digestion Our approach to obtaining a controlled cleavage of D N A at regions of local denaturation depends upon the extreme substrate specificity of the single strandspecific endonuclease first isolated from N. crassa by Linn and Lehman 14. Unfortunately the endonuclease digestion is very sensitive to formaldehyde, which must be used at sufficiently high concentrations to assure rapid kinetics. It can be shown, however, that the observed inhibition is not due to substrate effects, i.e. that a formaldehyde-reacted D N A molecule is not an unacceptable substrate for this enzyme. In Table I four samples are compared for their sensitivity to the Neurospora enzyme as measured by the rates of acid solubilization. In this experiment denatured D N A was rendered 50 ~ acid soluble by the enzyme in 40 min. After the D N A has been reannealed for 8 h it is completely refractile to solubilization by the Neurospora enzyme. If, however, the denatured D N A has been reacted with formaldehyde (which is then removed by dialysis) the process of reannealing has no effect on the rate of solubilization as compared to a control which has not been reannealed. These data do not indicate whether the Neurospora endonuclease is able to cleave directly at a base which has reacted with formaldehyde. However, it is clear that

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TABLE I EFFECT OF FORMALDEHYDE TREATMENT ON THE SENSITIVITY OF DENATURED E. COLI DNA TO NEUROSPORA ENDONUCLEASE DIGESTION DNA samples (a) and (b) (in I0 mM Tris buffer (pH 7.5) and 1 mM EDTA) were denatured at 100 °C for 10 min. DNA samples (c) and (d) were heat denatured in 6.7 mM KH2PO4, 0.I M NaCI, 3.4 mM EDTA, and 10 ~ formaldehyde. Formaldehyde was removed immediately by dialysis against 10 mM Tris buffer (pH 7.5) and 1 mM EDTA. Annealing was for 8 h at 60 °C in 10 mM Tris buffer, 0.9 M NaCI: and 1 mM EDTA. Acid solubilization at 37 °C by 0.3 unit of Neurospora endonuclease in 0.3 ml of standard assay mixture plus 0.3 M NaCI was measured over a 2-h period to ensure obtaining measurements for each of the substrates in a linear range. Sample (a) was rendered 50 ~ acid s~luble in 40 min. There was no detectable acid solubilization of sample (b) after 2 h of incubation. Treatment of denatured DNA

Relative rates of acid solubilization

(a) (b) (c) (d)

1.0 0 0.28 0.20

Untreated Reannealed + formaldehyde + reannealing + formaldehyde -- reannealing

D N A which has been sufficiently r e a c t e d with f o r m a l d e h y d e to p r e v e n t r e f o r m a t i o n o f a d o u b l e - s t r a n d e d structure can be r e n d e r e d acid soluble by the enzyme. Because o f the reversibility o f at least p a r t o f the f o r m a l d e h y d e r e a c t i o n 15 we have a s s u m e d t h a t it w o u l d be desirable to utilize a m o r e r a p i d m e t h o d than dialysis for r e m o v i n g the u n r e a c t e d f o r m a l d e h y d e p r i o r to e n z y m a t i c digestion. E t h a n o l p r e c i p i t a t i o n was also f o u n d u n s a t i s f a c t o r y in this respect. The m e t h o d finally chosen was t o pass the D N A - f o r m a l d e h y d e d e n a t u r i n g r e a c t i o n m i x t u r e t h r o u g h a spin-filter device l o a d e d with S e p h a d e x G-25 (in p r e p a r a t i o n ) . In a p e r i o d o f less t h a n 2 min the f o r m a l d e h y d e c o n c e n t r a t i o n is r e d u c e d f r o m 9.1 ~ to less than 0.05 ~ a n d the H ÷ c o n c e n t r a t i o n is r e d u c e d f r o m p H 11 to the precise p H at w h i c h it is desired to execute the e n z y m a t i c digestion, i.e. p H 8.7. W i t h this m e t h o d it has been possible to s i m u l t a n e o u s l y process m u l t i p l e samples as small as 4 0 p l with 75 ~ r e c o v e r y o f the D N A a n d zero d i l u t i o n . The effect o f limited denaturation on digestion The d e n a t u r a t i o n c o n d i t i o n s o f I n m a n a n d Schn6s were a d o p t e d as a starting p o i n t in o u r experiments. C o n s i d e r a b l e effort was m a d e to minimize the d u r a t i o n o f each o f the f o u r steps in the overall reaction. A n outline o f the e x p e r i m e n t a l p r o tocol is as follows. D N A is m i x e d with a 3 times f o l m a l d e h y d e - d e n a t u r i n g buffer at some specified p H between p H 10.90 a n d 11.40. A f t e r 5 m i n i n c u b a t i o n at 25 °C the s a m p l e is chilled quickly a n d n e u t r a l i z e d with p h o s p h a t e buffer. T r a n s t e r o f the r e a c t e d D N A to the d i g e s t i o n buffer is effected b y passage t h r o u g h S e p h a d e x G-25 as described a b o v e a n d is c o m p l e t e d within 5 m i n after the n e u t r a l i z a t i o n step. The e n z y m a t i c d i g e s t i o n o f the p a r t i a l l y d e n a t u r e d D N A is c a r r i e d o u t in 0.2 M Tris buffer ( p H 8.7) in o r d e r to m a x i m i z e the d i s c r i m i n a t i o n between single- a n d d o u b l e s t r a n d e d D N A 8. The r e a c t i o n is t e r m i n a t e d either b y the a d d i t i o n o f t r i c h l o r o a c e t i c or EDTA.

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The data in Table II show the percent of DNA rendered acid soluble by the enzymatic digestion after denaturation and fixation at several different pH values. The partial solubilization which is observed for the samples denatured and fixed in the range from pH 10.92 to 11.34 represents complete solubilization of all the available substrate, i.e. the digestion has yielded a limit product. This is evidenced by the absence of any trichloroacetic acid-insoluble material in those samples which have been denatured and fixed at pH 11.24 or greater. The complete acid solubilization of the samples treated at higher pH also indicates that passage through the Sephadex G-25 does remove all of the unreacted formaldehyde. We have observed that use of the spin-filter device in the denaturation procedure (either with or without the formaldehyde and phosphate neutralization step) results in considerably improved substrate for the Neurospora endonuclease.

Polyacrylamide agarose gel profiles of the Neurospora endonuclease digestion products In order to monitor the distribution of DNA cleavage sites the samples were subjected to electrophoresis in mixed gels of polyacrylamide-agarose immediately following the endonucleolytic digestion. Mixed gels of this composition have a very open structure and are able to fractionate particles in the size range of 100 S and larger 16. In these gels 4-S RNA migrates ahead of the blue indicator dye and there is very poor separation between the 4-S RNA and nucleotides which are acid soluble. The amounts of material moving ahead of the bromophenol blue marker in the top four panels of Fig. 1 agree closely with the amounts of acid-soluble DNA generated by the respective digestions shown in Table II. TABLE II E F F E C T OF P A R T I A L D E N A T U R A T I O N ON T H E TO N E U R O S P O R A E N D O N U C L E A S E D I G E S T I O N

F R A C T I O N OF D N A SENSITIVE

See Materials and Methods and text for protocol and conditions.

p H o f alkali-formaldehyde denaturation

~ a2p-labeled ~ 8 0 DNA rendered trichloroacetic acid soluble by endonuctease

10.92 11.04 11.10 11.14 11.24 11.34

19 19 31 54 98 98

In a DNA sample which has been denatured and fixed at pH 11.04, 32 ~ of the DNA fragments resistant to nuclease digestion migrate slower than the 23-S marker RNA. Nevertheless, only 1 ~ of the DNA migrates in the region of intact molecules (cf. bottom panel, Fig. 1). Raising the pH of denaturation and fixation by small increments results in a progressive decrease in the proportion of large DNA fragments until pH 11.24 when 84 ~ of the fragments move ahead of the 4-S RNA marker (cf. Table II).

D N A CLEAVAGE AT DENATURATION-SENSITIVE SITES

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Fig. 1. Gel profiles of Neurospora endonuclease limit digestion products of (P80 D N A denatured and fixed at subcritical pI-L All panels are from the same gel slab, electrophoresed 625 V - h (see Materials and Methods). D N A samples were denatured and fixed at the indicated pI~ before enzymatic digestion as described in Materials and Methods and text. Control sample of pI-[ 11.24 was denatured and fixed but not incubated with enzyme. Bottom panel shows profiles of undenatured D N A (-k) and ( - - ) incubation with Neurospora enzyme.

The profile of an aliquot removed from the pH 11.24 DNA sample just prior to the enzymatic digestion step shows no degradation of the DNA. A comparison of this profile with that of untreated native DNA (bottom panel, Fig. 1) shows that when high molecular weight DNA is denatured it migrates slower in these gels than the native parent molecules. This is not a consequence of denatured DNA "sticking" in the slot although aggregation may affect the mobility. We have observed this behavior under several conditions. Similar effects of secondary structure on the migration of nucleic acids in gel electrophoresis have also been reported by others 16. The bottom panel of Fig. 1 shows the gel profiles of undenatured DNA with and without exposure to the Neurospora enzyme. The small amount of breakdown observed in this sample is due mainly to nuclease-sensitive single-strand nicks which accumulate in the 3zp-labeled preparation. The level of this background cleavage is

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a function of both the enzyme preparation and the D N A substrate. Different p[eparations of the enzyme vary in the amount of contaminating double-strand nuclease activity. In the best preparations less than I0 ~ of native T7 DNA molecules suffer any single-strand nicks when incubated with an amount of enzyme sufficient to render denatured T7 D N A 40 ~ acid soluble a. The quality of the D N A is also important because single-strand nicks constitute foci for denaturation and formylation 17. Our results indicate that even without the formylation reaction the Neurospora enzyme will apparently attack D N A that is nicked. The cleavage profiles in Figs 1 and 2 were generated using an excess of Neurospora enzyme. In certain situations, however, it might prove necessary or beneficial to titrate for the minimum amount of enzyme required for a reproducible limit digestion at some specified pH. In order to monitor the larger D N A fragments generated as a consequence of partial denaturation at lower pH the electrophoresis time was increased by 60 O//o. In the profile of a D N A sample denatured and fixed under the mildest conditions reported here (pH 10.94, Fig. 2) the fraction of nuclease-resistant fragments falling in the region not resolvable from intact D N A (Slices 8 and 9) has increased to 49 % from 9 ~o at p H 11.02. Virtually all of the fragments are found to migrate slower in the gel than the 23-S r R N A marker. Oa the basis of our observations and reports from other laboratories on the relative behavior of single- and double-stranded nucleic acids in polyacrylamide gel electrophoresis 17, it is probable that those DNA fragments migrating behind or with the 23-S rRNA marker are larger than 1.2. 106, I0

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Fig. 2. Gel profiles o f large D N A f r a g m e n t s generated by Neurospora endonuclease following very mild d e n a t u r a t i o n . All panels are from the s a m e gel slab, electrophoresed 975 V • h. Conditions for d e n a t u r a t i o n , enzymatic digestign a n d electrophoresis are described in Materials and M e t h o d s a n d text. U n t r e a t e d native D N A migrates to slices 8 and 9.

As the milder denaturing conditions are approached it might be expected that the single-stranded regions would tend to be smaller as well as less frequent. Same indication of the smallest denatured regions which are cleaved by the Neurospora endonuclease can be inferred from the digestion of tRNA. At 27 °C the initial rate of hydrolysis decreases after approx. 20 ~ of the t R N A is rendered acid soluble 14, as if the enzyme was first recognizing and digesting the non-helical "loop" regions of the t R N A molecule which occur in sequences of 7-12 nucleotides.

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271

One limitation inherent in these experiments is the phenomenon of induction; formylated bases induce the denaturation of adjacent base pairs and there is a greater probability of a denatured loop opening further than of an identical loop (analogous in position) being opened in another molecule. The recent observations of Utiyama and Doty Is on the temperature dependence of this induction phenomenon may explain why the maps obtained by thecmal denaturation are less reproducible than those obtained by alkali-induced denaturation 1° and also imply appropriate measures to reduce this effect. The number-average histograms of Inman and Schn6s, which show the position and frequency of denatured sites along the molecule at a given pH, indicate that under the conditions used in these experiments the degree of homogeneity at a given pH is characteristic for different regions in the molecule a°. We have attempted to carry out the partial denaturation without the "fixation" step. In the absence of formaldehyde the denatured regions revert to a state that is refractile to Neurospora enzyme. This leads us to suggest, in agreement with the conclusions of Fuke et al.19, that in those studies of partially denatured DNA which do not include a "fixing" step the single-stranded regions which remain after quenching originate primarily at the termini of molecules or at single-strand nicks. The use of N. crassa endonuclease to cleave DNA molecules at early denaturing regions has possible application in several areas. At the analytical level it offers one approach to studying the chromosomal distribution of regions that are exceptionally sensitive to denaturation and which may be significant in the structure and/or regulation of the genome. In attempts to isolate and study specific regions of the chromosome the approach of cleaving DNA molecules at specific regions on the basis of their susceptibility to denaturation may provide a flexible complement to the sitespecific endonucleases (restriction enzymes) presently available (e.9. ref. 20). The experiments reported here have been carried out with ~80 DNA because of our interest in the specific transducing derivatives of this bacteriophage which carry the structural gene(s) for tyrosine tRNA. The ability to monitor a specific DNA fragment by hybridization with tRNA (refs 7, 21)should facilitate further characterization of the denaturation-dependent cleavage of DNA. ACKNOWLEDGEMENTS

We gratefully acknowledge the contribution made by Jan Gurgel in the early stages of this work. This work was supported by grant No. CAl1208 from the U.S.P.H.S. and grant No E-568 from the American Cancer Society. REFERENCES 1 Inman, R. B. (1967) J. Mol. Biol. 28, 103-116 2 Marmur, J., Round, R. and Schildkraut, C. L. (1963) Proy. Nucleic Acid Res. Mol. Biol. 1, 231-300 3 Roger, M. and Hotchkiss, R. D. (1961) Proc. NatL Acad. Sci. U.S. 47, 653-669 4 Ginoza, W. and Zimm, B. I4. (1961) Proc. Natl. Acad. Sci U.S. 47, 639-652 5 14irschman, S. Z., Gellert, M., Falkow, S. and Felsenfeld, G. (1967) J. MoL BioL 28, 469-477 6 Falkow, S. and Cowie, D. B. (1968) J. Bacteriol. 96, 777-784 7 Landy, A., Abelson, J., Goodman, H. M. and Smith, J. D. (1967) J. Mol. BioL 29, 457-471 8 Linn, S. (1967) in Methods in E n z y m o l o e y (Grossman, L. and Moldave, K., eds), Vol. XII, pp. 247-255, Academic Press, New York

272 9 10 11 12 13 14 15 16 17 18 19 20 21

A. L A N D Y et al. Vogel, 1-I. J. (1956) Microb. Gen. Bull. 13, 42-43 lnman, R. B. and Schn~Ss, M. (1970) J. Mol. Biol. 49, 93-98 Freifelder, D. and Davison, P. F. (1963) Biophys. J. 3, 49-63 Peacock, A. C. and Dingman, C. W. (1968) Biochemistry 7, 668-674 Loening, U. E. (1967) Biochem. J. 102, 251-257 Linn, S. and Lehman, I. R. (1965) J. Biol. Chem. 240, 1294-1304 Staehelin, M. (1958) Biochim. Biophys. Acta 29, 410-417 Dahlberg, A. E., Dingman, C. W. and Peacock, A. C. (1969) J. Mol. Biol. 41, 139-147 Fisher, M. P. and Dingman, C. W. (1971) Biochemistry 10, 1895-1899 Utiyama, H. and Doty, P. (1971) Biochemistry 10, 1254-1264 Fuke, M., Wada, A. and Tomizawa, J. (1970) J. Mol. Biol. 51,255-266 Danna, K. and Nathans, D. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2913-2917 Russell, R. L., Abelson, J. N., Landy, A., Gefter, M. L., Brenner, S. and Smith, J. D. (1970) J. Mol. Biol. 47, 1-13