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Properties of a new form of DNA from whole calf thymus nuclei: Evidence for reactive, special sites in DNA
ARCHIVES
OF
BIOCHEMISTRY
Properties
AND
BIOPHYSICS
of a New
Thymus
fi.ir Medizin,
Form of DNA
From Whole
Nuclei:
Evidence
Special
Sites in DNA
RICHARD Institut
132-143 (1971)
l&t,
S. WELSH
AND
Kernforschungsanlage
Calf
for Reactive,
KAREL VYSKA Jtilich
GmbH, Western Germany
Received July 7, 1970 A new method for DNA preparation from whole calf thymus nuclei, which was designed to minimize enzymatic activity throughout to the greatest possible extent, is presented. The isolated DNA shows a number of interesting properties differing from those of DNA prepared by standard literature methods. The most important property is that the DNA prepared from our whole, purified nuclei, having originally a high molecular weight of 20.5 million, cleaved on EDTA treatment into a number of relatively homogeneous DNA fractions with a mean molecular weight of 2.3 million. In contrast, the standard DNA did not cleave on EDTA treatment. On the basis of this finding it was concluded that in the DNA backbone special sites exist at which cleavage can occur. These sites were further shown to occur in one of two possible forms: (1) fully covalent and (2) noncovalent. The proportion of the sites in form 1 increases with increasing ATP level. In standard DNA all special sites are most likely in the covalent form. It was also shown that for the conversion from one form to the other enzvmes of the nucleus are necessary. The ATP level very probably controls this enzymatic activity in rive.
Recently, in numerous investigations, molecular changes in DNA in vitro by the action of such enzymes as endonucleases (1, 2), exonucleases (3), and ligases (4-6) have been described. These findings suggest the question as to what happens during nuclei rupture, when chromatin is exposed to cytoplasmic components. In order to answer this question, we developed a new method in which we minimized to the greatest possible extent enzymatic activity during preparation, first by means of prior separation of whole nuclei from all other cellular components at very low temperatures (- 10 f 59, and then by use of sodium dodecylsulfate (SDS) or Pronase, as enzymedestroying agents, after rupture of these nuclei. The DNA obtained by this method, having the same mean molecular weight as that for a standard preparation according to Zubay and Doty (7), showed several different properties which confirm our as-
sumption that somechangesoccur during the standard preparation. The main difference observed was that while our DNA cleaved on EDTA treatment into homogeneous subunits the standard DNA did not. In order to find the deciding factors causing these changes, as well as to show their characteristics, the second part of the investigation was carried out. This study showed that ATP and endonuclear enzymes are the main components which are necessary for the changes occurring during a standard preparation. METHODS
Basic Nuclei Preparation Suspension of tissue components. Calf thymus was ground in the frozen state and suspended in 40% glycerol-O.44 M sucrose-O.039 M sodium glycerophosphate-O.019 M sodium citrate-citric acid (GSGC) at subzero temperatures (-X--5’), with the pH maintained at 6.1 and the ionic strength
132
PROPERTIES
OF A NEW
at 0.15. The glycerophosphate (8) was included in the medium to protect the nuclei against enzymatic activity, the sucrose to prevent mitochondrial and nuclear rupture (9), and the glycerol to prevent clumping and freezing. ATucZei washing. The whole, unclumped nuclei in the suspension described above were purified of cytoplasmic components by centrifugation, as follows. After transferring the suspension, a gradient layer was set up at the bottom of each 250-ml centrifuge tube, by the use of a linear gradient maker,’ to prevent hard packing of nuclei, and centrifugation was carried out for 20 min at 65009. The pellets were resuspended by mild agitation in GSGC, and the procedure was repeated through four cycles with successive increases of centrifugation speed to 10,8OOg, as previously described (10). During this procedure the temperatures was maintained at -lO-5”, to minimize enzymatic activity, and to give purified whole nuclei. Nuclei e&action. In this step which was used for direct preparation of DNA of low molecular weight the nuclei were sedimented by centrifugation at 19,000g for 10 min and resuspended at once in water at 0” by motor-turning of the Potter homogenizer. They were then rapidly extracted by washing three times with water at O-4”, [to remove ATP, Mg ions, and a mixture of phosphopeptides (11, 12) by alternate centrifugation and resuspension with the Potter homogenizer in fresh portions of water]. By the third wash the nuclei were largely lysed to a gel state, such that it was necessary to increase the speed of centrifugation to about 33,000g to give adequate sedimentation of the gel. Saline extraction of precipitated DNP. To precipitate the deoxyribonucleoprotein (DNP), the sedimented gel of washed, lysed nuclei was suspended in the same way in water, and the suspension was adjusted to 0.14 M NaCl. The resulting DNP was dispersed by hand operation of a sintered glass homogenizer, at O-4”. After centrifuging down the dispersed and precipitated DNP, the saline extract, containing ribonucleoproteins, was removed, and the DNP pellet was again suspended by hand homogenization in 0.14M NaCl. DNP dissolution. The precipitated DNP prepared as above was dissolved in the high ionic * In the exit chamber of the gradient maker 25 ml of 80% glycerol (w/v)-0.44 M sucrose-O.039 M glycerophosphate-0.019 M citrate-citric acid was placed and in the other chamber 25 ml of a medium with the same composition except that the glycerol concentration was 40Yo (w/v) (GSGC).
FORM
OF DNA
133
mM strength buffer, 1 M NaCl-0.7 mM phosphate-l EDTA, pH 9.2 DNA preparation. DNA of low molecular weight was purified from the dissolved DNP by a clarifying centrifugation at 25,OoOg and by five to eight cycles of a method based on that of Kay et al. (14), modified (11) as follows: (1) For the first cycle, after dissolving the DNP and centrifuging, the gelatinous pellets were extracted with 2.5 M NaCl and centrifuged at 25,OOQgand the supernate pooled with the first 1 M NaCl extract. In all other cycles of purification, the final NaCl concentration in the SDSNaCl dispersion was 2.5 M instead of 1 M, except, the last cycle in which it was again 1 M. (2) Twice the volume of cold 95% ethanol instead of an equal volume was added to precipitate the DNA, if 1 M NaCl was used for the SDS-NaCl dispersion. If 2.5 M NaCl was used, it was necessary to add 2.5 times the volume of 95yo ethanol to precipitate the DNA. (3) Both types of DNA precipitates formed, fibrous and flocculent,3 were collected, the fibrous one by removal on the stirring rod and the flocculent one by centrifugation. (4) The solvent B (15) (0.0075 M phosphate-l mM EDTA, pH 7) instead of water was used to dissolve the precipitated DNA. For the physical chemical determinations, the precipitated DNAs were finally dialyzed against 0.7 mM phosphate-l mM EDTA, pH 7. This buffer, which has a total ionic strength of 0.0079, i.e., appreciably lower than the value of 0.0276 for solvent B, was used to overcome possible aggregation effects.4 In order to avoid EDTA treatment and thus DNA cleavage throughout in the preparation of DNA of high molecular weight (see Table I, 2 To rule out the possibility of occurrence of shear as well as enzymatic degradation of DNA during any of the treatments required to disperse nuclei, a parallel experiment (13) was set up to effect lysis of protect,ed nuclei by the action of Pronase, a powerful proteolytic enzyme. This treatment yielded DNA, initially of high molecular weight, which after dialysis against 1 M NaCl-0.024 M EDTA, pH 8.1, gave a banding pattern showing the same DNA components of low molecular weight as found in our N-DNA after special EDTA treatment. 3 These flocculent precipitates were found only for the control samples from whole, protect,ed nuclei. *For highly purified DNA, such aggregation was not found to occur at ionic strengths of 0.00792.5 in agreement with Rosenberg and St,udier (16), but for impure DNA aggregation can result from increasing the ionic strength.
134
WELSH AND VYSKA
Exp. 1 and 2), the procedure described above was modified as follows. The whole nuclei were dispersed in 0.14 M NaCl by motor-homogenization and then the DNP dissolved by dispersing in the required volume of 1 M NaCl. During “DNA preparation” after alcohol precipitation, the DNA fibers were at each cycle worked up in 0.14 M NaCl or 0.01 M phosphate, pH 7, instead of solvent B. The precipitates in this case were always entirely in the fibrous form and could not be so easily dissolved. More cycles of purification were required than for all DNA preparations of low molecular weight, since, without the presence of EDTA in the medium, it was much more difficult to split the protein from the DNA.6 For the final dialysis, in order to break up reversible complexes formed through metal ion interaction, it was also necessary to dilute the DNA to an OD at 260 w of 10 or less, and then dialyze against the medium used for the ultracentrifuge runs, 0.5 M NaC1-0.7 mM phosphate-l mM EDTA, pH 7. Special EDTA treatment. During this treatment, the dialyzed DNA samples were adjusted to 0.01 M EDTA, pH 7, stored for l-2 days at 4’, and finally dialyzed against nine times the total volume of 0.7 mM phosphate, pH 7 (to restore the original buffer). Ultracentrifuge rate runs. The boundary runs were made at 50,740 rpm in a temperature range of 5-lo”, with thermostatic regulation, in a Spinco Model E analytical ultracentrifuge, equipped with a uv optical system and a Cl-Br filter. The photographs were scanned with a recording microdensitometer and baseline corrections made, as previously described (10). The integral distribution curves of sedimentation coefficients were computed by the method of Shooter and Butler (17). For each of two concentrations for each sample, reciprocals of &o.~ values were taken at steps of 10% of the total composition (or every 5% at the extremities), and extrapolations to zero concentration made. For some curves (Figs. 1, 2, and 7), extrapolations were made mathematically by the use of known relationships (see figure legends). From the S,“,,, values thus obtained, a complete extrapolated curve was constructed for each sample. The banding runs were made at 48,000 rpm in a Spinco Model E, using a monochromator set at 265 rnE.cand the Beckman Dynagraph double beam 5 If the DNA precipitate from alcohol is placed in 2.5M NaCl and dispersed by gently pressing it against the walls of the container with a rubber policeman, with subsequent addition of SDS, the DNA is more effectively dissociated from the protein and dissolves more completely.
scanner. The temperature of each run was regulated to 3~0.1” within the range 5-lo”, by the method of Studier (18). The banding method of Vinograd et al. (19) was applied, using a 12-mm double sector centerpiece. From banding run patterns the distributions of sedimentation coefficients were obtained as follows. From the plots of density, automatically corrected for solvent absorption, as a function of distance obtained by use of the scanner during banding runs, the densities were taken at distances increased by a small increment from the meniscus and corrected for sectorial dilution (17). For each of these distances the corresponding 820,~ was calculated. For the concentrations used in these banding runs the S20.w values directly obtained were considered to be equivalent to the values at . . . infinite dllutlon (S~o.,), according to Vinograd et al. (20). The plot for corrected densities as a function of S:,., was then constructed. Since according to Vinograd et al. (19) banding runs give directly concentration distributions in differential form, this plot was considered to be equivalent to the distribution of sedimentation coefficients. To obtain the integral distribution curve (Fig. l), the S:,,, scale was divided into steps of 2S, and for each point the sum of all densities from the meniscus up to the point was plotted against the corresponding S&.,. Intrinsic viscosity determinations. The intrinsic viscosities at zero shear rate ([v]G = 0) were determined in two four-bulbed Ubbelohde viscometers, as described previously (10). The measurements were made at 25.00 f O.Ol”, at four or five DNA concentrations. Molecular weight determinations. The weightaverage molecular weight was finally calculated from the value of &., at 50yo of total concentration and the [7]G = 0 value for each sample, using the Mandelkern-Flory equation (21) for random coils, assuming a p of 2.27 X 104, according to Crothers and Zimm (22). EXPERIMENTAL
AND RESULTS
The method described above produces a new form of DNA (N-DNA). In order to point out very interesting properties of this N-DNA and also to make the necessary comparisons between it and standard DNA (S-DNA) described in the literature (7, 15), the following parallel studies were carried out on these two DNA forms. First,, the behavior of the two types of DNA before and after EDTA treatment is compared in Fig. 1, in which the integral distributions of sedimentation coefficients
PROPERTIES
OF A NEW F0R.M OF DNA
FIG. 1. Integral distribution curves of sedimentation coefficients for N-DNA and S-DNA before and after the special EDTA treatment (0.01 M EDTA, pH 8.8 for 24 hr and for standard S-DNA, afte enzymatic degradation. Extrapolations of the curves to zero concentration were done using the relationship l/S = l/iY(l + K[TJ]c), and the data of Eigner and Doty (15) for 8 as a function of [ql, at ionic strengths of 0.2 or higher, K = 0.79 (from our data), c = 0.022 mg/ml. Curve 1’: Boundary run, in 0.5 M NaCl-0.7 mu phosphate-l mu EDTA, pH 7, for NDNA (O&~o = 0.45) after EDTA treatment; 0 and X at 2328 and 2568 set total times of centrifugation, respectively. Curve 1: Same as curve 1’ after extrapolation to zero concentration. Curve 2’: Boundary run, in 0.5 M NaClLO.7 mM phosphate, pH 7, for N-DNA before EDTA treatment; X, 0, and 0 at 1537, 1914, and 2255 set total times of centrifugation, respectively. Curve 2: Same as curve 2’, after extrapolation to zero concentration. Curve 3: q Standard S-DNA, prepared according to Zubay and Doty (7), before and after the special EDTA treatment, obtained by integration of the curve 1 in Fig. 2. Curve 4: A Endonuclease-degraded thymus DNA, data interpolated to 14.6 min of enzyme degradation and extrapolated to zero concentration.
are plotted. The curves 2’ and 1’ represent the original unextrapolated data from boundary runs for our N-DNA before and after EDTA treatment, respectively (see Table I, Expts. 1 and 2). The corresponding distributions after extrapolation to zero concentrat,ion are plotted as curves 2 and 1, respectively. The curve 3 was derived by integration (see Methods) of the common curve obtained from banding runs on standard DNA before and after EDTA treatment’ (original data plotted in Fig. 2). The comparison of curves 2 and 3 in Fig. 1 for N-DNA and S-DNA before EDTA treatment reveals that, while the mean Sk .Wvalues are roughly the same (26s and 3OS), the pattern for the S-DNA (curve 3)
shows a broad continuous distribution6 in contrast to the more homogeneous one for N-DNA (curve 2). The curve 1 in this figure demonstrates that N-DNA, having originally a mean S,“, ,W of 26S, gives after EDTA treatment a greatly reduced mean of 11s. %I #TV In contrast, the data for S-DNA before and after EDTA treatment define a single curve (curve 3, Fig. 1, original data given in Fig. 2), which shows that in this case EDTA treatment has no effect. In addition, curve 4, depicting the integral distribution of enzymatically degraded 6 Similar to the derivative curve of Schumaker and Schachman (23) from boundary runs.
ii
Flow
x0
Mean
Preparation
equation
’ Linear
c hdd .024 .012 .006
by M.F.
equation
b +’
for log Sio w 88 B function
was employed.
I
2.27
+
10.98
1: +
so 88 to eliminate
20.49
+
26.24
+‘” +’
c
+
4-
+
+
2
+
3
+
4
data
for nine
use of EDTA
+ from
3.25
f
23.0 1.47 5.21 3.01
of our
most
2.84
24.5 1.90 4.44 2.69
+
11.68
11.25
throughout
7.19
1.58
+
17.12
12.39 +
13.60 15.24
11.48 11.90
6.20
45.5 1.54 5.55 5.93
+
16.12
13.47 14.57 15.40
+ + + +
+
+
1
+
+
7
+
+
8
PAR.&METERS
9
+
no.
FOR
+
Expt.
7.C2
45.5 1.50 1.74 5.23
+
16.95
13.60 15.20
homogeneous
(see Methods).
+
3.08
3.96
29.9
-I-
12.12
11.56 12.18
DNA
+
+
f
+
11
+
+
+
+
12
DNA
+
+
f
+
13
SAMPLES
+
+
14
12.60
2.19
+
preparations
15.20 16.CO
+
5.15
5.45
43.0 1.59
+
14.95
13.64 14.15
M = molecular
of Sueoka
6.91
7.11
51.0 1.78
+
16.86
here),
relationship
6.40
1.72 1.60
+
16.32
(six shown
by the
12.60
71.1 2.19 9.36 11.65
+
20.68 21.50
20.80 21.50
14.82 15.55 15.98
+ + + + + 1 : 1+ : : +
+
+
+
+
10
DESCRIBED
c Calculated
2.20
1.85 2.69
+
10.58
10.28 10.42
+ + + + + + + z+ T + :+ :+
+
6
+
5
AND
TABLE PROCEDURES
+ + T- 1
+
+
PREPARATION
+=
1
OF
of log M, determined
Modified
121) 2 Calcd. by linear log r&tic& used for computation a and Kf
C&d.
0.7 rn~ phosphate, pH 7.0 0.7 rn~ phosphate-l rn~~ EDTA, pH7 0.5 M NaCl phosphate1 mu EDTA, pH7
ODao .50 .25 ,125
OUTLINE
a GSGC b SalineEDTA B GSGC b SalineEDTA a Control b Ruptured -4” b +4’ a Control bl hr c5 hr d 24 hr
(21). CY = 0.4068, K’ = 1.5560.
relationship
item
Mw/MN~ M, x lo-’
1
3
ltll G = c
m/s20
1 2
1 2 3
solvent
w
A% w
Mean
n + Indicated
IC ID IE IIC DATA
Unextnrcted Water washed Control Mg Clz Mg ATP of DNP
1 2 1 2 4
1 Mitochondria effect 2 Temperatwe’j-a effect 3 Time effect
2 Washing
1 Suspension
PRocmuRE
Saline extraction DNP dissolution DNA preparation Special EDTA treatment AND PARAMETERS
Basic nuclei Extractions Special nuclei extractions
IB
IIB
Special nuclei treatments
IIA
PREPARATION IA Basic nuclei preparaton
SUMMARY
(31). weight
5.47
1.70
+
15.34
13.13 14.15
:
+
+
+
+
f
15
+
3.97
3.89
30.0 1.57
+
13.46
12.93
+ + +
+
+
17
:
+
+
+
18
+
3.31
25.4 1.64 1.98 3.20
+
12.48
11.98 12.18
by the Mahdelkerh-
5.45
1.70 1.92
+
15.30
14.32 14.80
Tc
+
+
+
16
PROPERTIES
OF A NEW FORM OF DNA
137
FIG. 2. Curve 1: Banding runs on S-DNA prepared according to Zubay and Doty (7), before and after special EDTA treatment. Before EDTA: l and 0 at 759 and 989 set total times of centrifugaion, respectively. After EDTA: A and v at 1291 and 1995 set total times of centrifugation, respectively.
S-DNA, was compared with curve 1’ for EDTA treated N-DNA. The comparison shows that enzymatically degraded DNA has, in contrast to EDTA-treated N-DNA, much higher heterogeneity in the region of 50-100% of total concentration and a smooth distribution without steps. In order to find the most important factors in DNA preparation which determined whether the isolated DNA of high molecular weight cleaved during EDTA treatment, as in t,he case of N-DNA, or did not, as in the case of S-DI\JA, we did the following experiments. (I) Effect of mitochondria. In this experiment, a tissue suspension (see Methods) was centrifuged at 14,500 rpm for 10 min. Two pellets from this centrifugation, containing
essentially
nuclei plus mitochondria,
were taken. The first pellet was blended in saline-EDTA (7) at top speed for 3 min, to rupture nuclei, permitting release of chromatin threads. Subsequently, these ruptured nuclei were washed five times in the same medium at 4”, according to Zubay and Doty (7) (Expt. 3). The other pellet was washed four times at 4’ in GSGC (see Methods), as a control (Expt. 4). All other steps in the DNA isolation were maintained the same as in our basic method. Figure 3 gives t,he int,egral distribution
curves of sedimentation coefficients derived from boundary runs, extrapolated to zero concentration. The extrapolated curve 1, for DNA from the control nuclei showed a greater homogeneity and a lower mean Sk ,w value’ (12.395) than for curve 2 for DNA from nuclei rupt’ured in the presence of mitochondria (mean Si, ,Wof 16.125) .* The molecular weight, calculated from the mean Xi, ,Wand intrinsic viscosity for DNA from ruptured nuclei (5.9 X 106), was about twice that of the DNA from control nuclei (3.0 X 106) (see Table I, Expts. 3 and 4). (2) Efect of MgATP. In this experiment the suspension of whole nuclei (Methods IA, 2) was centrifuged at 12,000 rpm and subdivided into three pellets. The first pellet was homogenized by blend7 This lower mean S&., was obtained directly without special EDTA treatment in the control and in those for subsequent experiments, because of the water washing, which removed ATP, and the dilute EDTA treatment during DNA preparation (see Methods and Discussion). 8 The lower value of S&., for the DNA from ruptured nuclei than that for S-DNA results from the fact that the nuclei, before rupturing in the presence of mitochondria, were washed once with GSGC, which removed some ATP and inhibited enzymatic activity (see Discussion).
138
WELSH AND VYSKA
FIG. 3. Integral distribution of DNA sedimentation coefficients illustrating mitochondria effect. Curve 1’: DNA from control nuclei, at OD 260= 0.50; 0 and 0 at 2670 and 3150 set total times of centrifugation, respectively. Curve 1”: Same at ODzso = 0.25; X at 1902set total time of centrifugation. Curve 1: Curve for this DNA extrapolated to infinite dilution. Curve 2’: DNA from nuclei ruptured in presence of mitochondria, OD260 = 0.25; q at 987 set total time of centrifugation. Curve 2”: Same at OD260 = 0.125; A at 714sec total time of centrifugation. Curve 2: Curve for this DNA extrauolated to infinite dilution, using data for OD~W of 0.50 (curve not shown) and of 0.125. L
ing in 0.01 M MgATP, pH 7, and then washed three times in the same medium. All other conditions and further steps were maintained the same as in our basic DNA preparation (see Table I, Exp. 5). The second pellet, which was used as a control, was washed with water, instead of MgATP, maintaining all other conditions the same as above. In Fig. 4, the curve 2 for DNA from control nuclei shows a much lower mean Sk ,w (11.75) than that (17.15) found in curve 4 for DNA from nuclei ruptured in the presence of MgATP.g This means that the mean DNA molecular weight (see Table I) increased from 2.84 X lo6 (11.7s) to 7.19 X lo6 (17.15), as seen in Table I, Expts. 6 and 5, respectively. g The lower mean S:,,, observed for the DNA from MgATP-treated nuclei than for S-DNA can be explained by the shorter time (see kinetic study) needed for the MgATP treatment than for the washing of ruptured nuclei in a standard preparation (7), and possibly by an inhibition by the glycerophosphate (24) of endonuclear enzymatic activity involved in the proposed closing of sites (see Discussion).
In order to show that the main effect was due to ATP and not Mg ion, the third pellet was washed three times with 0.01 M MgClz , maintaining all other steps the same as in the MgATP-treated sample (see Table I, Expt. 7). The comparison of the patterns for DNA from MgATP-treated and MgClz-treated pellets (Fig. 4, curves 4 and 3, respectively) confirmed that the ATP and not the Mg ion is responsible for the high molecular weight of the resulting DNA. Furthermore, whereas special EDTA treatment (see Methods) of the DNA from MgATP-treated nuclei produced no appreciable change in the Si,,, , this treatment lowered the mean St,,, of the DNA from MgC&-treated nuclei from 12.15 to 10.6s (see Expts. 7 and 9, Fig. 4, curves 1 and 3, respectively). After such EDTA treatment, the molecular weight of DNA from MgATPtreated nuclei was 7.0 X lo6 (16.9S), more than three times the molecular weight, 2.2 X lo6 (10.75), for DNA from MgChtreated nuclei (see Table I, Expts. 8 and 9). In order to show that enzymatic activity is
PROPERTIES
OF A NEW FORM OF DNA
0
FIG. 4. Integral distributions of DNA sedimentation coefficients illustrating MgATP effect. The curves 1, 2, 3, and 4 are the ones extrapolated to infinite dilution corresponding to curves l’, 2’, 3’, and 4’. Curve I’: EDTA-treated DNA from MgClt-treated nuclei OD200 = 0.25; q and n at 726 and 966 set total times of centrifugation, respectively. Curve 2’: DNA from water-extracted nuclei, ODzso = 0.50; V and A at 990 and 1230 set total times of centrifugation, respectively. Curve 3’: DNA from MgCL-treated nuclei, OD 260= 0.50; 0, 0 at 957 and 1197 set total times of centrifugation, respectively. Curve 4’ : EDTA-treated DNA from MgATP-treated nuclei ODscO= 0.25; X and l at 781 and 1261 set total times of centrifugation, respectively.
FIG. 5. Integral distributions of DNA sedimentation coefficients, illustrating temperature effect; curves 1,2,3, and 4 are the ones extrapolated to infinite dilution, corresponding to curves l’, 2’, 3’, and 4’, respectively. Curve 1’: DNAfrom ruptured nucleistored at -4--2”, ODtco = 0.50; 0 and 0 at 956 and 1194 set total times of centrifugation, respectively. Curve 2’: EDTA-treated DNA from ruptured nuclei stored at -4--2”, ODnso = 0.50; S and n at 964 and 1204set total times of centrifugation, respectively. Curve 3’, 4’: DNAs from ruptured nuclei stored at +4”. A and v EDTA-treated DNA, ODxco = 0.125, at 474 and 954 set total times of centrifugation, respectively. X, DNA (not EDTA-treated), ODzao = 0.092, values obtained graphically from data at 456, 696, and 936 set total times of centrifugation.
139
140
WELSH AND VYSKA
5/ :’ 1
4
2
1
FIG. 6a. Integral distributions of DNA sedimentation coefficients in the time effect experiment. Curves 1 and 2 are the ones extrapolated to infinite dilution corresponding to curves 1’ and 2’, respectively. Curve 1’: DNA from control nuclei at 0 time of dialysis, ODtaa = 0.25; 0 and 0 at 714 and 954 set total times of centrifugation, respectively. Curve 2’ : DNA from ruptured nuclei at 24 hr of dialysis OD26o = 0.25. q , before EDTA treatment, at 956 set total time of centrifugation. A and V after EDTA treatment, at 750 and 990 set total times of centrifugation, respectively. FIG. 6b. Plot to illustrate time effect: Mean DNA molecular weights, computed by the Mandelkern-Flory equation (see Table I), as a function of the time of dialysis.
involved in the processes which lead to the described differences between S-DNA and N-DNA we did the following experiment. (3) The temperature effect. For this experiment, frozen calf thymus was ground and homogenized by blending for 5 min at - 2- - lo’, directly in the saline-EDTA medium of Zubay and Doty (7), containing also 40% glycerol, to prevent freezing. This suspension was subdivided into two portsions: (a) The first portion was washed
in this medium at 4’, according to the Zubay-Doty procedure (7) (Expt. 10); (b) the second portion was washed in the same way, except t,hat the temperature was maintained at -4” (Expts. 11 and 12). All other steps in the DNA isolations were again maintained the same as in our basic method. In Fig. 5 curve 1 represents the pattern for DNA prepared from ruptured nuclei st’ored at the lovver temperature, while
PROPERTIES
OF A NEW
curve 3 represents t’he pattern for DNA prepared from nuclei stored at the higher temperature. From this figure and from the comparison of M,/M, value@ (see Table I, Expts. 12 and lo), it is evident that use of a lower temperature during the entire purification resulted in a significant decrease in the mean Si,,, of from 21.5s to 16.3s and an increase in homogeneity. The mean molecular weight of the DNA prepared from nuclei washed at the higher t’emperature” was 12.6 X lo6 and that for DNA from control nucleiI stored at the lower temperature was 6.9 X 106. The EDTA treatment in both cases (see Expts. 13 and 11, Fig. 4, curves 4 and 2) is seen to have no appreciable effect on the mean Si, ,w values. (4) The time efect. For t’his experiment, t.he tissue suspension in GSGC (see Methods) was adjusted to a total NaCl concentration of 0.34 M and a pH of 6.8, and the nuclei in it were ruptured by blending at maximum speed for 15 sec. The resulting ruptured nuclei were subdivided into five portions: The first, second, and third portions were dialyzed at 4” against 0.34 M NaCl for 1, 5, and 24 hr (see Table I, Expts. 17, 14, 15, Fig. 6b, points 2, 3, 4, respectively). During the dialysis of these portions, the pH of the dialyzed nuclei was periodically adjusted upward to 6.8 (at 4-lo-hr intervals). At the appropriate time, each of these portions was blended, as described above, centrifuged, the pellets from each resuspended, frozen rapidly to stop all enzymatic reactions, and st,ored at -20”. As a control,
FORM
OF DNA
141
a fourth portion (Expt,. lS, Fig. 6b, point 1) was taken immediately after the first blending, frozen very rapidly in liquid nikogen, and then treahed as above except that it was not, dialyzed. As an unruptured control, an aliquot was taken before the first blending, and was centrifuged, t,he pellet)s from it resuspended in GSGC, and stored at -20”. All of these samples were thawed and subsequently treated as in our basic D,NA preparation (described in Methods) . In Fig. Gb point 1 represent’s the common mean DNA molecular weight, for t’he controls for ruptured and unrupt’ured nuclei, respect,ively. Points 2, 3, and 4 represent the mean molecular weights for the DNAs from nuclei stored for different periods of time. The curve defined by these points shows t,he DNA molecular weight increased successively with time from a value of about 3.2 X lo6 at 0 hr to a plat’eau value of 5.44 X 10” (15.3s) for 12-24 hr of storage. DISCUSSION
The new method described in t,his report minimizes enzymatic activity on DNA during preparation. Shear degradation was minimized at, least to the same extent as described in t)he literature. The properties of the new DNA (N-DNA) obtained by this method were studied and compared with those of DNA obtained by a sbandard preparation (S-DNA) (7). We found the following differences. 1. While the mean molecular weight of N-DNA, 20.5 X 106, and Sf,,,, of 26.23 agree with values reported for S-DNA (15, 17), the homogeneity of N-DNA was lo M w/MN, the ratio of the weight average to much great,er than t,hat of S-DNA, as judged the number average molecular weight, provides a by the comparat,ive widths of the sedimenquantitative description of the heterogeneity of patterns. the sample, and a rough one is given by ~W/LS’~, tation coefficient distribution 2. As a result of dialyzing against’ EDTA, the ratio of the sedimentation coefficient at 80% DNA of high molecular weight cleaved into composition to that at 2OLjb. I1 This value corresponds to those reported several fractions of low molecular weight. in the literature for standard preparations (15). These fractions of low molecular weight are 12The relatively high mean molecular weight seen to be native from their melting profiles observed for this control, as compared to results (13). In contrast, S-DNA, after equivalent for the other DNA control samples, is to be exEDTA treatment, did not cleave at all. plained by the initial rupturing of nuclei in salineMoreover, this S-DNA was treated with EDTA before any removal of cytoplasmic comsome of the reagents required to rupture ponents, as normally done in preparations described in the literature. all bonds other than fully covalent ones
142
WELSH AND VYSKA
[SDS, salt, and EDTA at high concentration (13)]. Again no rupturing effect was observed. 3. While the distribution pattern for N-DNA after EDTA treatment showed distinct steps and a high degree of homogeneity, the pattern for a standard, polymerized DNA, randomly degraded with endonuclease (17), showed a broad smooth spectrum without distinct steps (Fig. 2). The finding that the new N-DNA, originally of high molecular weight, cleaves on EDTA treatment suggests that in the DNA backbone special sites, spaced at regular intervals, exist at which cleavage can take place. Since cleavage at these sites is realized by EDTA treatment, the bonds at the sites are probably those of metal ion chelation, or noncovalent in nature. On the other hand, the fact that the standard DNA does not cleave with this or any other treatment which ruptures noncovalent bonds (13) suggests that during the preparation primary noncovalent the special sites are permanently converted to fully covalent ones. After considering that, in contrast to our preparation of N-DNA from whole nuclei, all cellular components are permitted to react with released chromatin threads during nuclei rupture in standard literature preparations, we assumed that reaction between some of these components and DNA at the special sites in the DNA backbone occurred. Following this assumption, we ruptured the protected nuclei in the presence of only mitochondria. As the molecular weight of the DNA obtained from these nuclei was more than twice that for DNA from protected nuclei, we concluded that mitochondria must be the source of an agent causing conversion at the special sites. ATP, being a major component of mitochondria, was therefore suspected as being the active agent. The assumption was confirmed by the fact that the DNA prepared from protected nuclei ruptured in the presence of MgATP not only gave a higher molecular weight than the DNA from the corresponding control nuclei without ATP but also did not cleave on EDTA treatment. This means that ATP is one of the most important agents required for the conversion. This ATP effect is also most likely the
explanation for the effect of nuclei extraction which was observed during the direct preparation of DNA of low molecular weight. If whole nuclei prepared by our basic method are further extracted to remove ATP, most effectively by washing with water, EDTA, and then dialysis against the anion exchange resin (1 l), DNA having still lower mean Si,,, values is obtained than observed for our N-DNA after EDTA treatment. On the other hand, an intermediate Si, ,w was observed for the DNA from nuclei ruptured in the presence of mitochondria, the lowering of the value from that for S-DNA being explained by a similar ATP effect. A lower ATP level in the mitochondria study than that for a standard preparation would result from the one wash in GSGC, which removes all cell components except mitochondria. These findings suggest the number of converted sites is related to the concentration of ATP present in the suspension after nuclei rupture. The kinetic study demonstrated that the number of converted sites, as measured by the mean DNA molecular weight after EDTA treatment, increased with time to a plateau value, in a manner suggestive of enzyme kinetics. Further confirmation for the necessity of enzymatic activity during conversion was provided by the results of the temperature study. These results showed that with increasing temperature of ruptured nuclei storage, the Si, ,,,.of the isolated DNA and thus the number of converted sites increased rapidly, which is typical of enzymatic activity. The reason for the observed marked changes with temperature is probably not only just changes in reaction rate but also, as described in the literature (8, 9), the fact that the DNA in chromatin after lysis of nuclei (a process favored by higher temperatures) assumes a more linear configuration which would expose more special sites for reaction. By consideration of the results of the temperature and kinetics studies and t’hose for the experiment on purified, whole nuclei plus MgATP, we can conclude that for conversion at the special sites enzymatic activity is necessary, and that the enzymes required must be present in the nuclei. All the mentioned facts can be summarized by saying that in vivo in high-molecular-
PROPERTIES
OF A NEW
weight DNA special sites exist by which low-molecular-weight DNA blocks are connected. These sites have a different character than the usual phosphate ester bond between nucleotides. By our hypothesis, the bonds in these sites may occur in either of two forms: (1) fully covalent, and (2) ionic, which is probably stabilized in vivo by means of metal ion chelation. Both of these are probably interconvertible in vivo. The interconversion is controlled by the ATP level and requires the presence of nuclear enzymes. Finally, it is appropriate to draw attention to parallel findings, which also show the necessity of the presence of active, special sites in the DNA backbone. Some findings of especial interest are those which demonstrate single-stranded breaks in DNA, as required for repair after irradiation damage (25, 26), or occurring after thymine starvation (27), and those which describe multiple site replication in the polytene chromosome (28) or, more recently, the replication of DKA as small, tandemly joined units (29) or replicons (30). ACKNOWLEDGMENTS The research by one of us (R.S.W.) presented here was carried out during the tenure of an Established Investigatorship of the American Heart Association, Inc., at the University of Redlands, Redlands, California; University of California, Riverside, California; Laboratory of Molecular Biology, NINDB, National Institutes of Health, Bethesda, Maryland; and Medical Research Center, Brookhaven National Laboratory, Upton, L.I., New York. The capable technical assistance of Miss Judit,h Baron is gratefully acknowledged. The authors express their appreciation to Dr. L. E. Feinendegen for his interest, encouragement, and helpful suggestions, and to Dr. Kenneth V. Shooter for his valuable help in the preparation of the manuscript. REFERENCES 1. TAK.~GI, Y., SEKIGUCHI, M., OKU~A, S., NAK~YAMI, H., SHIMADA, K., YASUDA, S., MISHIMOTO, T., AND YOSHIHARA, II., Cold Spring Harbor Symp. Qua&. Biol. 33, 219 (1968). 2. BUTTIN, G., AND WRIGHT, M. R., Cold Spring Harbor Symp. Qua&. Biol. 33, 259 (1968). 3. LEHMAN, I. R., Progr. i~ucl. Acid Res. 2, 84 (1963).
FORM
OF DNA
143
4. OLIVERA, B. M., AND LICHMBN, I. IL., Proc. Nat. Acad. Sci. U.S.A. 67, 1426 (1967). 5. WEISS, B., AND RICHARDSON, C. C., Proc. Nat. Acad. Sci. U.S.A. 67, 1021 (1967). 6. ZIMMERMAN, S. B., AND OSHINSKY, C. K., J. Biol. Chem. 244, 4689 (1969). 7. ZUBAY, G., AND DOTY, P., J. Mol. Biol. 1, 1 (1959). 8. PHILPOT, J. ST. L., AND ST.~NIER, J. E., Biochem. J. 63, 214 (1956). 9. DOUNCE, A. L., WITTER, R. F., MONTY, K. J., PETE, S., AND COTTONE, M. S., J. Biophys. Biochem. Cytol. 1, 139 (1955). 10. WELSH, R. S., Proc. Nat. Acad. Sci. U.S.A. 48, 887 (1962). 11. WELSH, R. S., Report of the Kernforschungsanlage Jiilich GmbH, Western Germany (1969). 12. WELSH, It. S., Second International Biophysics Congress Abstracts 159 (1966). 13. WELSH, R. S., resultIs to be published. 14. K.~Y, E. It. M., SIMMONS, N. S., AND DOUNC~, A. L., J. Amer. Chem. Sot. 74, 1724 (1952). 15. EIGNEIL, J., AND DOTY, P., J. Mol. Biol. 12, 549 (1965) . 16. ROSENBFJRG, A. H., AND STUDIER, F. W., Biopolymers 7, 765 (1969). 17. SHOOTER, K. V., AND BUTLER, J. A. V., Trans. Faraday Sot. 62, 734 (1956). 18. STUDIER, F. W., J. Mol. Biol. 11, 373 (1965). 19. VINOGRAD, J., BRUNER, B., KENT, R., AND WEIGLE, J., Proc. Xat. Acad. Sci. U.S.A. 49, 902 (1963). 20. VINOGRAD, J., AND BRUNER, R., Biopolymers 4, 131 (1966). 21. MANUELKERN, L., SCHERAGA, H. A., KRIGBAUM, W. B., AND FLORY, P. J., J. Chem. Phys. 20, 1932 (1952). 22. CROTHERS, D. M., AND ZIMY, B., J. Mol. Biol. 12, 525 (1965). 23. SCHUMAKER, V. N., AND SCHSCHMAN, H. K., Biochim. Biophys. Acta 23, 628 (1957). 24. ZADRAiIL, S., PIV~X, L., SPONrlR, J., AND ~ORMOV~, Z., Collect. Czech. Chem. Commun. 30, 3920 (1965). 25. BRUNH, C. F., AND HANAXVALT, P. C., Radiat. Res. 38, 285 (1969). 26. SI”TLOIV, J. K., RANDOLPH, M. L., BOLING, M. E., MATTINGLY, A., PRICE, G., AND GORDON, M. P., Cold Spring Harbor Symp. Qua&. Biol. 28, 209 (1968). 27. FREIFELDER, I)., J. Mol. Biol. 46, 1 (1969). 28. PLAUT, W., NASH, I)., SND FANNING, T., J. Mol. Biol. 16, 85 (1965). 29. HURI<;RMAN, J. A., ASD RIGGS, A. D., J. Mol. BioZ. 32, 327 (1968). 30. OK.~DA, S., Biophys. J. 8, 650 (1968). 31. SUEOKA, N., Proc. *Vat. Acad. Sci. U.S.A. 46, 1480 (1959).
OF
BIOCHEMISTRY
Properties
AND
BIOPHYSICS
of a New
Thymus
fi.ir Medizin,
Form of DNA
From Whole
Nuclei:
Evidence
Special
Sites in DNA
RICHARD Institut
132-143 (1971)
l&t,
S. WELSH
AND
Kernforschungsanlage
Calf
for Reactive,
KAREL VYSKA Jtilich
GmbH, Western Germany
Received July 7, 1970 A new method for DNA preparation from whole calf thymus nuclei, which was designed to minimize enzymatic activity throughout to the greatest possible extent, is presented. The isolated DNA shows a number of interesting properties differing from those of DNA prepared by standard literature methods. The most important property is that the DNA prepared from our whole, purified nuclei, having originally a high molecular weight of 20.5 million, cleaved on EDTA treatment into a number of relatively homogeneous DNA fractions with a mean molecular weight of 2.3 million. In contrast, the standard DNA did not cleave on EDTA treatment. On the basis of this finding it was concluded that in the DNA backbone special sites exist at which cleavage can occur. These sites were further shown to occur in one of two possible forms: (1) fully covalent and (2) noncovalent. The proportion of the sites in form 1 increases with increasing ATP level. In standard DNA all special sites are most likely in the covalent form. It was also shown that for the conversion from one form to the other enzvmes of the nucleus are necessary. The ATP level very probably controls this enzymatic activity in rive.
Recently, in numerous investigations, molecular changes in DNA in vitro by the action of such enzymes as endonucleases (1, 2), exonucleases (3), and ligases (4-6) have been described. These findings suggest the question as to what happens during nuclei rupture, when chromatin is exposed to cytoplasmic components. In order to answer this question, we developed a new method in which we minimized to the greatest possible extent enzymatic activity during preparation, first by means of prior separation of whole nuclei from all other cellular components at very low temperatures (- 10 f 59, and then by use of sodium dodecylsulfate (SDS) or Pronase, as enzymedestroying agents, after rupture of these nuclei. The DNA obtained by this method, having the same mean molecular weight as that for a standard preparation according to Zubay and Doty (7), showed several different properties which confirm our as-
sumption that somechangesoccur during the standard preparation. The main difference observed was that while our DNA cleaved on EDTA treatment into homogeneous subunits the standard DNA did not. In order to find the deciding factors causing these changes, as well as to show their characteristics, the second part of the investigation was carried out. This study showed that ATP and endonuclear enzymes are the main components which are necessary for the changes occurring during a standard preparation. METHODS
Basic Nuclei Preparation Suspension of tissue components. Calf thymus was ground in the frozen state and suspended in 40% glycerol-O.44 M sucrose-O.039 M sodium glycerophosphate-O.019 M sodium citrate-citric acid (GSGC) at subzero temperatures (-X--5’), with the pH maintained at 6.1 and the ionic strength
132
PROPERTIES
OF A NEW
at 0.15. The glycerophosphate (8) was included in the medium to protect the nuclei against enzymatic activity, the sucrose to prevent mitochondrial and nuclear rupture (9), and the glycerol to prevent clumping and freezing. ATucZei washing. The whole, unclumped nuclei in the suspension described above were purified of cytoplasmic components by centrifugation, as follows. After transferring the suspension, a gradient layer was set up at the bottom of each 250-ml centrifuge tube, by the use of a linear gradient maker,’ to prevent hard packing of nuclei, and centrifugation was carried out for 20 min at 65009. The pellets were resuspended by mild agitation in GSGC, and the procedure was repeated through four cycles with successive increases of centrifugation speed to 10,8OOg, as previously described (10). During this procedure the temperatures was maintained at -lO-5”, to minimize enzymatic activity, and to give purified whole nuclei. Nuclei e&action. In this step which was used for direct preparation of DNA of low molecular weight the nuclei were sedimented by centrifugation at 19,000g for 10 min and resuspended at once in water at 0” by motor-turning of the Potter homogenizer. They were then rapidly extracted by washing three times with water at O-4”, [to remove ATP, Mg ions, and a mixture of phosphopeptides (11, 12) by alternate centrifugation and resuspension with the Potter homogenizer in fresh portions of water]. By the third wash the nuclei were largely lysed to a gel state, such that it was necessary to increase the speed of centrifugation to about 33,000g to give adequate sedimentation of the gel. Saline extraction of precipitated DNP. To precipitate the deoxyribonucleoprotein (DNP), the sedimented gel of washed, lysed nuclei was suspended in the same way in water, and the suspension was adjusted to 0.14 M NaCl. The resulting DNP was dispersed by hand operation of a sintered glass homogenizer, at O-4”. After centrifuging down the dispersed and precipitated DNP, the saline extract, containing ribonucleoproteins, was removed, and the DNP pellet was again suspended by hand homogenization in 0.14M NaCl. DNP dissolution. The precipitated DNP prepared as above was dissolved in the high ionic * In the exit chamber of the gradient maker 25 ml of 80% glycerol (w/v)-0.44 M sucrose-O.039 M glycerophosphate-0.019 M citrate-citric acid was placed and in the other chamber 25 ml of a medium with the same composition except that the glycerol concentration was 40Yo (w/v) (GSGC).
FORM
OF DNA
133
mM strength buffer, 1 M NaCl-0.7 mM phosphate-l EDTA, pH 9.2 DNA preparation. DNA of low molecular weight was purified from the dissolved DNP by a clarifying centrifugation at 25,OoOg and by five to eight cycles of a method based on that of Kay et al. (14), modified (11) as follows: (1) For the first cycle, after dissolving the DNP and centrifuging, the gelatinous pellets were extracted with 2.5 M NaCl and centrifuged at 25,OOQgand the supernate pooled with the first 1 M NaCl extract. In all other cycles of purification, the final NaCl concentration in the SDSNaCl dispersion was 2.5 M instead of 1 M, except, the last cycle in which it was again 1 M. (2) Twice the volume of cold 95% ethanol instead of an equal volume was added to precipitate the DNA, if 1 M NaCl was used for the SDS-NaCl dispersion. If 2.5 M NaCl was used, it was necessary to add 2.5 times the volume of 95yo ethanol to precipitate the DNA. (3) Both types of DNA precipitates formed, fibrous and flocculent,3 were collected, the fibrous one by removal on the stirring rod and the flocculent one by centrifugation. (4) The solvent B (15) (0.0075 M phosphate-l mM EDTA, pH 7) instead of water was used to dissolve the precipitated DNA. For the physical chemical determinations, the precipitated DNAs were finally dialyzed against 0.7 mM phosphate-l mM EDTA, pH 7. This buffer, which has a total ionic strength of 0.0079, i.e., appreciably lower than the value of 0.0276 for solvent B, was used to overcome possible aggregation effects.4 In order to avoid EDTA treatment and thus DNA cleavage throughout in the preparation of DNA of high molecular weight (see Table I, 2 To rule out the possibility of occurrence of shear as well as enzymatic degradation of DNA during any of the treatments required to disperse nuclei, a parallel experiment (13) was set up to effect lysis of protect,ed nuclei by the action of Pronase, a powerful proteolytic enzyme. This treatment yielded DNA, initially of high molecular weight, which after dialysis against 1 M NaCl-0.024 M EDTA, pH 8.1, gave a banding pattern showing the same DNA components of low molecular weight as found in our N-DNA after special EDTA treatment. 3 These flocculent precipitates were found only for the control samples from whole, protect,ed nuclei. *For highly purified DNA, such aggregation was not found to occur at ionic strengths of 0.00792.5 in agreement with Rosenberg and St,udier (16), but for impure DNA aggregation can result from increasing the ionic strength.
134
WELSH AND VYSKA
Exp. 1 and 2), the procedure described above was modified as follows. The whole nuclei were dispersed in 0.14 M NaCl by motor-homogenization and then the DNP dissolved by dispersing in the required volume of 1 M NaCl. During “DNA preparation” after alcohol precipitation, the DNA fibers were at each cycle worked up in 0.14 M NaCl or 0.01 M phosphate, pH 7, instead of solvent B. The precipitates in this case were always entirely in the fibrous form and could not be so easily dissolved. More cycles of purification were required than for all DNA preparations of low molecular weight, since, without the presence of EDTA in the medium, it was much more difficult to split the protein from the DNA.6 For the final dialysis, in order to break up reversible complexes formed through metal ion interaction, it was also necessary to dilute the DNA to an OD at 260 w of 10 or less, and then dialyze against the medium used for the ultracentrifuge runs, 0.5 M NaC1-0.7 mM phosphate-l mM EDTA, pH 7. Special EDTA treatment. During this treatment, the dialyzed DNA samples were adjusted to 0.01 M EDTA, pH 7, stored for l-2 days at 4’, and finally dialyzed against nine times the total volume of 0.7 mM phosphate, pH 7 (to restore the original buffer). Ultracentrifuge rate runs. The boundary runs were made at 50,740 rpm in a temperature range of 5-lo”, with thermostatic regulation, in a Spinco Model E analytical ultracentrifuge, equipped with a uv optical system and a Cl-Br filter. The photographs were scanned with a recording microdensitometer and baseline corrections made, as previously described (10). The integral distribution curves of sedimentation coefficients were computed by the method of Shooter and Butler (17). For each of two concentrations for each sample, reciprocals of &o.~ values were taken at steps of 10% of the total composition (or every 5% at the extremities), and extrapolations to zero concentration made. For some curves (Figs. 1, 2, and 7), extrapolations were made mathematically by the use of known relationships (see figure legends). From the S,“,,, values thus obtained, a complete extrapolated curve was constructed for each sample. The banding runs were made at 48,000 rpm in a Spinco Model E, using a monochromator set at 265 rnE.cand the Beckman Dynagraph double beam 5 If the DNA precipitate from alcohol is placed in 2.5M NaCl and dispersed by gently pressing it against the walls of the container with a rubber policeman, with subsequent addition of SDS, the DNA is more effectively dissociated from the protein and dissolves more completely.
scanner. The temperature of each run was regulated to 3~0.1” within the range 5-lo”, by the method of Studier (18). The banding method of Vinograd et al. (19) was applied, using a 12-mm double sector centerpiece. From banding run patterns the distributions of sedimentation coefficients were obtained as follows. From the plots of density, automatically corrected for solvent absorption, as a function of distance obtained by use of the scanner during banding runs, the densities were taken at distances increased by a small increment from the meniscus and corrected for sectorial dilution (17). For each of these distances the corresponding 820,~ was calculated. For the concentrations used in these banding runs the S20.w values directly obtained were considered to be equivalent to the values at . . . infinite dllutlon (S~o.,), according to Vinograd et al. (20). The plot for corrected densities as a function of S:,., was then constructed. Since according to Vinograd et al. (19) banding runs give directly concentration distributions in differential form, this plot was considered to be equivalent to the distribution of sedimentation coefficients. To obtain the integral distribution curve (Fig. l), the S:,,, scale was divided into steps of 2S, and for each point the sum of all densities from the meniscus up to the point was plotted against the corresponding S&.,. Intrinsic viscosity determinations. The intrinsic viscosities at zero shear rate ([v]G = 0) were determined in two four-bulbed Ubbelohde viscometers, as described previously (10). The measurements were made at 25.00 f O.Ol”, at four or five DNA concentrations. Molecular weight determinations. The weightaverage molecular weight was finally calculated from the value of &., at 50yo of total concentration and the [7]G = 0 value for each sample, using the Mandelkern-Flory equation (21) for random coils, assuming a p of 2.27 X 104, according to Crothers and Zimm (22). EXPERIMENTAL
AND RESULTS
The method described above produces a new form of DNA (N-DNA). In order to point out very interesting properties of this N-DNA and also to make the necessary comparisons between it and standard DNA (S-DNA) described in the literature (7, 15), the following parallel studies were carried out on these two DNA forms. First,, the behavior of the two types of DNA before and after EDTA treatment is compared in Fig. 1, in which the integral distributions of sedimentation coefficients
PROPERTIES
OF A NEW F0R.M OF DNA
FIG. 1. Integral distribution curves of sedimentation coefficients for N-DNA and S-DNA before and after the special EDTA treatment (0.01 M EDTA, pH 8.8 for 24 hr and for standard S-DNA, afte enzymatic degradation. Extrapolations of the curves to zero concentration were done using the relationship l/S = l/iY(l + K[TJ]c), and the data of Eigner and Doty (15) for 8 as a function of [ql, at ionic strengths of 0.2 or higher, K = 0.79 (from our data), c = 0.022 mg/ml. Curve 1’: Boundary run, in 0.5 M NaCl-0.7 mu phosphate-l mu EDTA, pH 7, for NDNA (O&~o = 0.45) after EDTA treatment; 0 and X at 2328 and 2568 set total times of centrifugation, respectively. Curve 1: Same as curve 1’ after extrapolation to zero concentration. Curve 2’: Boundary run, in 0.5 M NaClLO.7 mM phosphate, pH 7, for N-DNA before EDTA treatment; X, 0, and 0 at 1537, 1914, and 2255 set total times of centrifugation, respectively. Curve 2: Same as curve 2’, after extrapolation to zero concentration. Curve 3: q Standard S-DNA, prepared according to Zubay and Doty (7), before and after the special EDTA treatment, obtained by integration of the curve 1 in Fig. 2. Curve 4: A Endonuclease-degraded thymus DNA, data interpolated to 14.6 min of enzyme degradation and extrapolated to zero concentration.
are plotted. The curves 2’ and 1’ represent the original unextrapolated data from boundary runs for our N-DNA before and after EDTA treatment, respectively (see Table I, Expts. 1 and 2). The corresponding distributions after extrapolation to zero concentrat,ion are plotted as curves 2 and 1, respectively. The curve 3 was derived by integration (see Methods) of the common curve obtained from banding runs on standard DNA before and after EDTA treatment’ (original data plotted in Fig. 2). The comparison of curves 2 and 3 in Fig. 1 for N-DNA and S-DNA before EDTA treatment reveals that, while the mean Sk .Wvalues are roughly the same (26s and 3OS), the pattern for the S-DNA (curve 3)
shows a broad continuous distribution6 in contrast to the more homogeneous one for N-DNA (curve 2). The curve 1 in this figure demonstrates that N-DNA, having originally a mean S,“, ,W of 26S, gives after EDTA treatment a greatly reduced mean of 11s. %I #TV In contrast, the data for S-DNA before and after EDTA treatment define a single curve (curve 3, Fig. 1, original data given in Fig. 2), which shows that in this case EDTA treatment has no effect. In addition, curve 4, depicting the integral distribution of enzymatically degraded 6 Similar to the derivative curve of Schumaker and Schachman (23) from boundary runs.
ii
Flow
x0
Mean
Preparation
equation
’ Linear
c hdd .024 .012 .006
by M.F.
equation
b +’
for log Sio w 88 B function
was employed.
I
2.27
+
10.98
1: +
so 88 to eliminate
20.49
+
26.24
+‘” +’
c
+
4-
+
+
2
+
3
+
4
data
for nine
use of EDTA
+ from
3.25
f
23.0 1.47 5.21 3.01
of our
most
2.84
24.5 1.90 4.44 2.69
+
11.68
11.25
throughout
7.19
1.58
+
17.12
12.39 +
13.60 15.24
11.48 11.90
6.20
45.5 1.54 5.55 5.93
+
16.12
13.47 14.57 15.40
+ + + +
+
+
1
+
+
7
+
+
8
PAR.&METERS
9
+
no.
FOR
+
Expt.
7.C2
45.5 1.50 1.74 5.23
+
16.95
13.60 15.20
homogeneous
(see Methods).
+
3.08
3.96
29.9
-I-
12.12
11.56 12.18
DNA
+
+
f
+
11
+
+
+
+
12
DNA
+
+
f
+
13
SAMPLES
+
+
14
12.60
2.19
+
preparations
15.20 16.CO
+
5.15
5.45
43.0 1.59
+
14.95
13.64 14.15
M = molecular
of Sueoka
6.91
7.11
51.0 1.78
+
16.86
here),
relationship
6.40
1.72 1.60
+
16.32
(six shown
by the
12.60
71.1 2.19 9.36 11.65
+
20.68 21.50
20.80 21.50
14.82 15.55 15.98
+ + + + + 1 : 1+ : : +
+
+
+
+
10
DESCRIBED
c Calculated
2.20
1.85 2.69
+
10.58
10.28 10.42
+ + + + + + + z+ T + :+ :+
+
6
+
5
AND
TABLE PROCEDURES
+ + T- 1
+
+
PREPARATION
+=
1
OF
of log M, determined
Modified
121) 2 Calcd. by linear log r&tic& used for computation a and Kf
C&d.
0.7 rn~ phosphate, pH 7.0 0.7 rn~ phosphate-l rn~~ EDTA, pH7 0.5 M NaCl phosphate1 mu EDTA, pH7
ODao .50 .25 ,125
OUTLINE
a GSGC b SalineEDTA B GSGC b SalineEDTA a Control b Ruptured -4” b +4’ a Control bl hr c5 hr d 24 hr
(21). CY = 0.4068, K’ = 1.5560.
relationship
item
Mw/MN~ M, x lo-’
1
3
ltll G = c
m/s20
1 2
1 2 3
solvent
w
A% w
Mean
n + Indicated
IC ID IE IIC DATA
Unextnrcted Water washed Control Mg Clz Mg ATP of DNP
1 2 1 2 4
1 Mitochondria effect 2 Temperatwe’j-a effect 3 Time effect
2 Washing
1 Suspension
PRocmuRE
Saline extraction DNP dissolution DNA preparation Special EDTA treatment AND PARAMETERS
Basic nuclei Extractions Special nuclei extractions
IB
IIB
Special nuclei treatments
IIA
PREPARATION IA Basic nuclei preparaton
SUMMARY
(31). weight
5.47
1.70
+
15.34
13.13 14.15
:
+
+
+
+
f
15
+
3.97
3.89
30.0 1.57
+
13.46
12.93
+ + +
+
+
17
:
+
+
+
18
+
3.31
25.4 1.64 1.98 3.20
+
12.48
11.98 12.18
by the Mahdelkerh-
5.45
1.70 1.92
+
15.30
14.32 14.80
Tc
+
+
+
16
PROPERTIES
OF A NEW FORM OF DNA
137
FIG. 2. Curve 1: Banding runs on S-DNA prepared according to Zubay and Doty (7), before and after special EDTA treatment. Before EDTA: l and 0 at 759 and 989 set total times of centrifugaion, respectively. After EDTA: A and v at 1291 and 1995 set total times of centrifugation, respectively.
S-DNA, was compared with curve 1’ for EDTA treated N-DNA. The comparison shows that enzymatically degraded DNA has, in contrast to EDTA-treated N-DNA, much higher heterogeneity in the region of 50-100% of total concentration and a smooth distribution without steps. In order to find the most important factors in DNA preparation which determined whether the isolated DNA of high molecular weight cleaved during EDTA treatment, as in t,he case of N-DNA, or did not, as in the case of S-DI\JA, we did the following experiments. (I) Effect of mitochondria. In this experiment, a tissue suspension (see Methods) was centrifuged at 14,500 rpm for 10 min. Two pellets from this centrifugation, containing
essentially
nuclei plus mitochondria,
were taken. The first pellet was blended in saline-EDTA (7) at top speed for 3 min, to rupture nuclei, permitting release of chromatin threads. Subsequently, these ruptured nuclei were washed five times in the same medium at 4”, according to Zubay and Doty (7) (Expt. 3). The other pellet was washed four times at 4’ in GSGC (see Methods), as a control (Expt. 4). All other steps in the DNA isolation were maintained the same as in our basic method. Figure 3 gives t,he int,egral distribution
curves of sedimentation coefficients derived from boundary runs, extrapolated to zero concentration. The extrapolated curve 1, for DNA from the control nuclei showed a greater homogeneity and a lower mean Sk ,w value’ (12.395) than for curve 2 for DNA from nuclei rupt’ured in the presence of mitochondria (mean Si, ,Wof 16.125) .* The molecular weight, calculated from the mean Xi, ,Wand intrinsic viscosity for DNA from ruptured nuclei (5.9 X 106), was about twice that of the DNA from control nuclei (3.0 X 106) (see Table I, Expts. 3 and 4). (2) Efect of MgATP. In this experiment the suspension of whole nuclei (Methods IA, 2) was centrifuged at 12,000 rpm and subdivided into three pellets. The first pellet was homogenized by blend7 This lower mean S&., was obtained directly without special EDTA treatment in the control and in those for subsequent experiments, because of the water washing, which removed ATP, and the dilute EDTA treatment during DNA preparation (see Methods and Discussion). 8 The lower value of S&., for the DNA from ruptured nuclei than that for S-DNA results from the fact that the nuclei, before rupturing in the presence of mitochondria, were washed once with GSGC, which removed some ATP and inhibited enzymatic activity (see Discussion).
138
WELSH AND VYSKA
FIG. 3. Integral distribution of DNA sedimentation coefficients illustrating mitochondria effect. Curve 1’: DNA from control nuclei, at OD 260= 0.50; 0 and 0 at 2670 and 3150 set total times of centrifugation, respectively. Curve 1”: Same at ODzso = 0.25; X at 1902set total time of centrifugation. Curve 1: Curve for this DNA extrapolated to infinite dilution. Curve 2’: DNA from nuclei ruptured in presence of mitochondria, OD260 = 0.25; q at 987 set total time of centrifugation. Curve 2”: Same at OD260 = 0.125; A at 714sec total time of centrifugation. Curve 2: Curve for this DNA extrauolated to infinite dilution, using data for OD~W of 0.50 (curve not shown) and of 0.125. L
ing in 0.01 M MgATP, pH 7, and then washed three times in the same medium. All other conditions and further steps were maintained the same as in our basic DNA preparation (see Table I, Exp. 5). The second pellet, which was used as a control, was washed with water, instead of MgATP, maintaining all other conditions the same as above. In Fig. 4, the curve 2 for DNA from control nuclei shows a much lower mean Sk ,w (11.75) than that (17.15) found in curve 4 for DNA from nuclei ruptured in the presence of MgATP.g This means that the mean DNA molecular weight (see Table I) increased from 2.84 X lo6 (11.7s) to 7.19 X lo6 (17.15), as seen in Table I, Expts. 6 and 5, respectively. g The lower mean S:,,, observed for the DNA from MgATP-treated nuclei than for S-DNA can be explained by the shorter time (see kinetic study) needed for the MgATP treatment than for the washing of ruptured nuclei in a standard preparation (7), and possibly by an inhibition by the glycerophosphate (24) of endonuclear enzymatic activity involved in the proposed closing of sites (see Discussion).
In order to show that the main effect was due to ATP and not Mg ion, the third pellet was washed three times with 0.01 M MgClz , maintaining all other steps the same as in the MgATP-treated sample (see Table I, Expt. 7). The comparison of the patterns for DNA from MgATP-treated and MgClz-treated pellets (Fig. 4, curves 4 and 3, respectively) confirmed that the ATP and not the Mg ion is responsible for the high molecular weight of the resulting DNA. Furthermore, whereas special EDTA treatment (see Methods) of the DNA from MgATP-treated nuclei produced no appreciable change in the Si,,, , this treatment lowered the mean St,,, of the DNA from MgC&-treated nuclei from 12.15 to 10.6s (see Expts. 7 and 9, Fig. 4, curves 1 and 3, respectively). After such EDTA treatment, the molecular weight of DNA from MgATPtreated nuclei was 7.0 X lo6 (16.9S), more than three times the molecular weight, 2.2 X lo6 (10.75), for DNA from MgChtreated nuclei (see Table I, Expts. 8 and 9). In order to show that enzymatic activity is
PROPERTIES
OF A NEW FORM OF DNA
0
FIG. 4. Integral distributions of DNA sedimentation coefficients illustrating MgATP effect. The curves 1, 2, 3, and 4 are the ones extrapolated to infinite dilution corresponding to curves l’, 2’, 3’, and 4’. Curve I’: EDTA-treated DNA from MgClt-treated nuclei OD200 = 0.25; q and n at 726 and 966 set total times of centrifugation, respectively. Curve 2’: DNA from water-extracted nuclei, ODzso = 0.50; V and A at 990 and 1230 set total times of centrifugation, respectively. Curve 3’: DNA from MgCL-treated nuclei, OD 260= 0.50; 0, 0 at 957 and 1197 set total times of centrifugation, respectively. Curve 4’ : EDTA-treated DNA from MgATP-treated nuclei ODscO= 0.25; X and l at 781 and 1261 set total times of centrifugation, respectively.
FIG. 5. Integral distributions of DNA sedimentation coefficients, illustrating temperature effect; curves 1,2,3, and 4 are the ones extrapolated to infinite dilution, corresponding to curves l’, 2’, 3’, and 4’, respectively. Curve 1’: DNAfrom ruptured nucleistored at -4--2”, ODtco = 0.50; 0 and 0 at 956 and 1194 set total times of centrifugation, respectively. Curve 2’: EDTA-treated DNA from ruptured nuclei stored at -4--2”, ODnso = 0.50; S and n at 964 and 1204set total times of centrifugation, respectively. Curve 3’, 4’: DNAs from ruptured nuclei stored at +4”. A and v EDTA-treated DNA, ODxco = 0.125, at 474 and 954 set total times of centrifugation, respectively. X, DNA (not EDTA-treated), ODzao = 0.092, values obtained graphically from data at 456, 696, and 936 set total times of centrifugation.
139
140
WELSH AND VYSKA
5/ :’ 1
4
2
1
FIG. 6a. Integral distributions of DNA sedimentation coefficients in the time effect experiment. Curves 1 and 2 are the ones extrapolated to infinite dilution corresponding to curves 1’ and 2’, respectively. Curve 1’: DNA from control nuclei at 0 time of dialysis, ODtaa = 0.25; 0 and 0 at 714 and 954 set total times of centrifugation, respectively. Curve 2’ : DNA from ruptured nuclei at 24 hr of dialysis OD26o = 0.25. q , before EDTA treatment, at 956 set total time of centrifugation. A and V after EDTA treatment, at 750 and 990 set total times of centrifugation, respectively. FIG. 6b. Plot to illustrate time effect: Mean DNA molecular weights, computed by the Mandelkern-Flory equation (see Table I), as a function of the time of dialysis.
involved in the processes which lead to the described differences between S-DNA and N-DNA we did the following experiment. (3) The temperature effect. For this experiment, frozen calf thymus was ground and homogenized by blending for 5 min at - 2- - lo’, directly in the saline-EDTA medium of Zubay and Doty (7), containing also 40% glycerol, to prevent freezing. This suspension was subdivided into two portsions: (a) The first portion was washed
in this medium at 4’, according to the Zubay-Doty procedure (7) (Expt. 10); (b) the second portion was washed in the same way, except t,hat the temperature was maintained at -4” (Expts. 11 and 12). All other steps in the DNA isolations were again maintained the same as in our basic method. In Fig. 5 curve 1 represents the pattern for DNA prepared from ruptured nuclei st’ored at the lovver temperature, while
PROPERTIES
OF A NEW
curve 3 represents t’he pattern for DNA prepared from nuclei stored at the higher temperature. From this figure and from the comparison of M,/M, value@ (see Table I, Expts. 12 and lo), it is evident that use of a lower temperature during the entire purification resulted in a significant decrease in the mean Si,,, of from 21.5s to 16.3s and an increase in homogeneity. The mean molecular weight of the DNA prepared from nuclei washed at the higher t’emperature” was 12.6 X lo6 and that for DNA from control nucleiI stored at the lower temperature was 6.9 X 106. The EDTA treatment in both cases (see Expts. 13 and 11, Fig. 4, curves 4 and 2) is seen to have no appreciable effect on the mean Si, ,w values. (4) The time efect. For t’his experiment, t.he tissue suspension in GSGC (see Methods) was adjusted to a total NaCl concentration of 0.34 M and a pH of 6.8, and the nuclei in it were ruptured by blending at maximum speed for 15 sec. The resulting ruptured nuclei were subdivided into five portions: The first, second, and third portions were dialyzed at 4” against 0.34 M NaCl for 1, 5, and 24 hr (see Table I, Expts. 17, 14, 15, Fig. 6b, points 2, 3, 4, respectively). During the dialysis of these portions, the pH of the dialyzed nuclei was periodically adjusted upward to 6.8 (at 4-lo-hr intervals). At the appropriate time, each of these portions was blended, as described above, centrifuged, the pellets from each resuspended, frozen rapidly to stop all enzymatic reactions, and st,ored at -20”. As a control,
FORM
OF DNA
141
a fourth portion (Expt,. lS, Fig. 6b, point 1) was taken immediately after the first blending, frozen very rapidly in liquid nikogen, and then treahed as above except that it was not, dialyzed. As an unruptured control, an aliquot was taken before the first blending, and was centrifuged, t,he pellet)s from it resuspended in GSGC, and stored at -20”. All of these samples were thawed and subsequently treated as in our basic D,NA preparation (described in Methods) . In Fig. Gb point 1 represent’s the common mean DNA molecular weight, for t’he controls for ruptured and unrupt’ured nuclei, respect,ively. Points 2, 3, and 4 represent the mean molecular weights for the DNAs from nuclei stored for different periods of time. The curve defined by these points shows t,he DNA molecular weight increased successively with time from a value of about 3.2 X lo6 at 0 hr to a plat’eau value of 5.44 X 10” (15.3s) for 12-24 hr of storage. DISCUSSION
The new method described in t,his report minimizes enzymatic activity on DNA during preparation. Shear degradation was minimized at, least to the same extent as described in t)he literature. The properties of the new DNA (N-DNA) obtained by this method were studied and compared with those of DNA obtained by a sbandard preparation (S-DNA) (7). We found the following differences. 1. While the mean molecular weight of N-DNA, 20.5 X 106, and Sf,,,, of 26.23 agree with values reported for S-DNA (15, 17), the homogeneity of N-DNA was lo M w/MN, the ratio of the weight average to much great,er than t,hat of S-DNA, as judged the number average molecular weight, provides a by the comparat,ive widths of the sedimenquantitative description of the heterogeneity of patterns. the sample, and a rough one is given by ~W/LS’~, tation coefficient distribution 2. As a result of dialyzing against’ EDTA, the ratio of the sedimentation coefficient at 80% DNA of high molecular weight cleaved into composition to that at 2OLjb. I1 This value corresponds to those reported several fractions of low molecular weight. in the literature for standard preparations (15). These fractions of low molecular weight are 12The relatively high mean molecular weight seen to be native from their melting profiles observed for this control, as compared to results (13). In contrast, S-DNA, after equivalent for the other DNA control samples, is to be exEDTA treatment, did not cleave at all. plained by the initial rupturing of nuclei in salineMoreover, this S-DNA was treated with EDTA before any removal of cytoplasmic comsome of the reagents required to rupture ponents, as normally done in preparations described in the literature. all bonds other than fully covalent ones
142
WELSH AND VYSKA
[SDS, salt, and EDTA at high concentration (13)]. Again no rupturing effect was observed. 3. While the distribution pattern for N-DNA after EDTA treatment showed distinct steps and a high degree of homogeneity, the pattern for a standard, polymerized DNA, randomly degraded with endonuclease (17), showed a broad smooth spectrum without distinct steps (Fig. 2). The finding that the new N-DNA, originally of high molecular weight, cleaves on EDTA treatment suggests that in the DNA backbone special sites, spaced at regular intervals, exist at which cleavage can take place. Since cleavage at these sites is realized by EDTA treatment, the bonds at the sites are probably those of metal ion chelation, or noncovalent in nature. On the other hand, the fact that the standard DNA does not cleave with this or any other treatment which ruptures noncovalent bonds (13) suggests that during the preparation primary noncovalent the special sites are permanently converted to fully covalent ones. After considering that, in contrast to our preparation of N-DNA from whole nuclei, all cellular components are permitted to react with released chromatin threads during nuclei rupture in standard literature preparations, we assumed that reaction between some of these components and DNA at the special sites in the DNA backbone occurred. Following this assumption, we ruptured the protected nuclei in the presence of only mitochondria. As the molecular weight of the DNA obtained from these nuclei was more than twice that for DNA from protected nuclei, we concluded that mitochondria must be the source of an agent causing conversion at the special sites. ATP, being a major component of mitochondria, was therefore suspected as being the active agent. The assumption was confirmed by the fact that the DNA prepared from protected nuclei ruptured in the presence of MgATP not only gave a higher molecular weight than the DNA from the corresponding control nuclei without ATP but also did not cleave on EDTA treatment. This means that ATP is one of the most important agents required for the conversion. This ATP effect is also most likely the
explanation for the effect of nuclei extraction which was observed during the direct preparation of DNA of low molecular weight. If whole nuclei prepared by our basic method are further extracted to remove ATP, most effectively by washing with water, EDTA, and then dialysis against the anion exchange resin (1 l), DNA having still lower mean Si,,, values is obtained than observed for our N-DNA after EDTA treatment. On the other hand, an intermediate Si, ,w was observed for the DNA from nuclei ruptured in the presence of mitochondria, the lowering of the value from that for S-DNA being explained by a similar ATP effect. A lower ATP level in the mitochondria study than that for a standard preparation would result from the one wash in GSGC, which removes all cell components except mitochondria. These findings suggest the number of converted sites is related to the concentration of ATP present in the suspension after nuclei rupture. The kinetic study demonstrated that the number of converted sites, as measured by the mean DNA molecular weight after EDTA treatment, increased with time to a plateau value, in a manner suggestive of enzyme kinetics. Further confirmation for the necessity of enzymatic activity during conversion was provided by the results of the temperature study. These results showed that with increasing temperature of ruptured nuclei storage, the Si, ,,,.of the isolated DNA and thus the number of converted sites increased rapidly, which is typical of enzymatic activity. The reason for the observed marked changes with temperature is probably not only just changes in reaction rate but also, as described in the literature (8, 9), the fact that the DNA in chromatin after lysis of nuclei (a process favored by higher temperatures) assumes a more linear configuration which would expose more special sites for reaction. By consideration of the results of the temperature and kinetics studies and t’hose for the experiment on purified, whole nuclei plus MgATP, we can conclude that for conversion at the special sites enzymatic activity is necessary, and that the enzymes required must be present in the nuclei. All the mentioned facts can be summarized by saying that in vivo in high-molecular-
PROPERTIES
OF A NEW
weight DNA special sites exist by which low-molecular-weight DNA blocks are connected. These sites have a different character than the usual phosphate ester bond between nucleotides. By our hypothesis, the bonds in these sites may occur in either of two forms: (1) fully covalent, and (2) ionic, which is probably stabilized in vivo by means of metal ion chelation. Both of these are probably interconvertible in vivo. The interconversion is controlled by the ATP level and requires the presence of nuclear enzymes. Finally, it is appropriate to draw attention to parallel findings, which also show the necessity of the presence of active, special sites in the DNA backbone. Some findings of especial interest are those which demonstrate single-stranded breaks in DNA, as required for repair after irradiation damage (25, 26), or occurring after thymine starvation (27), and those which describe multiple site replication in the polytene chromosome (28) or, more recently, the replication of DKA as small, tandemly joined units (29) or replicons (30). ACKNOWLEDGMENTS The research by one of us (R.S.W.) presented here was carried out during the tenure of an Established Investigatorship of the American Heart Association, Inc., at the University of Redlands, Redlands, California; University of California, Riverside, California; Laboratory of Molecular Biology, NINDB, National Institutes of Health, Bethesda, Maryland; and Medical Research Center, Brookhaven National Laboratory, Upton, L.I., New York. The capable technical assistance of Miss Judit,h Baron is gratefully acknowledged. The authors express their appreciation to Dr. L. E. Feinendegen for his interest, encouragement, and helpful suggestions, and to Dr. Kenneth V. Shooter for his valuable help in the preparation of the manuscript. REFERENCES 1. TAK.~GI, Y., SEKIGUCHI, M., OKU~A, S., NAK~YAMI, H., SHIMADA, K., YASUDA, S., MISHIMOTO, T., AND YOSHIHARA, II., Cold Spring Harbor Symp. Qua&. Biol. 33, 219 (1968). 2. BUTTIN, G., AND WRIGHT, M. R., Cold Spring Harbor Symp. Qua&. Biol. 33, 259 (1968). 3. LEHMAN, I. R., Progr. i~ucl. Acid Res. 2, 84 (1963).
FORM
OF DNA
143
4. OLIVERA, B. M., AND LICHMBN, I. IL., Proc. Nat. Acad. Sci. U.S.A. 67, 1426 (1967). 5. WEISS, B., AND RICHARDSON, C. C., Proc. Nat. Acad. Sci. U.S.A. 67, 1021 (1967). 6. ZIMMERMAN, S. B., AND OSHINSKY, C. K., J. Biol. Chem. 244, 4689 (1969). 7. ZUBAY, G., AND DOTY, P., J. Mol. Biol. 1, 1 (1959). 8. PHILPOT, J. ST. L., AND ST.~NIER, J. E., Biochem. J. 63, 214 (1956). 9. DOUNCE, A. L., WITTER, R. F., MONTY, K. J., PETE, S., AND COTTONE, M. S., J. Biophys. Biochem. Cytol. 1, 139 (1955). 10. WELSH, R. S., Proc. Nat. Acad. Sci. U.S.A. 48, 887 (1962). 11. WELSH, R. S., Report of the Kernforschungsanlage Jiilich GmbH, Western Germany (1969). 12. WELSH, It. S., Second International Biophysics Congress Abstracts 159 (1966). 13. WELSH, R. S., resultIs to be published. 14. K.~Y, E. It. M., SIMMONS, N. S., AND DOUNC~, A. L., J. Amer. Chem. Sot. 74, 1724 (1952). 15. EIGNEIL, J., AND DOTY, P., J. Mol. Biol. 12, 549 (1965) . 16. ROSENBFJRG, A. H., AND STUDIER, F. W., Biopolymers 7, 765 (1969). 17. SHOOTER, K. V., AND BUTLER, J. A. V., Trans. Faraday Sot. 62, 734 (1956). 18. STUDIER, F. W., J. Mol. Biol. 11, 373 (1965). 19. VINOGRAD, J., BRUNER, B., KENT, R., AND WEIGLE, J., Proc. Xat. Acad. Sci. U.S.A. 49, 902 (1963). 20. VINOGRAD, J., AND BRUNER, R., Biopolymers 4, 131 (1966). 21. MANUELKERN, L., SCHERAGA, H. A., KRIGBAUM, W. B., AND FLORY, P. J., J. Chem. Phys. 20, 1932 (1952). 22. CROTHERS, D. M., AND ZIMY, B., J. Mol. Biol. 12, 525 (1965). 23. SCHUMAKER, V. N., AND SCHSCHMAN, H. K., Biochim. Biophys. Acta 23, 628 (1957). 24. ZADRAiIL, S., PIV~X, L., SPONrlR, J., AND ~ORMOV~, Z., Collect. Czech. Chem. Commun. 30, 3920 (1965). 25. BRUNH, C. F., AND HANAXVALT, P. C., Radiat. Res. 38, 285 (1969). 26. SI”TLOIV, J. K., RANDOLPH, M. L., BOLING, M. E., MATTINGLY, A., PRICE, G., AND GORDON, M. P., Cold Spring Harbor Symp. Qua&. Biol. 28, 209 (1968). 27. FREIFELDER, I)., J. Mol. Biol. 46, 1 (1969). 28. PLAUT, W., NASH, I)., SND FANNING, T., J. Mol. Biol. 16, 85 (1965). 29. HURI<;RMAN, J. A., ASD RIGGS, A. D., J. Mol. BioZ. 32, 327 (1968). 30. OK.~DA, S., Biophys. J. 8, 650 (1968). 31. SUEOKA, N., Proc. *Vat. Acad. Sci. U.S.A. 46, 1480 (1959).