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Analysis of DNA bearing single-chained terminals by BNC chromatography
ANALYTICAL
BIOCHEMISTRY
Analysis
!j@
of DNA
5.58568 (1972)
Bearing
by BNC ROBERT Department
Single-Chained
Terminals
Chromatography
A. SCHLEGEL,l REED E. PYERITZ, CHARLES A. THOMAS, JR. of Biological Boston,
Chemistry, Massachusetts
Harvard 02116
Medical
AND
School,
Received May 17, 1972; accepted July 17, 1972
The chromatography of DNA on hydroxyapatite (HA) and benzoylated-naphthoylated DEAE-cellulose (BNC) both depend on the secondary structure of the polynucleotide chains, yet operate in a precisely opposite manner. While both fractionations are almost completely independent of molecular weight, HA retains structures having regions of double-helical structure. While the length of such double-helical regions necessary for retention on HA are not known precisely, it is likely that a few hundred nucleotide pairs will suffice (1). In contrast BNC retains single chains as well as double-helical pieces of DNA provided that they bear single-chained regions of sufficient length. The relationship between the length of the single-chain region(s) and the concentration of caffeine required for elution from BNC columns is the topic of this communication. BNC has proved valuable in the isolation of intermediates of DNA replication (2-5), bacteriophage RNA replication (6), and DNA repair (7). Fragments of DNA produced by hydrodynamic shear-breakage have been shown to often have single-chained terminals which have been characterized on BNC (8). Though the production of this type of damaged fragment is often undesired, their properties have been useful in the study of eukaryotic chromosomal organization (9). Partially or totally single-chained molecules elute from BNC in the presence of caffeine. Iyer and Rupp (7) found a linear relationship between the length of the single chain attached to the duplex DNA and the concentration of caffeine necessary for elution. We have confirmed and extended this observation. We present here a more detailed technique that makes possible the study of mechanically or enzymically altered DNA molecules bearing single chains. ‘Present address: The Walter and Eliza Hall Institute Royal Melbourne Hospital, Melbourne, Australia. 558 Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
of Medical
Research, The
DNA
BEARING
SINGLE-CHAINED
559
TERMINALS
METHODS
Preparation of DNA. Bacteriophage strain T7L (Luria), obtained from W. S. Studier, was 3H- or 32P-labeled and purified as described by Kelly and Thomas (10). Sedimentation of the T7 DNA through alkaline sucrose revealed that an average of one single chain in eight was broken, indicating the presence of 0.25 single-chain nicks per molecule. The best preparations of T7 DNA from this laboratory contain an average of 0.1 nicks per molecule. Benxoylated-naphthoylated DEAE-cellulose chromatography. BNC was prepared by the procedure of Gillam et al. (11) and stored as a suspension in 0.001 ib! EDTA/O.Ol M Tris-HCl (pH 7.4) at 4°C with a drop of toluene. Columns were prepared just prior to use by suspending about 1 ml of BNC slurry in 0.3 NET2 (0.3M NaCb’0.001 M EDTAJO.01 M Tris-HCI (pH 7.4)) and introducing enough of this SUSpension to a 1 X 5 cm column to give a gravity-packed bed volume of 0.5 ml supported on a plate of porous polyethylene. The column was then washed with at least 15 ml of 0.3 NET followed by addition of the DNA sample, also in 0.3 NET. At least 24 mg of DNA could be adsorbed to this small column, though we routinely worked with 1-5 pg of sample of minimum specific activity lo4 cpm/pg. The flow rate was adjusted to 0.5 ml/min and the adsorbed sample was washed with 15 ml of 0.3 NET. Double-chained molecules were eluted by washing with 15 ml of 1.0 NET. The elution of molecules with single-chained regions was accomplished with a 40 ml O-l% linear caffeine gradient in 1.0 NET. All operations were carried out at room temperature. Assay of column fractions. Fractions of 12 drops (0.65 ml) were collected directly into miniscintillation vials (Nuclear Associates), mixed with 5.5 ml of a scintillation cocktail (15.0 gm PPO, 0.325 gm POPOP, 950 ml Triton X-100, and 1900 ml toluene), capped, shaken, placed inside the conventional glass scintillation vials, and counted in a Packard Tri-Carb liquid scintillation spectrometer. The counting efficiency of this procedure was less than 10% inferior to placing a sample (10 ~1) of a column fraction directly into a normal glass vial containing 10 ml cocktail. Caffeine concentration was determined by diluting 10 ~1 aliquots of fractions with 2.0 ml 1.0 NET and determining the absorbance at 260 nm. Velocity sedimentation. Neutral sedimentations were made through 5-25% linear sucrose gradients in 1.0 NET. Alkaline sedimentations were made through 5-25s linear sucrose gradients in 0.3 M NaOH, 0.7 NET. *The number (0.3) preceding NET refers to the which is the only variable in this solution throughout
molar concentration these experiments.
of
NaCl,
560
SCHLEGEL,
PYERITZ,
AND
THOMAS
Centrifugation was at 50,000 rpm for 7&120 min at 20°C in cellulose nitrate tubes in a swinging-bucket rotor. Nine-drop fractions were collected from the bottom in the conventional manner. Exonuclease treatments. The h-exonuclease was purified according to Radding (12) and E. coli exonuclease III was purified according to Richardson (13). Both enzymes were prepared and generously donated by Dr. M. Fuke. The reaction mixture for X-exonuclease contained 3 mM MgCl, and 10 n& glycine-KOH (pH 9.2). Under these conditions, X-exonuclease acts in a progressive, as opposed to a random, manner; therefore, for a given concentration of DNA, an enzyme saturation curve was plotted before doing an experiment. In this manner we ensured that a sufficient excess of enzyme was added to bind to each DNA terminal. Exonuclease III hydrolyzes nucleotides from 3’-terminals in a random manner, so that the enzyme concentration is not critical. The reaction mixture contained 7 m&f MgCl,, 8.5 mM p-mercaptoethanol, and 67 mM Tris-HCl (pH 8.0). Both nuclease digestions were stopped by the addition of EDTA to 5 mM and cooling to 0°C. The extent of digestion was determined by the release of trichloroacetic acid soluble radioactivity, which was presumed to be free nucleotides. Shear-breakage of DNA. Preparations of DNA were broken by stirring as described by Hershey, Goldberg, Burgi, and Ingraham (14) and modified by Schlegel (15). Shear-breakage under these conditions does not produce fragments bearing significant single-chained tails as judged by their failure to bind to BNC (8). RESULTS
T7 DNA molecules. Radiolabeled T7 DNA was treated with either X-exonuclease or exonuclease III as described above. Samples removed from the digestion mixtures at various times were diluted with an equal volume of 0.1 M EDTA at 0°C and the extent of degradation determined. The number of nucleotides released from each 5’-terminus (h-exo) or 3’-terminus (exo III) was calculated assuming that T7L contained 38,000 nucleotide pairs. Samples of the exonuclease III treated T7 DNA, which ranged from an average of 240 to 1330 nucleotides removed per 3’-terminus, were annealed in 2 X SSC3 at 65°C for 2 hr. As measured by sedimentation in neutral sucrose gradients, greater than 75% of the treated T7 molecules cyclized. Preparation
of partially-digested
Chromatography
of p&idly
Samples of each partially
digested T7 DNA
molecules on BNC.
digested sample were loaded onto BNC
’ SSC is 0.15 M NaCl, 0.015 M sodium citrate.
col-
DNA
BEARING
SINGLE-CHAINED
TEXMINALS
561
umns, washed, and eluted as described under “Methods.” A typical elution profile, this one for 1.5% digestion (570 nucleotides per 5’-terminus) by X-exonuclease, is shown in Fig. la. Recoveries of input radioactivity were rarely less than 80% after final elution with 1.0% caffeine in 1.0 NET. As expected, all of the undegraded T7 DNA added as a control was eluted in the salt peak, while most of the degraded DNA appeared in the caffeine gradient. For samples with less than an average of 1000 nucleotides exposed per 5’-terminus, we consistently observed two peaks in the caffeine gradient. The smaller earlier peak (peak I in Fig. la) always occurred at 0.1% caffeine. Peak I. The fraction of the DNA which eluted in peak I varied inversely with the degree of degradation. For example, peak I contained 35% of the input label when 240 nucleotides had been removed. This was reduced to 5% when an average of 1500 nucleotides had been removed from each end. Considerably less DNA eluted in peak I when this DNA was treated with exonuclease III rather than with X-exonuclease (Fig. lb). T7 DNA, from which an average of 900 nucleotides had been removed from each 5’-termini by X-exonuclease, was chromatographed; 91% of the input eluted with caffeine and, of this, 8% was in peak I. An aliquot of the pooled fractions of peak I was rechromatographed and again it eluted at 0.1% caffeine. The fractions containing peak I and the pooled fractions comprising peak II were dialyzed separately against 2 X SSC, and then annealed at 65°C. No cyclization occurred in material from peak I: all molecules were linear monomers of T7 size. After annealing, most of the DNA molecules from peak II were in the form of rings. It must be remembered that, while a total of TR + 12 nucleotides must be removed from both ends to provide for a stable cyclization event (when TR is the number of nucleotides in the terminal repetition), this resection can be arranged in TR-11 ways. One possible configuration could have TR nucleotides removed from one end and 12 nucleotides from the other. Therefore, we conclude that few if any nucleotides are removed from either terminal of the material in peak I by A-exonuclease, while all molecules in Peak II have at least one terminal and possibly two terminals significantly single-chained (Fig. la). When the resection of the terminals was carried out by exonuclease III, only a small percentage of the input eluted in 0.1% caffeine, even for very limited digestions (Fig. lb). This contrast between the exonuclease III resected T7 and the X-exonuclease resected T7 was entirely reproducible, and suggests a fundamental difference in the mechanism of action of these two exonucleases. Exonuclease III initiates digestion
562
SCHLEGEL,
PYERITZ,
AND
THOMAS
0 10
0.3
NETA I-
20
1.0 NET
30 Fraction
40
L T
50
O-LO%
60
70
CAFFEINE 4
Fraction
Fro. 1. BNC chromatography of resected T7 DNA molecules: (a) ‘H-T7 DNA WETIS treated with A-exonuclease as described in the text so that 1.5% of the input radioactivity was rendered TCA-soluble, corresponding to an average resection per 5’-terminal of 570 nucleotides. A sample of this resected DNA was diluted with 0.3 NET along with a sample of unresected *‘P-T7 DNA. Chromatography was carried out as described in the text. Open circles (O), resected “H-T7 DNA; closed circles (a), unresected “P-T7 DNA; triangles (A), absorbancy at 260 nm of a 1: 200 dilution of alternate fractions. (b) *H-T7 DNA was treated with exonuclease III so that 1.1% of the input radioactivity was rendered acid-soluble, corresponding to an average resection per 3’-terminal of 420 nucleotides. Chromatography was carried out as described in the text. The peak fraction used in estimating the caffeine concentration needed for elution is indicated in this figure and Fig. la by an arrow. Open circles (0)) resected aH-T7 DNA; triangles (A), absorbancy at 260 nm of a 1:200 dilution of alternate fractions.
DNA
BEARING
SINGLE-CHAINED
TERMINALS
563
with either 3’-OH or 3’-PO, groups present since it is also a 3’-phosphatase as well as an exonuclease (16). The 3’-terminus need not even be base-paired, though the double-chain conformation is preferred (17). On the other hand, h-exonuclease specifically requires the terminal 5’-PO4 group of base-paired DNA to initiate action (18). Therefore, during the course of treatment with these nucleases (220 min), exonuclease III begins to digest nucleotides from all 3’-termini. If the 5’-PO4 group is missing, however, h-exonuclease initiates with difficulty if at all. If degradation does occur at 5’-OH terminals, the extent might be much lower than at 5’-PO4 terminals. Another known difference exists between these two enzymes that may explain the presence of peak I. Under the stated pH conditions, exonuclease III acts in a random manner (each enzyme molecule freely moves from substrate to substrate removing one nucleotide at a time) whereas h-exonuclease acts progressively (once bound to the 5’-terminal, the X-exonuclease molecule degrades only a single DNA molecule) (19). If a h-exonuclease molecule can no longer continue resection, further degradation from that point is effectively blocked, until the original enzyme molecule falls off. Since X-exonuclease is fairly unstable under reaction conditions, degradation at some terminals may be blocked for this reason. Thus, we suggest that the DNA eluting in peak I consists of those molecules with few nucleotides removed from both terminals. This is supported by the fact that none of this peak I DNA is capable of cyclization, and that peak I almost disappears with exonuclease III degradation. Peak II. From elution profiles such as in Fig. 1 it is possible to estimate the caffeine concentration of the peak fraction of the radioactivity eluting in greater than 0.1% caffeine. These approximate caffeine concentrations obtained from numerous experiments employing both A-exonuclease and exonuclease III are plotted versus the average number of nucleotides resected in Fig. 2. We find that the caffeine concentration necessary to elute peak II steadily increases for molecules with approximately 200 nucleotides removed per terminal to molecules with an average of 1300 nucleotides resected per terminal. For resections greater than 1300-1500 nucleotides per terminal, the caffeine concentration is inversely related to the single-chain length. The results of Iyer and Rupp (7) are also plotted in Fig, 2. Their results are similar to ours, and the slopes of both parts of the biphasic curves are similar. However, our curve is located about 1000 nucleotides to the left of that determined in the study already published (7). In addition, Iyer and Rupp are able to extrapolate their curve through the
564
SCHLEGEL,
1
I
I
PYFXITZ,
I
I
AND
I
THOMAS
I
I
I
I
I
0.60
0.40 .-I c z u 8
0.20
0 Nucleotides
Resected
FIQ. 2. Relationship between degree of resection and caffeine concentration needed to elute peak II from BNC. Estimation of peak elution is described in the text and Fig. lb. Open circles (O), T7 DNA treated with A-exonuclease; closed circles (O), T7 DNA treated with exonuclesse III; vertical bars indicate data of Iyer and Rupp (7) for Pz2 DNA treated with A-exonuclease. The lines through the data points (indicated by circles) were drawn by means of a least-square analysis by computer. The broken lines represent degrees of resection not explored by these experiments.
origin, though the chromatographic properties of molecules possessing single chains less than 800 nucleotides long were not examined. Differences such as this probably reflect variations in the BNC resin; it appears that our preparation of BNC binds single chains more tightly in the range of O-2000 nucleotides resected, since a higher caffeine concentration is needed in our experiments to elute molecules of a given degree of resection. Results with our preparation of BNC have been entirely reproducible over a period of at Ieast three months. Both extremes of exonuclease resection are of interest in Figure 2. Peak I elutes entirely at 0.1% caffeine, probably as a result of very limited resection by the exonuclease. We have not attempted to determine the extent of resection of the peak I molecules. However, linear monomers of phage X DNA are eluted completely with 1.0 NET (3), and, we have confirmed that fact with our BNC preparation. Therefore, either a single chain 12 nucleotides in length is not sufficient to bind to BNC, or the cohesive terminals of h are internally complementary and have assumed a partially duplex form that does not interact with BNC (20,21). The minimum length of single chain required to bind to BNC might depend on the cellulose particle size and shape, and on the extent of benzoylation and naphthoylation.
DNA
BEARING
SINGLE-CHAINED
565
TERMINALS
The decrease in caffeine concentration required to elute single chains greater than 2000 nucleotides can be explained if one remembers that, under these salt and temperature conditions, nonspecific hydrogen bonding of bases is promoted (22). The formation of “hairpin” structures by the long single chains would effectively reduce the number of nucleotides available for binding to BNC. The cooperation of both single-chained terminals in binding. Peak II in elutions of samples with an average of lOOC~2000 nucleotides removed per terminus generally appears as a double peak (see Fig. 1). This suggested to us that molecules with both ends resected bind more tightly to BNC than molecules with only one end bearing a single chain. To test this hypothesis, exonuclease III treated T7 DNA was sheared to half-molecules by stirring. Table 1 lists the elution data for sheared and unsheared molecules, first digested with exonuclease III. It is apparent that approximately 2&25% of the material which eluted in caffeine before shearing eluted in NET after breakage to half-molecules. Those half-molecules which continue to elute in caffeine now do so at a lower
Elution
TABLE 1 of Exonuclease III Treated T7 DNA before and after Shearing y0 of input counts
Substrate Whole molecules
Half-molecules
Av. length of single chains (nut.)
0.3 NET
240 420 1000 1300 420 1000 1300
0.4 0.9 2.7 4.5 1.3 4.2 4.1
1.0 NET
O-l% caffeine
y. caffeine of peak II elution
0.2 0.2 0.4 1.2 24.7 21.7 19.5
84.3 86.0 80.9 80.2 57.2 61.3 66.3
0.27 0.31 0.37 0.47 0.25 0.33 0.33
Whole T7 molecules were resected with exonuclease III to the extents shown. Aliquots of three of the resected preparations were sheared to half-molecules by stirring as described in the text. The caffeine concentration in each fraction was measured by determining the absorbancy at 260 nm of dilutions of 10 ~1 samples. The caffeine concentration necessary for elution of partially single-chained molecules wss estimated by determining the per cent caffeine in the peak fraction of peak II (see text). One-quarter to one-fifth of the half-molecules elute in 1.0 NET, indicating an absence cf single-chained terminals. Those half-molecules continuing to elute in caffeine do so at a significantly lower caffeine concentration than do whole molecules resected to the same extent. For example, the decrease in caffeine concentration necessary to elute molecules with only one single-chained terminal 1300 nucleotides long from that needed to elute a whole molecule with two such single chains is from 0.47y0 to 0.3397,, a shit of 8 fractions in the gradient.
566
SCHLEGEL,
PYERITZ,
AND
THOMAS
T7 DNA Shearing
Exonuclease
I
BNC
I
BNC
= GE
=
I
BNC
FIG. 3. Schematic representation of results and conclusions of experiments described in the text. The lower portion of the figure contains examples of BNC chromatographic elution profiles which were reproducibly obtained after performing the procedures described on the top line. We interpret these results as suggesting the formation of the molecular configurations drawn on the top line.
caffeine concentration. This suggests that the presence of significant single chains on both ends of a molecule results in tighter binding to BNC than does the presence of only one single-chained end. The shearing process does not remove single chains from the molecules, because in unrelated experiments, we have been able to form “joined halves” with T7 by first resecting and then shearing (23). Our interpretation of these results is depicted in Fig. 3. In the experiment just described, exonuclease III failed to digest approximately 25% of the 3’-terminals. This finding is not typical of many previous exonuclease III digestions in which all terminals are digested (15). Why this occurred in this case is unclear. Generally, shear breakage to half-molecules and BNC-chromatography of T7 DNA after exonuclease III treatment does not result in any DNA eluting in 1.0 NET, indicating the presence of single chains at all of the 5’-terminals (15). Repeating this experiment with T7 DNA treated with X-exonuclease invariably yields 15-40s of the half-molecules elutable by l.OM NaCl. In addition, the shear-broken molecules in peak II elute at a lower caffeine concentration than do the whole molecules. These results support the model shown in Fig. 3 and again suggest a difference in the mode of action of the two exonucleases. SUMMARY
T7 DNA was t,reated with X-exonuclease or exonuclease III in order to produce single-chained terminals on predominately duplex molecules. DNA treated in this fashion binds to columns of benzoylatedNative
DNA
BEARING
SINGLE-CHAINED
567
TERMINALS
naphthoylated DEAE-cellulose (BNC) in the presence of l.OM NaCl and is elutable by a linear gradient of O-1.00/0 caffeine. The concentration of caffeine needed to elute resected molecules is proportional to the length of the single-chained terminals for single chains ranging from 200 to 1300 nucleotides in length. A gradual decrease in the caffeine concentration necessary to elute molecules with terminals longer than 1500 nucleotides is observed. The BNC column therefore provides a simple method for the fractionation of DNA molecules based on the length of exposed single chains. Shear-breakage of resected molecules reveals that DNA possessing two single-chained terminals binds more tightly to BNC than molecules with only one resected terminal. Based on these results, a comparison was made between DNA resected by one exonuclease and the other. Significant differences were obtained which suggest that these methods should be useful for determining the mode of action of various nucleases. ACKNOWLEDGMENTS We thank Dr. Motohiro Fuke for gifts of A-exonuclease and exonuclease III. This investigation was generously supported by grants from the National Institutes of Health (5ROI-A1081861 and the National Science Foundation (GB-86111, as well as a National Science Foundation Predoctoral Fellowship to Reed Pyerite. REFERENCES 1. MIYAZAWA, 2. KNIPPERS,
Y., AND THOMAS, C. A., R., RAZIN, A., DAVIS, R.,
JR., J. Mol.
Biol. AND SINSHEIMER,
11, 223 (1965). R.
L., J. Mol.
Biol.
45,
237 (1969). 3. KIGER, J. A., JR., AND SINSHEIMER,
R. L., J. Mol. Biol. 40, 467 (1969). 4. SEDAT, J., LYON, A., AND SINSHEIMER, R. L., J. Mol. Biol. 44, 415 (1969). 5. SCHLEGEL, R. A., AND THOMAS, C. A., JR., J. Mol. Biol. 68, 319 (1972). 6. KELLY, R., AND SINSHEIMER, R. L., J. Mol. Biol. 29, 22 (1967). 7. IYER, V. N., AND RUPP, W. D., Biochim. Biophys. Acta 228, 117 (1971). 8. PYERITZ, R. E., SCHLEGEL, R. A., AND THOMAS, C. A., JR., B&him. Biophys. Acta 272, 504 (1972). ~..PYERITZ, R. E., LEE, C. S., AND THOMAS, C. A., JR., Chromosomu 33, 284 (1971). 10. KELLY, T. J., JR., AND THOMAS, C. A., JR., J. Mol. Biol. 44, 459 (1969). 11. GILLAM, I., MILLWARD, S., BLEW, D., VON TIGERSTROM, M., ‘WIMMER, E., AND TENER, G. M., Biochemistry 6, 3043 (1967). 12. RADDING, C. M., J. Mol. Biol. 18, 235 (1966). 13. RICHARDSON, C. C., in “Procedures in Nucleic Acid Research” (G. L. Cantoni and D. R. Davies, eds.), p. 212. Harper & Row, New York, 1966. 14. HERSHEY, A. D., GOLDBERG, E., BURGI, E., AND INGRAHAM, L., J. Mol. E&l. 6,
230 (1963). 15. SCHLEGEL, R. A., Ph.D. Thesis, Harvard University, Cambridge, Mass., 16. RICHARDSON, C. C., AND KORNBERQ, A., J. Biol. Chem. 239, 242 (1964). Ii’. RICHARDSON, C. C., LEHMAN, I. R., AND KORNBERG, A., J. Biol. Chem.
( 1969) 18. LITTLE,
J. W.,
J. Biol.
Chem.
242,
679
(1967).
1971.
239, 251
568
SCHLEGEL,
PYERITZ,
AND
THOMAS
19. CARTER, D. M., ANTI RADDINQ, C. M., J. Biol. Chem. 246, 2502 (1971). 20. WANO, J. C., AND DAVIDEON, N., Cold Spring Harbor Symp. Quant. Biol. 33, 409 (1968). 21. WV, R., AND TAYLOR, E., J. Mol. Biol. 57, 491 (1971). 22. STUDIER, F. W., J. Mol. Biol. 41, 189 (1969). 23. LEE, C. S., PYERITZ, R. E., AND THOMAS, C. A., JR., in preparation.
BIOCHEMISTRY
Analysis
!j@
of DNA
5.58568 (1972)
Bearing
by BNC ROBERT Department
Single-Chained
Terminals
Chromatography
A. SCHLEGEL,l REED E. PYERITZ, CHARLES A. THOMAS, JR. of Biological Boston,
Chemistry, Massachusetts
Harvard 02116
Medical
AND
School,
Received May 17, 1972; accepted July 17, 1972
The chromatography of DNA on hydroxyapatite (HA) and benzoylated-naphthoylated DEAE-cellulose (BNC) both depend on the secondary structure of the polynucleotide chains, yet operate in a precisely opposite manner. While both fractionations are almost completely independent of molecular weight, HA retains structures having regions of double-helical structure. While the length of such double-helical regions necessary for retention on HA are not known precisely, it is likely that a few hundred nucleotide pairs will suffice (1). In contrast BNC retains single chains as well as double-helical pieces of DNA provided that they bear single-chained regions of sufficient length. The relationship between the length of the single-chain region(s) and the concentration of caffeine required for elution from BNC columns is the topic of this communication. BNC has proved valuable in the isolation of intermediates of DNA replication (2-5), bacteriophage RNA replication (6), and DNA repair (7). Fragments of DNA produced by hydrodynamic shear-breakage have been shown to often have single-chained terminals which have been characterized on BNC (8). Though the production of this type of damaged fragment is often undesired, their properties have been useful in the study of eukaryotic chromosomal organization (9). Partially or totally single-chained molecules elute from BNC in the presence of caffeine. Iyer and Rupp (7) found a linear relationship between the length of the single chain attached to the duplex DNA and the concentration of caffeine necessary for elution. We have confirmed and extended this observation. We present here a more detailed technique that makes possible the study of mechanically or enzymically altered DNA molecules bearing single chains. ‘Present address: The Walter and Eliza Hall Institute Royal Melbourne Hospital, Melbourne, Australia. 558 Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
of Medical
Research, The
DNA
BEARING
SINGLE-CHAINED
559
TERMINALS
METHODS
Preparation of DNA. Bacteriophage strain T7L (Luria), obtained from W. S. Studier, was 3H- or 32P-labeled and purified as described by Kelly and Thomas (10). Sedimentation of the T7 DNA through alkaline sucrose revealed that an average of one single chain in eight was broken, indicating the presence of 0.25 single-chain nicks per molecule. The best preparations of T7 DNA from this laboratory contain an average of 0.1 nicks per molecule. Benxoylated-naphthoylated DEAE-cellulose chromatography. BNC was prepared by the procedure of Gillam et al. (11) and stored as a suspension in 0.001 ib! EDTA/O.Ol M Tris-HCl (pH 7.4) at 4°C with a drop of toluene. Columns were prepared just prior to use by suspending about 1 ml of BNC slurry in 0.3 NET2 (0.3M NaCb’0.001 M EDTAJO.01 M Tris-HCI (pH 7.4)) and introducing enough of this SUSpension to a 1 X 5 cm column to give a gravity-packed bed volume of 0.5 ml supported on a plate of porous polyethylene. The column was then washed with at least 15 ml of 0.3 NET followed by addition of the DNA sample, also in 0.3 NET. At least 24 mg of DNA could be adsorbed to this small column, though we routinely worked with 1-5 pg of sample of minimum specific activity lo4 cpm/pg. The flow rate was adjusted to 0.5 ml/min and the adsorbed sample was washed with 15 ml of 0.3 NET. Double-chained molecules were eluted by washing with 15 ml of 1.0 NET. The elution of molecules with single-chained regions was accomplished with a 40 ml O-l% linear caffeine gradient in 1.0 NET. All operations were carried out at room temperature. Assay of column fractions. Fractions of 12 drops (0.65 ml) were collected directly into miniscintillation vials (Nuclear Associates), mixed with 5.5 ml of a scintillation cocktail (15.0 gm PPO, 0.325 gm POPOP, 950 ml Triton X-100, and 1900 ml toluene), capped, shaken, placed inside the conventional glass scintillation vials, and counted in a Packard Tri-Carb liquid scintillation spectrometer. The counting efficiency of this procedure was less than 10% inferior to placing a sample (10 ~1) of a column fraction directly into a normal glass vial containing 10 ml cocktail. Caffeine concentration was determined by diluting 10 ~1 aliquots of fractions with 2.0 ml 1.0 NET and determining the absorbance at 260 nm. Velocity sedimentation. Neutral sedimentations were made through 5-25% linear sucrose gradients in 1.0 NET. Alkaline sedimentations were made through 5-25s linear sucrose gradients in 0.3 M NaOH, 0.7 NET. *The number (0.3) preceding NET refers to the which is the only variable in this solution throughout
molar concentration these experiments.
of
NaCl,
560
SCHLEGEL,
PYERITZ,
AND
THOMAS
Centrifugation was at 50,000 rpm for 7&120 min at 20°C in cellulose nitrate tubes in a swinging-bucket rotor. Nine-drop fractions were collected from the bottom in the conventional manner. Exonuclease treatments. The h-exonuclease was purified according to Radding (12) and E. coli exonuclease III was purified according to Richardson (13). Both enzymes were prepared and generously donated by Dr. M. Fuke. The reaction mixture for X-exonuclease contained 3 mM MgCl, and 10 n& glycine-KOH (pH 9.2). Under these conditions, X-exonuclease acts in a progressive, as opposed to a random, manner; therefore, for a given concentration of DNA, an enzyme saturation curve was plotted before doing an experiment. In this manner we ensured that a sufficient excess of enzyme was added to bind to each DNA terminal. Exonuclease III hydrolyzes nucleotides from 3’-terminals in a random manner, so that the enzyme concentration is not critical. The reaction mixture contained 7 m&f MgCl,, 8.5 mM p-mercaptoethanol, and 67 mM Tris-HCl (pH 8.0). Both nuclease digestions were stopped by the addition of EDTA to 5 mM and cooling to 0°C. The extent of digestion was determined by the release of trichloroacetic acid soluble radioactivity, which was presumed to be free nucleotides. Shear-breakage of DNA. Preparations of DNA were broken by stirring as described by Hershey, Goldberg, Burgi, and Ingraham (14) and modified by Schlegel (15). Shear-breakage under these conditions does not produce fragments bearing significant single-chained tails as judged by their failure to bind to BNC (8). RESULTS
T7 DNA molecules. Radiolabeled T7 DNA was treated with either X-exonuclease or exonuclease III as described above. Samples removed from the digestion mixtures at various times were diluted with an equal volume of 0.1 M EDTA at 0°C and the extent of degradation determined. The number of nucleotides released from each 5’-terminus (h-exo) or 3’-terminus (exo III) was calculated assuming that T7L contained 38,000 nucleotide pairs. Samples of the exonuclease III treated T7 DNA, which ranged from an average of 240 to 1330 nucleotides removed per 3’-terminus, were annealed in 2 X SSC3 at 65°C for 2 hr. As measured by sedimentation in neutral sucrose gradients, greater than 75% of the treated T7 molecules cyclized. Preparation
of partially-digested
Chromatography
of p&idly
Samples of each partially
digested T7 DNA
molecules on BNC.
digested sample were loaded onto BNC
’ SSC is 0.15 M NaCl, 0.015 M sodium citrate.
col-
DNA
BEARING
SINGLE-CHAINED
TEXMINALS
561
umns, washed, and eluted as described under “Methods.” A typical elution profile, this one for 1.5% digestion (570 nucleotides per 5’-terminus) by X-exonuclease, is shown in Fig. la. Recoveries of input radioactivity were rarely less than 80% after final elution with 1.0% caffeine in 1.0 NET. As expected, all of the undegraded T7 DNA added as a control was eluted in the salt peak, while most of the degraded DNA appeared in the caffeine gradient. For samples with less than an average of 1000 nucleotides exposed per 5’-terminus, we consistently observed two peaks in the caffeine gradient. The smaller earlier peak (peak I in Fig. la) always occurred at 0.1% caffeine. Peak I. The fraction of the DNA which eluted in peak I varied inversely with the degree of degradation. For example, peak I contained 35% of the input label when 240 nucleotides had been removed. This was reduced to 5% when an average of 1500 nucleotides had been removed from each end. Considerably less DNA eluted in peak I when this DNA was treated with exonuclease III rather than with X-exonuclease (Fig. lb). T7 DNA, from which an average of 900 nucleotides had been removed from each 5’-termini by X-exonuclease, was chromatographed; 91% of the input eluted with caffeine and, of this, 8% was in peak I. An aliquot of the pooled fractions of peak I was rechromatographed and again it eluted at 0.1% caffeine. The fractions containing peak I and the pooled fractions comprising peak II were dialyzed separately against 2 X SSC, and then annealed at 65°C. No cyclization occurred in material from peak I: all molecules were linear monomers of T7 size. After annealing, most of the DNA molecules from peak II were in the form of rings. It must be remembered that, while a total of TR + 12 nucleotides must be removed from both ends to provide for a stable cyclization event (when TR is the number of nucleotides in the terminal repetition), this resection can be arranged in TR-11 ways. One possible configuration could have TR nucleotides removed from one end and 12 nucleotides from the other. Therefore, we conclude that few if any nucleotides are removed from either terminal of the material in peak I by A-exonuclease, while all molecules in Peak II have at least one terminal and possibly two terminals significantly single-chained (Fig. la). When the resection of the terminals was carried out by exonuclease III, only a small percentage of the input eluted in 0.1% caffeine, even for very limited digestions (Fig. lb). This contrast between the exonuclease III resected T7 and the X-exonuclease resected T7 was entirely reproducible, and suggests a fundamental difference in the mechanism of action of these two exonucleases. Exonuclease III initiates digestion
562
SCHLEGEL,
PYERITZ,
AND
THOMAS
0 10
0.3
NETA I-
20
1.0 NET
30 Fraction
40
L T
50
O-LO%
60
70
CAFFEINE 4
Fraction
Fro. 1. BNC chromatography of resected T7 DNA molecules: (a) ‘H-T7 DNA WETIS treated with A-exonuclease as described in the text so that 1.5% of the input radioactivity was rendered TCA-soluble, corresponding to an average resection per 5’-terminal of 570 nucleotides. A sample of this resected DNA was diluted with 0.3 NET along with a sample of unresected *‘P-T7 DNA. Chromatography was carried out as described in the text. Open circles (O), resected “H-T7 DNA; closed circles (a), unresected “P-T7 DNA; triangles (A), absorbancy at 260 nm of a 1: 200 dilution of alternate fractions. (b) *H-T7 DNA was treated with exonuclease III so that 1.1% of the input radioactivity was rendered acid-soluble, corresponding to an average resection per 3’-terminal of 420 nucleotides. Chromatography was carried out as described in the text. The peak fraction used in estimating the caffeine concentration needed for elution is indicated in this figure and Fig. la by an arrow. Open circles (0)) resected aH-T7 DNA; triangles (A), absorbancy at 260 nm of a 1:200 dilution of alternate fractions.
DNA
BEARING
SINGLE-CHAINED
TERMINALS
563
with either 3’-OH or 3’-PO, groups present since it is also a 3’-phosphatase as well as an exonuclease (16). The 3’-terminus need not even be base-paired, though the double-chain conformation is preferred (17). On the other hand, h-exonuclease specifically requires the terminal 5’-PO4 group of base-paired DNA to initiate action (18). Therefore, during the course of treatment with these nucleases (220 min), exonuclease III begins to digest nucleotides from all 3’-termini. If the 5’-PO4 group is missing, however, h-exonuclease initiates with difficulty if at all. If degradation does occur at 5’-OH terminals, the extent might be much lower than at 5’-PO4 terminals. Another known difference exists between these two enzymes that may explain the presence of peak I. Under the stated pH conditions, exonuclease III acts in a random manner (each enzyme molecule freely moves from substrate to substrate removing one nucleotide at a time) whereas h-exonuclease acts progressively (once bound to the 5’-terminal, the X-exonuclease molecule degrades only a single DNA molecule) (19). If a h-exonuclease molecule can no longer continue resection, further degradation from that point is effectively blocked, until the original enzyme molecule falls off. Since X-exonuclease is fairly unstable under reaction conditions, degradation at some terminals may be blocked for this reason. Thus, we suggest that the DNA eluting in peak I consists of those molecules with few nucleotides removed from both terminals. This is supported by the fact that none of this peak I DNA is capable of cyclization, and that peak I almost disappears with exonuclease III degradation. Peak II. From elution profiles such as in Fig. 1 it is possible to estimate the caffeine concentration of the peak fraction of the radioactivity eluting in greater than 0.1% caffeine. These approximate caffeine concentrations obtained from numerous experiments employing both A-exonuclease and exonuclease III are plotted versus the average number of nucleotides resected in Fig. 2. We find that the caffeine concentration necessary to elute peak II steadily increases for molecules with approximately 200 nucleotides removed per terminal to molecules with an average of 1300 nucleotides resected per terminal. For resections greater than 1300-1500 nucleotides per terminal, the caffeine concentration is inversely related to the single-chain length. The results of Iyer and Rupp (7) are also plotted in Fig, 2. Their results are similar to ours, and the slopes of both parts of the biphasic curves are similar. However, our curve is located about 1000 nucleotides to the left of that determined in the study already published (7). In addition, Iyer and Rupp are able to extrapolate their curve through the
564
SCHLEGEL,
1
I
I
PYFXITZ,
I
I
AND
I
THOMAS
I
I
I
I
I
0.60
0.40 .-I c z u 8
0.20
0 Nucleotides
Resected
FIQ. 2. Relationship between degree of resection and caffeine concentration needed to elute peak II from BNC. Estimation of peak elution is described in the text and Fig. lb. Open circles (O), T7 DNA treated with A-exonuclease; closed circles (O), T7 DNA treated with exonuclesse III; vertical bars indicate data of Iyer and Rupp (7) for Pz2 DNA treated with A-exonuclease. The lines through the data points (indicated by circles) were drawn by means of a least-square analysis by computer. The broken lines represent degrees of resection not explored by these experiments.
origin, though the chromatographic properties of molecules possessing single chains less than 800 nucleotides long were not examined. Differences such as this probably reflect variations in the BNC resin; it appears that our preparation of BNC binds single chains more tightly in the range of O-2000 nucleotides resected, since a higher caffeine concentration is needed in our experiments to elute molecules of a given degree of resection. Results with our preparation of BNC have been entirely reproducible over a period of at Ieast three months. Both extremes of exonuclease resection are of interest in Figure 2. Peak I elutes entirely at 0.1% caffeine, probably as a result of very limited resection by the exonuclease. We have not attempted to determine the extent of resection of the peak I molecules. However, linear monomers of phage X DNA are eluted completely with 1.0 NET (3), and, we have confirmed that fact with our BNC preparation. Therefore, either a single chain 12 nucleotides in length is not sufficient to bind to BNC, or the cohesive terminals of h are internally complementary and have assumed a partially duplex form that does not interact with BNC (20,21). The minimum length of single chain required to bind to BNC might depend on the cellulose particle size and shape, and on the extent of benzoylation and naphthoylation.
DNA
BEARING
SINGLE-CHAINED
565
TERMINALS
The decrease in caffeine concentration required to elute single chains greater than 2000 nucleotides can be explained if one remembers that, under these salt and temperature conditions, nonspecific hydrogen bonding of bases is promoted (22). The formation of “hairpin” structures by the long single chains would effectively reduce the number of nucleotides available for binding to BNC. The cooperation of both single-chained terminals in binding. Peak II in elutions of samples with an average of lOOC~2000 nucleotides removed per terminus generally appears as a double peak (see Fig. 1). This suggested to us that molecules with both ends resected bind more tightly to BNC than molecules with only one end bearing a single chain. To test this hypothesis, exonuclease III treated T7 DNA was sheared to half-molecules by stirring. Table 1 lists the elution data for sheared and unsheared molecules, first digested with exonuclease III. It is apparent that approximately 2&25% of the material which eluted in caffeine before shearing eluted in NET after breakage to half-molecules. Those half-molecules which continue to elute in caffeine now do so at a lower
Elution
TABLE 1 of Exonuclease III Treated T7 DNA before and after Shearing y0 of input counts
Substrate Whole molecules
Half-molecules
Av. length of single chains (nut.)
0.3 NET
240 420 1000 1300 420 1000 1300
0.4 0.9 2.7 4.5 1.3 4.2 4.1
1.0 NET
O-l% caffeine
y. caffeine of peak II elution
0.2 0.2 0.4 1.2 24.7 21.7 19.5
84.3 86.0 80.9 80.2 57.2 61.3 66.3
0.27 0.31 0.37 0.47 0.25 0.33 0.33
Whole T7 molecules were resected with exonuclease III to the extents shown. Aliquots of three of the resected preparations were sheared to half-molecules by stirring as described in the text. The caffeine concentration in each fraction was measured by determining the absorbancy at 260 nm of dilutions of 10 ~1 samples. The caffeine concentration necessary for elution of partially single-chained molecules wss estimated by determining the per cent caffeine in the peak fraction of peak II (see text). One-quarter to one-fifth of the half-molecules elute in 1.0 NET, indicating an absence cf single-chained terminals. Those half-molecules continuing to elute in caffeine do so at a significantly lower caffeine concentration than do whole molecules resected to the same extent. For example, the decrease in caffeine concentration necessary to elute molecules with only one single-chained terminal 1300 nucleotides long from that needed to elute a whole molecule with two such single chains is from 0.47y0 to 0.3397,, a shit of 8 fractions in the gradient.
566
SCHLEGEL,
PYERITZ,
AND
THOMAS
T7 DNA Shearing
Exonuclease
I
BNC
I
BNC
= GE
=
I
BNC
FIG. 3. Schematic representation of results and conclusions of experiments described in the text. The lower portion of the figure contains examples of BNC chromatographic elution profiles which were reproducibly obtained after performing the procedures described on the top line. We interpret these results as suggesting the formation of the molecular configurations drawn on the top line.
caffeine concentration. This suggests that the presence of significant single chains on both ends of a molecule results in tighter binding to BNC than does the presence of only one single-chained end. The shearing process does not remove single chains from the molecules, because in unrelated experiments, we have been able to form “joined halves” with T7 by first resecting and then shearing (23). Our interpretation of these results is depicted in Fig. 3. In the experiment just described, exonuclease III failed to digest approximately 25% of the 3’-terminals. This finding is not typical of many previous exonuclease III digestions in which all terminals are digested (15). Why this occurred in this case is unclear. Generally, shear breakage to half-molecules and BNC-chromatography of T7 DNA after exonuclease III treatment does not result in any DNA eluting in 1.0 NET, indicating the presence of single chains at all of the 5’-terminals (15). Repeating this experiment with T7 DNA treated with X-exonuclease invariably yields 15-40s of the half-molecules elutable by l.OM NaCl. In addition, the shear-broken molecules in peak II elute at a lower caffeine concentration than do the whole molecules. These results support the model shown in Fig. 3 and again suggest a difference in the mode of action of the two exonucleases. SUMMARY
T7 DNA was t,reated with X-exonuclease or exonuclease III in order to produce single-chained terminals on predominately duplex molecules. DNA treated in this fashion binds to columns of benzoylatedNative
DNA
BEARING
SINGLE-CHAINED
567
TERMINALS
naphthoylated DEAE-cellulose (BNC) in the presence of l.OM NaCl and is elutable by a linear gradient of O-1.00/0 caffeine. The concentration of caffeine needed to elute resected molecules is proportional to the length of the single-chained terminals for single chains ranging from 200 to 1300 nucleotides in length. A gradual decrease in the caffeine concentration necessary to elute molecules with terminals longer than 1500 nucleotides is observed. The BNC column therefore provides a simple method for the fractionation of DNA molecules based on the length of exposed single chains. Shear-breakage of resected molecules reveals that DNA possessing two single-chained terminals binds more tightly to BNC than molecules with only one resected terminal. Based on these results, a comparison was made between DNA resected by one exonuclease and the other. Significant differences were obtained which suggest that these methods should be useful for determining the mode of action of various nucleases. ACKNOWLEDGMENTS We thank Dr. Motohiro Fuke for gifts of A-exonuclease and exonuclease III. This investigation was generously supported by grants from the National Institutes of Health (5ROI-A1081861 and the National Science Foundation (GB-86111, as well as a National Science Foundation Predoctoral Fellowship to Reed Pyerite. REFERENCES 1. MIYAZAWA, 2. KNIPPERS,
Y., AND THOMAS, C. A., R., RAZIN, A., DAVIS, R.,
JR., J. Mol.
Biol. AND SINSHEIMER,
11, 223 (1965). R.
L., J. Mol.
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237 (1969). 3. KIGER, J. A., JR., AND SINSHEIMER,
R. L., J. Mol. Biol. 40, 467 (1969). 4. SEDAT, J., LYON, A., AND SINSHEIMER, R. L., J. Mol. Biol. 44, 415 (1969). 5. SCHLEGEL, R. A., AND THOMAS, C. A., JR., J. Mol. Biol. 68, 319 (1972). 6. KELLY, R., AND SINSHEIMER, R. L., J. Mol. Biol. 29, 22 (1967). 7. IYER, V. N., AND RUPP, W. D., Biochim. Biophys. Acta 228, 117 (1971). 8. PYERITZ, R. E., SCHLEGEL, R. A., AND THOMAS, C. A., JR., B&him. Biophys. Acta 272, 504 (1972). ~..PYERITZ, R. E., LEE, C. S., AND THOMAS, C. A., JR., Chromosomu 33, 284 (1971). 10. KELLY, T. J., JR., AND THOMAS, C. A., JR., J. Mol. Biol. 44, 459 (1969). 11. GILLAM, I., MILLWARD, S., BLEW, D., VON TIGERSTROM, M., ‘WIMMER, E., AND TENER, G. M., Biochemistry 6, 3043 (1967). 12. RADDING, C. M., J. Mol. Biol. 18, 235 (1966). 13. RICHARDSON, C. C., in “Procedures in Nucleic Acid Research” (G. L. Cantoni and D. R. Davies, eds.), p. 212. Harper & Row, New York, 1966. 14. HERSHEY, A. D., GOLDBERG, E., BURGI, E., AND INGRAHAM, L., J. Mol. E&l. 6,
230 (1963). 15. SCHLEGEL, R. A., Ph.D. Thesis, Harvard University, Cambridge, Mass., 16. RICHARDSON, C. C., AND KORNBERQ, A., J. Biol. Chem. 239, 242 (1964). Ii’. RICHARDSON, C. C., LEHMAN, I. R., AND KORNBERG, A., J. Biol. Chem.
( 1969) 18. LITTLE,
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19. CARTER, D. M., ANTI RADDINQ, C. M., J. Biol. Chem. 246, 2502 (1971). 20. WANO, J. C., AND DAVIDEON, N., Cold Spring Harbor Symp. Quant. Biol. 33, 409 (1968). 21. WV, R., AND TAYLOR, E., J. Mol. Biol. 57, 491 (1971). 22. STUDIER, F. W., J. Mol. Biol. 41, 189 (1969). 23. LEE, C. S., PYERITZ, R. E., AND THOMAS, C. A., JR., in preparation.