Determination of the Number of Superhelical Turns in Simian Virus 40 DNA by Gel Electrophoresis

Determination of the Number of Superhelical Turns in Simian Virus 40 DNA by GelElectrophoresisAuthor(s): Walter KellerSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 72, No. 12 (Dec., 1975), pp. 4876-4880Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/65307 .

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Proc. Nat. Acad. Sci. USA Vol. 72, No. 12, pp. 4876-4880, December 1975 Biochemistry

Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis

(DNA-relaxing enzyme/ethidium bromide/helix unwinding angle/chromatin structure)

WALTER KELLER

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724

Communicated by Barbara McClintock, September 30, 1975

ABSTRACT Closed-circular, superhelical DNA from simian virus 40 (SV40 DNA I) was treated with an excess of DNA-relaxing enzyme in the presence of increasing amounts of ethidium bromide (EtdBr). After removal of the ethidium, each sample consisted of a group of closed-circular DNA molecules differing in their number of superhelical turns (r) around a mean value of r in a Gaussian-like distribution. The DNA samples were analyzed by electrophoresis in agarose gels under conditions where the electrophoretic mobility was a function of the number of superhelical turns. Since the distributions around r of DNA molecules of different samples overlapped, the difference in the mean number of superhelical turns from sample to sample, Ar, could be determined and used to measure the mean number (1) for native SV40 DNA I. By this criterion, SV40 DNA I contains a Gaussian- like distribution of molecules differing by integral numbers around a mean value of r =-24 i 2 at 37?C [in 0.2 M NaCl, 10 mM Tris-HCI (pH 7.9), and 0.2 mM EDTA]. The heterogeneity in r is probably a consequence of thermal fluctuations in the DNA helix at the time when the last phosphodiester bond is closed in vivo.

When correlated to the buoyant shift of completely relaxed SV40 DNA in a CsCl-propidium diiodide gradient, the number of Ar = 24 i 2 of superhelical versus relaxed DNA implies an unwinding of the DNA helix by 26-28? upon intercalation of one molecule of EtdBr.

The presence of superhelical turns in all naturally occurring closed-circular duplex DNAs has long been an intriguing puzzle. (See ref. 1 for review.) The recent work of Germond et al. (2) provides the first plausible explanation for the exis- tence of superhelices in the DNAs of simian virus 40 (SV40) and polyoma virus. In these particles, DNA is associated with histones that are derived from their host cells (3-7). Such viral DNA-protein complexes have structural features similar to those of cellular chromatin (8, 2). In the electron microscope, they appear as open-circular (relaxed) rings containing approximately twenty protein beads [v-bodies (9) or nucleosomes (10)]. Removal of the protein causes the DNA to assume superhelical configuration (2). After depro- teinization, superhelices occur only in closed-circular DNA and are a consequence of a topological constraint (1), which prevents rotation of the strands in the double helix around the helix axis. Determination of the number of superhelical turns in SV40 DNA may help us to understand the organiza- tion of DNA in chromatin.

To this end, I have prepared a series of DNA samples with various degrees of superhelicity, by reacting SV40 DNA I with an excess of mammalian DNA-relaxing enzyme (11- 15) in the presence of increasing concentrations of the inter- calating dye ethidium bromide (EtdBr). EtdBr reduces the average rotation angle in the double helix (16-19). Removal of the dye forces the enzymatically relaxed DNA to acquire

Abbreviations: SV40, simian virus 40; EtdBr, ethidium bromide; PrpI2, propidium diiodide.

negative superhelical turns whose number is determined by the concentration of EtdBr present during relaxation. This procedure is analogous to the method of closing circular DNA containing at least one broken phosphodiester bond (nicked DNA, form II) by treatment with DNA ligase in the presence of various amounts of EtdBr (20).

SV40 DNA, enzymatically relaxed in the presence of EtdBr, does not migrate as a single homogeneous species during electrophoresis in agarose gels (21). Instead, this DNA is distributed into a group of about 10 closely spaced bands, with the most intense bands located at the middle of the distribution. This phenomenon was initially believed to be a consequence of the presence of EtdBr during relaxation. However, as Depew and Wang (22) and Pulleyblank et al. (23) have demonstrated, Gaussian-type distributions of DNA bands are also observed in circular DNA closed by DNA ligase or by DNA-relaxing enzyme in the absence of EtdBr and are the result of thermal fluctuation in the DNA helix which leads to rotation of the two strands around the strand opposite the nicks. Ligation locks the DNA into a set of molecules differing in their topological winding number, a (1), by small positive as well as negative integral values (22, 23). Molecules differing in their topological winding number will also differ in their number of superhelical turns, T- (see Results and Discussion). The shape of the resulting distribution is determined by the free energy of superhelix formation and has been used to measure this quanti- ty (22, 23). In the experiments described here, I have taken advantage of the distributions of DNA around T to count the number of superhelical turns in native SV40 DNA. The uncertainties of our earlier attempts of measuring this number by gel electrophoretic methods (13) were mainly due to insufficient resolution. This difficulty has now been over- come.

MATERIALS AND METHODS

SV40 virus was propagated in African green monkey cells (CV-1) and purified by standard procedures. Plaque-purified wild-type virus (strain 777), was used at 0.4-1 plaque- forming units per cell. DNA was prepared from virions as described (24). Radioactively labeled SV40 DNA was obtained by growing the virus in the presence of [3lH]thym- idine or [32P]orthophosphate. DNA was stored in 0.01 M Tris-HCl, pH 7.9; 0.2 M NaCl; 0.2 mM EDTA (buffer A) at 0'C or at -70? C. DNA concentrations were determined spectrophotometrically. DNA-relaxing enzyme was purified from human tissue culture cells (KB-3) as previously described (15). For the experiments reported here, an aliquot of fraction 6 (15) was dialyzed into buffer A containing 10% (vol/vol) glycerol and 1 mM dithiothreitol. Ethidium bromide (EtdBr) and propidium diiodide (PrpI2) were pur-

4876



Biochemistry: Keller Proc. Nat. Acad. Sci. USA 72(1975) 4877

chased from Calbiochem. EtdBr concentrations were measured spectrophotometrically at two different wavelengths. At 460 nm the specific absorptivity was taken as 4220 M-1 cm-l and 287 nm as 5.39 X 104 M-1 cm-1 (25).

Treatment of SV40 DNA with DNA-relaxing enzyme was carried out in reaction mixtures of 0.1 ml containing 10 mM Tris-HCI (pH 7.9); 0.2 M NaCl; 0.2 mM Na2-EDTA; 0.05 mM dithiothreitol; 0.5% (vol/vol) glycerol; 0.1 mM (nucleotide) SV40 DNA; approximately 100 units of DNA-relaxing enzyme (15); and EtdBr ranging from 0 to 6.9 X 10-6 M (see Fig. 3). A control mixture was included which did not contain relaxing enzyme and EtdBr. After incubation at 37?C for 30 min, the mixtures were extracted twice with phenol and once with chloroform at 37?C. The aqueous phases were recovered, diluted with H20, and adjusted to 10% (weight/volume) of sucrose and 0.008% (weight/volume) of bromophenol blue in a final volume of 0.2 ml. Aliquots of 20 Al were analyzed by gel electrophoresis.

Gel Electrophoresis. Electrophoresis of DNA in agarose (1.4% weight/volume; Sigma, no. A-6877) was performed in slabs (16 X 12 X 0.3 cm) as previously described (13, 15) ex- cept that the gels were formed between two glass plates. Sample wells were obtained by inserting a toothed plexiglass comb into the top of the agarose immediately after pouring. The electrophoresis buffer was 40 mM Tris-HCI (pH 7.9), 5 mM sodium acetate, and 1 mM Na2-EDTA. When appropri- ate, EtdBr was added to the melted agarose before the gel was cast and to the electrophoresis buffer at concentrations indicated in the legend to Fig. 1, from a 0.02 mg/ml of stock solution (in H20) kept in the dark at 40. The electrophoresis buffer was circulated between the electrode compartments at a rate of 2-5 ml/min. A constant voltage of 4 V/cm was applied for 18 hr. Unless otherwise indicated, electrophoresis was carried out at room temperature. After electrophoresis, the gels were incubated for at least 1 hr in 500 ml of electrophoresis buffer containing 0.5 ,ug/ml of EtdBr. Photographs of fluorescent DNA bands were taken as described earlier (15). Each gel was photographed three times with exposure times of 15 sec, 30 sec, and 60 sec. Negatives were traced with a Joyce-Loebl microdensitometer.

Mixed agarose (0.5% weight/volume)-polyacrylamide (1.9% weight/volume) gels were run in slabs (17 X 18 X 0.3 cm) at 2.5 V/cm for 85 hr in the buffer reported by Ger- mond et al. (2) and analyzed as described above. Additional procedures are detailed in Results and in the legends of Figs. 1-5.

RESULTS AND DISCUSSION The Measurement of Ir by Agarose Gel Electrophoresis.

The topological properties of closed-circular duplex DNA are defined by the relationship a = f + r (Eq. 1; see ref. 1 for definitions). In brief, the topological winding number a of a DNA molecule is determined by the sum of its helical turns f and its superhelical turns r. As shown by Depew and Wang (22) and by Pulleyblank et al. (23), thermal fluctuation in the DNA helix at the time of ring closure causes heterogeneity in the topological winding number a of the resulting closed-circular molecules. After closure, the individual DNA molecules within the distribution around a may still fluctuate in their values for ,B and r, but the sum (a) of these two quantities will always be a constant and integral number. According to Depew and Wang (22), the electrophoretic mobility of closed-circular DNA molecules is determined by the time average of the absolute value of T, because fluctuations around T are fast compared to the electro-

1 2 3 4 5 6 7 8 910 11 1 2 3 4 5 6 7 8 9 10 11 ,,,,,,III II I I, I ,,,, I l,,,I

u b a - w -~~~~~~~~~~~~~~~~~.. ........ .... .. ........................ . ....

... ...... ..

Illlli~~~~~~~~~~~~~~~~~~~~~~~~iiiPii

I* -

.- . ,...,. ~~~~~~~~~~~~~~~~~ ~ ~ ~ ~ i4.O4......

FIG. 1. Agarose gel electrophoresis of SV4O DNA containing increasing numbers of superhelical turns. The DNA samples were prepared as described in Materials and Methods. EtdBr concen trations in the reaction mixtures are indicated in the lower abscis sa of Fig. 3. Sample 11 was untreated SV40 DNA I. Gel b was run in a cold room at 4?C; all other gels were run at room temperature. Gels a and b had no EtdBr. Gels c-f contained the following con centrations of EtdBr: c, 0.012 gg/ml; d, 0.016 rg/ml; e, 0.024 ,ug/ml; f, 0.06 ,ug/ml. Staining and photography of the fluorescent DNA bands were performed as described in Materials and Methods. The slow migrating topmost band present in all samples is nicked- circular SV40 DNA.

phoresis time. Molecules differing in ax have different values of T and will form separate bands during gel electrophoresis

A series of SV4O DNA samples differing in their average number of superhelical turns X was prepared by treatment of the DNA with an excess of DNA-relaxing enzyme in the presence of increasing concentrations of EtdBr and subse quent removal of the dye as described in Materials and Methods. At the time of ring closure, the average value of the topological winding number az will be different in different samples, depending on the amount of EtdBr interca lated into the DNA. Intercalation reduces the number of he lical turns. Since ax = ,B in a relaxed DNA, upon removal of EtdBr after relaxation, the number of helical turns willin crease. This increase must be compensated for by a corresponding number of negative superhelical turns because ae A3 + T (1). As a consequence of thermal fluctuations around az during ring closure, each DNA sample consists of a set of molecules, differing in ax and therefore in T around a mean value (T). After gel electrophoresis, adjacent bands within a given set differ by one superhelical turn (22, 23), as suggest- ed earlier (13). The middle of the distributions of samples that differ in i will be found at different positions in a gel. If the dif.ferences in 7 of samples in neighboring lanes of a gel are not too large, their distributions of bands around T- will



4878 Biochemistry: Keller Proc. Nat. Acad. Sci. USA 72(1975)

AT= 4.5

b3

b4

b5

e Distance Migrated _-

FIG. 2. Densitometer tracings of lanes 3-5 of gel b (Fig. 1). See text for explanation.

overlap. DNA molecules in neighboring samples which have the same electrophoretic mobility are identical in a and therefore in ir. Therefore, the difference in the mean number of superhelical turns AT- in DNA samples from neighboring lanes of an agarose gel can be determined by measuring the distances from center to center of the various band sets in units of r.

The electrophoretic analysis of SV40 DNA molecules containing different mean numbers of superhelical turns is illustrated in Fig. 1. In each of the six panels, sample 1 repre- sents DNA which had been relaxed in the absence of EtdBr; samples 2-10 contained increasing concentrations of EtdBr during enzymatic relaxation; sample 11 was untreated SV40 DNA I. (Sample 1 consisted of molecules with a mean number of T~ = 0, since its pattern upon electrophoresis was indis- tinguishable from the pattern obtained by treating nicked- circular SV40 DNA II with DNA-ligase.) From the pattern of the gel in Fig. la, run under standard electrophoresis conditions (1.4% agarose, room temperature), it is clear that only some of the DNA samples (nos. 5-7) were resolved into their constituent Gaussian band sets. DNA samples whose value of I- was below a certain level (nos. 1 and 2) ran as a single band, together with nicked-circular (form II) DNA, which is the topmost band. Likewise, molecules whose T was above a certain range (nos. 8-11) formed a single fast migrating band during electrophoresis. To resolve the individual DNA species in such samples, two modifications of the standard electrophoresis conditions were employed: (a) lowering the temperature during electrophoresis, and (b) addition of various amounts of EtdBr to the gel matrix and the electrophoresis buffer. Any change in temperature and/or ionic strength will alter the average rotation angle (00) between adjacent base pairs in the DNA helix (26-28). In closed-circular DNA this will cause an alteration in the helix winding number f and lead to a corresponding shift in T

(Eq. 1). Therefore, any change in f, from the reaction conditions during relaxation to the analysis conditions, will shift the mean number of superhelical turns T by some positive or negative value. In Fig. lb, the effect of lowering the temperature during electrophoresis can be seen. Since a decrease in temperature leads to an increase in the average value of the helix rotation angle 00, all samples become more negatively supercoiled (Eq. 1), with a corresponding downfield shift of the DNA bands during electrophoresis (compare, for example, samples 2 and 3 in g3els a and b of Fig. 1). The ef-

Sample number 12345 67 8 911 10

35 -

30

25 ________ j/

,420 X

15 -

l0

5

3 4 5 1

7 Ethidium bromide concentration (juM)

FIG. 3. Plot of the differences in the mean number of superhelical turns (Ai) in DNA samples of Fig. 1 as a function of EtdBr concentration during enzymatic relaxation. The line through the data points was calculated by the least squares method. The arrow indicates the relative position of untreated SV40 DNA I (sample I1).

fect of adding EtdBr to the gels on the electrophoretic mobility of superhelical DNA is shown in gels c-f of Fig. 1. EtdBr unwinds the DNA helix upon intercalation. Thus, the helix winding number f was lowered, causing a corresponding positive shift in the superhelix winding number r of all closed-circular DNA molecules. Therefore, DNA samples with low numbers of negative superhelical turns eventually became positively supercoiled and those containing a higher number of negative turns became less negatively supercoiled.

Complete separation of all DNA samples into their com- ponent band sets could be achieved by combining the results of gel electrophoresis in the presence of four different concentrations of EtdBr (Fig. lc-f). The principle of the method used to measure AT-, between different band sets is shown in Fig. 2. Photographic negatives of the fluorescent DNA bands were traced with a microdensitometer. The middle of each distribution of bands was determined by calculating their average intensity location, with the relative peak heights serving as a measure of DNA mass. Because adjacent peaks within a group are separated by one superhelical turn, the difference between different DNA samples in the mean number of superhelical turns (ATi-) could be counted directly in units of turns. (Fractional values were rounded to the nearest half-turn.) Tracings of all six gels shown in Fig. 1 were used to perform this analysis. When AT- was plotted against the concentration of EtdBr present during relaxation, a straight line was obtained as illustrated in Fig. 3. The highest number of superhelical turns counted was T~ = -34 + 3 (sample 10). By comparison, native SV40 DNA I had a value of T =-24 + 2 (sample 11). The reference state for this value is 370C and buffer A (0.2 M NaCl, 10 mM Tris. HCI, pH 7.9, 0.2 mM EDTA). The error in determining Ai between two DNA samples was estimated to be of the order of +0.5-1.0 turn for each individual comparison. Thus the error in the determination of i for SV40 DNA depends on the number of overlaps that had to be counted from the middle of sample 1 (Fig. 1) to the middle of native SV40 DNA (sample 11, Fig. 1). As indicated by the data points containing error bars in Fig. 3, the number of superhelical turns in SV40 DNA could be determined by counting a min- imum of three overlaps: zr1-jl = Ai1-6 + AI6-8 ? A!T811

(subscript numerals indicate sample numbers in Fig. 1). An



Biochemistry: Keller Proc. Nat. Acad. Sci. USA 72(1975) 4879

lo n

8 - L-4 <8

U 10 20 30 4 <

Fraction number

FIG. 4. Analysis of superhelical and relaxed SV40 DNA by the "buoyant-separation" method. SV40 [32P]DNA (aPPrOXimatelY 1 ,ug, in buffer A) was treated with DNA-relaxing enzyme at 37?C as described in Materials and Methods. A control mixture containing approximately 1 ,ug of SV40 [3H]DNA was incubated without enzyme. After incubation, the mixtures were phenol-extracted, mixed, and diluted to 3.25 ml with 0.01 M Tris-HCl, pH 7.9, 1 mM EDTA. CsCl (3.40 g) and 1.0 ml of PrpI2 (2.5 mg/ml) were added and the solution was centrifuged at 32,000 rpm in a SW50.1 rotor at 20?C for 40 hr. Fractions were collected from the bottom of the centrifuge tube, and the DNA was precipitated with cold 5% tri- chloroacetic acid and collected on Whatman GF/C glass fiber filters. After drying, the filters were analyzed for radioactivity in Li- quifluor. A control gradient was run in parallel, containing a mixture of unreacted SV4O [32P]DNA and SV4O [3H]DNA (not shown). In the control, the form I bands of both DNAs formed pre- cisely superimposable peaks. Both DNA preparations contained an appreciable number of nicked-circular (form II) molecules, which served as marker. 1? = enzymatically relaxed SV4O DNA; I = untreated SV4O DNA; II = nicked-circular SV4O DNA.

additional source of error could have been the preferential binding of EtdBr to DNA molecules containing negative superhelical turns during electrophoresis, resulting in nonlin- ear shifts of T-. This possibility was eliminated by electrophoresis of samples 1-11 in a mixed agarose-polyacrylamide gel (see Materials and Methods) in the absence of EtdBr. The band sets of samples 7-11 were resolved in such a gel and exhibited the same values for ATi as those found in gels c-e of Fig. 1 (results not shown).

Several estimates of the number of superhelical turns in SV4O DNA have been reported earlier. They are all derived from measurements of the critical amount of EtdBr required to remove all supercoils. The number of superhelical turns in SV40 DNA obtained by the "velocity sedimentation-dye titration" method (29) was T = -15 + 3.5 (in 1.0 M NaCI at 200?C). Analysis by "buoyant density-dye titration" gave T = -14.7 i 1.7 (in 5.8 M CsCI at 200?C) [(29), corrected in (30)], whereas the "buoyant-separation" method (31) yielded T = -19 + 1 (in 5.8 M CsGI at 20?C). Several adjust- ments have to be made before these values can be compared to the number 2 =-24 i 2 (in 0.2 M NaCI at 370C) reported here. In contrast to the method described above, the estimates of T derived earlier required a knowledge of the value of the duplex unwinding angle ke of EtdBr. ,be was taken to be -12? (16) for these calculations. However, the 12? value of Xe has recently been challenged (17-19) and there is good evidence (17, 19) that Xe iS -26 i 2?. When corrected for this change, the earlier estimates of T for SV4O DNA become - =-35 determined by "velocity sedimentation-dye titration," T = -32 obtained by "buoyant density- dye titration", and T = -40 from "buoyant separation" measurements. To account for the difference in temperature at which these analyses were carried out (20?0C) and the reference temperature employed here (370C), two to three su-

FIG. 5. Pattern of native SV40 DNA after electrophoresis on an agarose-polyacrylamide gel. Electrophoresis of 0.25 jig of SV40 DNA I and densitometry of the stained gel were performed as described in Materials and Methods. The position of nicked-circular SV40 DNA is designated by "II"; the arrow indicates the average intensity location, calculated with the intensities of bands 1-18.

perhelical turns must be subtracted from the earlier numbers of r (22, 23). The remaining discrepancy of 6 to 14 turns between the older estimates and the number of T presented here can perhaps be accounted for by differences in ionic strength and counter ion (26-28). For example, by ex- trapolating from data obtained with circular phage X DNA (28) and correcting to an unwinding angle of 'ke = -260 for EtdBr, a change from 1.0 M NaCl to 0.2 M NaCl would result in a decrease of the average rotation angle (00) by 0.620 per base pair. For SV40 DNA this amounts to a reduction of T by -0.62.5200/360 =-9 turns. Thus, the value of 7 =

-35 (corrected for 'ke = 26?) obtained by velocity sedimentation in 1.0 M NaCl at 200C would become r = -24 when adjusted to the reference state (0.2 M NaCl and 370C) employed in my experiments. Whether or not the close corre- spondence of this corrected number with the value of X determined here is adventitious remains to be seen. According to sedimentation measurements on X DNA (J. C. Wang, per- sonal communication), the decrease in 00 accompanying the lowering of NaCl concentration from 1 M to 0.2 M is smaller than that reported by Hinton and Bode (28).

The Unwinding Angle of EtdBr. It is possible to deduce the angular change in the DNA helix resulting from the intercalation of EtdBr from the slope of the line in Fig. t3. However, this calculation would require measurements of the binding parameters (e.g., free versus bound dye, maxi- mum molar binding ratio) at each EtdBr concentration. In- stead, I have chosen to correlate the buoyant density difference in a CsCI-PrpI2 gradient of SV40 DNA I compared to completely relaxed DNA (in buffer A at 370 C) with the difference in the number of superhelical turns of these two samples A; = 24 : 2. The distance separation of these DNAs from a marker of nicked-circular SV4O DNA (form II) is shown in Fig. 4. The shift of this distance separation between native (Arl) and relaxed DNA (Arir) in CsCI-PrpI2 gradients is directly related to the difference in the molar binding ratio (per DNA nucleotide) (v) of EtdBr, by the following relationship (1, 31):

^O- I = 0.1[(Ar?/Arj) - 1]/0.67 [21

From the result presented in Fig. 4, vio - vI was found to be Av = 0.03 + 0.003. From this, the unwinding angle (0,e, in degrees) for EtdBr was obtained with the following equa- tion:

= 360;F/2N(vlo - vP) [3]

where N is the number of nucleotide pairs in the DNA. Tak- ing T as -24 i 2 as determined by electrophoresis, and N as 5200 for SV4O DNA, 'ke iS calculated to be -27.7 + 5?.



4880 Biochemistry: Keller Proc. Nat. Acad. Sci. USA 72(1975)

The value of AP on which our estimate of ke is based, obtained by the buoyant-separation method, is probably not very accurate and should be measured by a more sensitive procedure. Nevertheless, the estimate derived here strongly supports the value of ke = -26 i 2? for the unwinding of EtdBr reported by Wang (17, 19).

Heterogeneity of SV40 DNA I. Inspection of the electrophoresis pattern obtained with SV40 DNA I (Fig. 1; sample 11 in gels c, e, and d) reveals that this DNA did not migrate as a single band. Instead, it showed a distribution of bands quite similar to the patterns seen with enzymatically relaxed forms of DNA. That this was not caused by some artifact due to the presence of EtdBr during electrophoresis is demonstrated in Fig. 5. Here, SV40 DNA I was subjected to electrophoresis in the absence of EtdBr in a mixed agarose-polyacrylamide gel (2) as described in Materials and Methods. The DNA was distributed into a large number of bands. Bands 1-12 in Fig. 5 form a symmetrical Gaussian-like distribution of intensities around band 7. In addition, there are also some bands with very low intensities which correspond to DNA with a smaller number of superhelical turns (bands 13-20, Fig. 5). Heterogeneity of SV40 DNA was first reported by Germond et al. (2). We take this heterogeneity in T as evidence that at the time of ring-closure in vivo the DNA is undergoing thermal fluctuations in its topological winding number, a. Fluctuations in histone binding would tend to broaden the thermally induced distribution. This is in fact observed; compare, for example, the number of bands per Gaussian set in enzymatically relaxed DNA (Fig. 2) to the number of bands in SV40 DNA I in Fig. 5.

Chromatin Structure. Chromatin has been shown to con- sist of repeating units, each of which is thought to contain 180 to 200 base pairs of DNA (reviewed in ref. 32). If this is so, the SV40 mini-chromosome would have approximately 26 such repeats. By electron microscopy, Griffith (8) and Germond et al. (2) have counted 21 to 26 nucleosomes in nucleoprotein complexes containing SV40 DNA. There is clearly a conspicuous similarity in the number of repeat units, the number of nucleosomes, and the number of superhelical turns found in SV40 DNA after removal of the protein. Assuming that the pitch of the helix in chromatin remains unchanged from that of the B form of naked DNA, the latter finding could be interpreted to indicate that the DNA helix in such complexes is wound around a histone core as a condensed loop, giving rise to one negative superhelical turn per nucleosome. Whether the condensation is a consequence of bending, 4-folding, or kinking (33) of the DNA helix remains to be demonstrated.

I am grateful to Drs. J. Vinograd and J. C. Wang for sending me their manuscripts prior to publication. I thank Bill Bauer, Mavis Shure, and Jim Wang for discussions concerning the experiment re-

ported in Fig. 4. Ingrid Wendel provided invaluable technical assis- tance. The work was supported by Grant CA 13106 from the Na- tional Cancer Institute.

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