A study of DNA denaturation in the ultracentrifuge · 2008. 1. 5. · BIOPOLY MERS VOL. 15, 1591-1613 (1976) A Study of DNA Denaturation in the U1 t racent rif uge GARY WIESEHAHN,

BIOPOLY MERS VOL. 15, 1591-1613 (1976)

A Study of DNA Denaturation in the U1 t racent rif uge

GARY WIESEHAHN, THOMAS R. CECH,* and JOHN E. HEARST,? Department of Chemistry, University of California, Berkeley,

California 94720

Synopsis

The melting transition of DNA in alkaline CsCl can be followed in the analytical ultracentrifuge. Equilibrium partially denatured states can be observed. These partially denatured DNA bands have bandwidths of up to several times those of native DNA. Less stable molecules melt early and are found a t heavier densities in the melting region.

An idealized ultracentrifuge melting transition is described. The melting transition of singly nicked PM-2 DNA resembles the idealized curve. The DNA profile is a Gaussian band a t all points in the melt. DNA's from mouse, D. melanogaster, M. lysodeikticus, T4, and T7 also show equilibrium bands a t partially denatured densities, some of which are highly asymmetric. Simple sequence satellite DNA shows an all-or-none transition with no equilibrium bands a t partially denatured densities.

The temperature a t which a DNA denatures is an increasing function of the (G + C) content of the DNA. The T, does not show a molecular-weight dependence in the range 1.2 X 106-1.5 X loi daltons (single strand) for mouse, M. lysodeikticus, or T4 DNA. The mouse DNA partially denatured bands do not change shape as a function of molecular weight. The T4 DNA intermediate band develops a late-melting tail a t low molecular weight. M. lysodeikticus DNA bands a t partially denatured densities become broader as the molecular weight is decreased. Mouse DNA is resolved into six Gaussian components a t each point in the melting transition.

MATERIALS AND METHODS

Mouse DNA was extracted from SVT2 mouse tissue culture cells or from combined livers, brains, spleens, and testes of Balb/c mice as described previously.' Mouse main band and satellite were separated preparatively in Agf-Cs2S04 gradients according to Corneo et aL2 DNA from Drosophila melanogaster was isolated from 24-hr embryos essentially as described by Peacock et al.3 D. melanogaster 1.687 satellite DNA was purified by banding total DNA (150 pg/ml) in 1.70 g/cm3 CsCl a t 40,000 r/min for 60 hr in a Beckman 60Ti rotor. The tubes were dripped into 40 fractions, and the OD260 of each fraction was read on a Cary 15 spectrophotometer. Fractions on the light side of the band were pooled and dialyzed into 0.01 M Tris-HC1, 1 mM EDTA, pH 8.0. This light-side DNA (20 yglml) was then rebanded in Cs Formate (1.75 g/cm3) at 42,000 r/min for 60 hr in a fixed

* Present address: Cambridge, Mass. 02138.

Department of Biology, Massachusetts Institute of Technology,

To whom requests for reprint should be addressed. 1591

C 1976 by John Wiley & Sons, Inc.

1592

114-

11.3-

I 1 2-

I1

I a

WIESEHAHN, CECH, AND HEARST

l -

i i i h 5 b f E $ l b Na,HPO,: Na,PO, = 10- X

Fig. 1. Measured pH as a function of buffer acidsalt ratio: ( 0 ) measured in order of increasing pH; (0) measured in order of decreasing pH.

angle 50 rotor. The tubes were dripped and the OD260 of each fraction was taken. The fractions containing purified 1.687 DNA were pooled and dialyzed. The DNA’s of M. lysodeikticus, singly nicked PM-2, and T4 were gifts from P. Wollenzien, L. Liu, and A. Rosenfeld, respectively. PM-2 was nicked according to the procedure of Wang.4 Na2HP04 and Na3P04 (Mallinckrodt), CsCl and Cs Formate (both from Harshaw, optical grade), and Cs2SO4 (Alpha) were used without further purification. Glass-distilled water was used in all solutions.

Measurement of pH

A radiometer pH meter equipped with a GK2321C electrode was used for pH measurements. Stock solutions of 0.5 M Na2HP04 and 0.5 M Na3P04 were prepared. Solutions with Na2HP04:Na3P04 ratios from 102 to 1O:lO were made from the stock solutions. One part of each of these buffers was diluted into 11 parts, 1 mM EDTA, pH 8.5, and the pH was measured and plotted (Fig. 1). The meter was standardized with pH 10.00 buffer before each reading. Diluting into 1.70 g/cm3 CsC1,l mM EDTA, pH 8.5 had negligible effect on the pH reading. Therefore, the pH of each sample was taken to be the pH corresponding to the acidsalt buffer ratio (diluted 12:l) used to make the sample. Reproducibility in pH measurement of a buffer made from the same stock solutions on the same day was f O . O 1 pH unit. Over a period of time (90 days), however, the pH reading of a buffer made from the same stock solutions would drift by as much as 0.05 pH unit.

Density Gradient Centrifugation

A typical sample was made by mixing 460 p1 of saturated CsCl, 50 p1 of phosphate buffer, 34 p1 of H20, 11 p1 of 0.05 M EDTA, and 50 pl of DNA

DNA DENATURATION 1593

in 0.015 M NaC1,0.0015 M Na citrate, 1 mM EDTA (pH 7.6). The DNA solution was added last, and the entire procedure was carried out in an ice bucket to avoid partial denaturation of the DNA. The EDTA was found to be necessary to inhibit degradation of single-stranded DNA. The reference was prepared exactly as the sample with 0.015 M NaC1,0.0015 M Na citrate, 1 mM EDTA substituted for the DNA solution. The refractive index was measured at 25°C on a Zeiss refractometer, and the density calculated from the calibration curve of Ifft et al.5 The sample and reference were immediately loaded into a centrifuge cell with 12-mm double- sector titanium centerpieces. Typically four cells were run in the AN-F rotor with the rotor hole as reference edge. The rotor temperature was controlled with the RTIC unit. The CsCl density was chosen so that the native density of the DNA was in the top half of the cell. All of the runs were at 40,000 r/min. After the initial equilibrium was reached (-40 hr), the temperature was raised by 2°C or less. The new equilibrium position was reached in 12 hr. If the sample was low molecular weight or had moved a large distance in the cell, scans were taken at hourly intervals to assure that equilibrium was reached.

Calculation of Buoyant Density

The difference in CsCl concentration between the root mean square (rms) position (isoconcentration point)5 of the cell and band center was determined using the equation Apo = (w2/p)FAr, where i; is the arithmetic mean distance (cm) of the DNA band and the rms position from the center of rotation, Ar is the distance of the band center (cm) from the center of rotation minus the distance of the rms position from the center of rotation, o is the angular velocity, and p = 1.20 X lo9 cm5/(g-sec2) for CsCl at 25"C.5 No correction was made for the variation of p with temperature. The pressure at band center was calculated using the equation P = (02rlp)-(9.861 X 10-7) where r is the distance from band center to the center of rotation, 1 is the distance from the meniscus to band center, is the arithmetic mean of the density at band center and a t the meniscus, and w is the angular velocity. The factor 9.861 X converts dyn/cm2 into atmospheres. The effect of pressure on the buoyant density of DNA in CsCl is calculated from the equation App = 3.94 X P , where P is in atmospheres.6 The final equation needed to determine the buoyant density is p = po + Apo + A p p where p o is the starting density of the solution. The final densities are lighter than those observed in a pure CsCl density gradient (about 0.01 g/cm3 lighter) because of the presence of 0.1 M Na+ ions in the solution. This procedure is more laborious and tends to give less precise densities than those found using a marker DNA in the same cell. The marker DNA obscures intermediate states, however, so it was rarely used. When a marker DNA was used, densities were calculated according to Schmid and Hearst7 using IIPB = 9.35 X 10-lo (g sec2)/cm5.

1594 WIESEHAHN, CECH, AND HEARST

Molecular Weight Determination

DNA was sheared by forcing the DNA solution through a 22 or 27 gauge needle a t maximum thumb pressure. Smaller DNA was prepared in a decompression bomb. The DNA solution was pressurized with argon to 300 psi, equilibrated for 10 min, and then rapidly decompressed as it exited the bomb through a needle valve.

Single-stranded DNA molecular weights were determined by boundary sedimentation through 0.9 M NaC1,O.l M NaOH and correction to 5 ' 2 0 , ~

according to Studier.8 Alternatively, the DNA was band sedimented through 3 M CsC1, 0.1 M KOH. Correction to S ~ O , ~ and calculation of molecular weights were performed according to the equation S O ~ O , ~ (Cs DNA) = 0.079 M0.397 (Na DNA) (K. Martin, personal communication).

Calculation of Moments

Tracings of buoyant-density profiles were digitized manually by mea- suring the trace height a t l-mm intervals (0.05 mm in real space) on the chart paper. The narrowest distribution was described by 46 points, and the widest required 167 points. The moments of the distributions were calculated from the digitized data using a PDP-8/E computer. No correction was made for hyperchromicity of melted DNA or for radial dilution effect.

Curve Fitting

A nonlinear regression computer program (G2 CAL NLIN) was used to find the values of the parameters ui, pi, and Bi which minimize the sum of the squares

S = z [ Y j - F j ( u , ~ , B ) l ~ where yj are the digitized data points and

This is just a sum of N Gaussians with the standard deviations CT~, means ki, and relative areas proportional to Bi. The program uses a modified Gauss-Newton method to find a least squares fit.g

The value of u was set by finding the best least squares fi t to the mouse satellite peak (at 29'C so that the satellite is well separated from the main band). All of the native components were required to have this u (0.544). A least squares fit was found for the totally native tracings (22O and 24OC). The averages of the relative amounts of each component from these two fits were used as starting points for the 26°C tracing. With the u's and relative amounts fixed, the values of the means were found by the program. The u of any melting component was then allowed to vary. Finally, the relative amounts of the components were allowed to vary. The position


and (r of the small (G + C)-rich main-band component were held fixed in the middle of the melting transition. The program was unable to place this component unambiguously because it represented such a small fraction of the total sample. The (r for the totally denatured DNA was calculated to be 0.9 from the known single-strand molecular weight and from the assumption that all of the guanines and thymines were titrated. Since there could be some broadening due to a small density difference between separated strands, the program was allowed to vary u to find the best fit. The value that minimized the least squares function was 0.939.

RESULTS

In alkaline CsCl the buoyant density of denatured DNA is higher than that of native DNA due to the titration of guanine and thymine. Native DNA, destabilized by the replacement of H+ with Cs+, can be melted at a relatively low temperature. Alkaline titrations of native and denatured DNA have been done previously.1°-12 In each case the titration was done by banding samples of DNA a t different pH's and the same temperature. The double-strand to single-strand transition is such a sensitive function of pH that partially denatured bands are rarely observed.I2 If the starting pH is carefully chosen, however, the entire transition may be observed in an analytical ultracentrifuge operating in the range 20"-35°C.

The experiment is analogous to an optical melting curve. During the buoyant-density melt the shape and position of an equilibrium band are monitored. The position of the mean of the band distribution indicates the fraction of the base pairs which have been titrated, while the shape gives information about the base sequence heterogeneity of the DNA being melted. Since equilibrium must be attained at each point during the melt, an entire melt takes from five to seven days and typically contains 12 points.

An Idealized Melting Curve

Let us assume we have a collection of DNA molecules of identical molecular weight and identical base sequence. This DNA can be banded in alkaline CsCl that has been adjusted to a pH such that double-strand DNA is stable at 20"C, and single-strand DNA is stable at 30°C. Let us further assume tha't the DNA is equally stable at each point in the gradient at any given temperature (i.e., there is no stability gradient in the cell). Finally, let us assume that there is no temperature dependence on the buoyant density of the DNA.

The experiment is begun at 20"C, and an equilibrium band is formed with a Gaussian shape,13 with the variance of the DNA band being inversely proportional to the molecular weight. The temperature is raised, and the band is unchanged. The temperature is raised again, and the DNA begins to denature. The thymine and guanine bases of the melted region are ti-


trated by Cs+ causing the DNA to move to a higher buoyant density. If the DNA base sequence is homogeneous, then every base pair will break and the single strands will move to their entirely denatured positions. The bandwidth of the denatured peak will be larger than that of the native peak due to the halving of the molecular weight. This is an all-or-none transition (Fig. 2).

If there is heterogeneity in the DNA base sequence, only the least stable regions of the helix will denature. The DNA will move to a new equilibrium position, which is intermediate between its native position and its denatured position. This will be a stable equilibrium, and the DNA band will still be a Gaussian with the same variance as before (the molecular weight is the same as before if we neglect the contribution of the titrating Cs+ ions). The melting will continue as the temperature is again raised. Each higher temperature generates a higher buoyant-density DNA band with the same shape as before. When the temperature is high enough, the most stable regions of the molecule are melted, and the strands separate. The band moves to its totally denatured position, and the bandwidth increases due to the halving of the molecular weight (Fig. 3).

In reality none of the above assumptions is correct. The buoyant density of DNA is a function of temperature.14 It will be shown that there is a stability gradient in the cell, and DNA samples are not always made up of identical DNA molecules.

PM-2 DNA

PM-2 DNA, which has been nicked an average of once per molecule and separated from unnicked circles, is an example of a DNA sample with “identical” DNA molecules. Figure 4 shows analytical tracings of PM-2 DNA at different points in the melting region. Each of the partially denatured tracings is a stable, equilibrium tracing. PM-2 DNA, therefore, melts as expected for a DNA with sequence heterogeneity.

The PM-2 DNA can be melted with a marker DNA (M. Zysodeikticus), which is native throughout the PM-2 melt present in the same cell. Since the buoyant density of both the marker DNA and PM-2 DNA will increase as the temperature is raised, the buoyant density of PM-2 DNA relative to marker DNA will show no temperature dependence. Only the density increase due to denaturation will be seen. Figure 5a shows the plot of buoyant density of PM-2 in such an experiment. I t looks like the ideal curve.

Figure 5b is a plot of one-half the bandwidth at 0.606 of the peak maximum as a function of the fraction of single-stranded DNA. Clearly, this does not look like the similar plot in the ideal case. The PM-2 DNA peak broadens, then narrows, and finally broadens again as the DNA denatures. The maximum bandwidth is attained when the DNA is about one-half melted. The minimum is at about 80% melted, and the final bandwidth is 1.48 f 0.15 times the original, native bandwidth.

The explanation for this nonideal behavior is that the assumption of no


+-%--% Temperature ?C)

X

0 20 40 60 80 10 Fraction Single-Stranded

Fig. 2. Ideal case of all-or-none melt. (a) Buoyant density as a function of temperature. Function is discontinuous a t T, (25OC). (b) One-half bandwidth a t 0.606 of peak maximum u as a function of fraction single-stranded. Native D is taken to be 1.

20 25 3c (bi Temperature ('C)


Fig. 3. Ideal case of heterogeneous DNA melt. (a) Buoyant density as a function of temperature. T, is taken to be 25OC. (b) u as a function of fraction single-stranded. Native u is taken to be 1.

stability gradient in the cell is false. In the cell, there is not only a gradient of CsC1, but there are also gradients of pressure and pH. The reason for the pH gradient is that C S ~ P O ~ will redistribute differently than Cs2HP04 in a centrifugal field, causing a different acid:salt ratio a t each point in the


(a) ,, 22"

(C) 24 5'

A d !

248'

25 2"

1692 1748

Fig. 4. Analytical tracings of PM-2 melt. Buffer ratio = 10:6.3. Measured pH = 11.52. Calculated buoyant densities in this figure and in all other figures of analytical tracings are lighter than in pure CsCl gradients because of the presence of Na+ ions.

cell. In addition, the acid dissociation constant of HP04= is pressure dependent. The result is a measurable positive pH gradient.12 Since we cannot separate the effect of the pH gradient from the effect of the salt and pressure gradients,15J6 we will consider the combined stability gradient that they produce.

As the PM-2 molecule begins to melt, it moves to a higher density. Be- cause of the stability gradient, the molecule is less stable at this new position and more base pairs break. The molecule therefore moves to an even higher density. The band will continue to move until the DNA (even a DNA with heterogeneous sequence) is totally denatured unless the density of the solution increases more (as a function of radius) than the DNA increases in density because of melting. In the P04-CsC1-PM-2 DNA system the CsCl density gradient is steep enough to stabilize intermediate states. In these intermediate bands, however, the DNA on the high-density side of the band will have a larger fraction of titrated base pairs than the DNA on the low- density side of the band. This results in the broadening of the interme-



Fig. 5. PM-2 melt. (a) Buoyant density as a function of temperature: (0) buffer ratio = 10:5.4, T, is 28.5O, and the 10-9096 width is 2.2OC; (0 ) buffer ratio = 106.3, actual T, was 24.7"C, each point was increased by 3.8"C so that these data could be compared with the 10:5.4 ratio data. (b) One-half bandwidth (cm f 0.0512) a t 0.606 of peak maximum as a function of fraction single-stranded. Fraction single-stranded was calculated assuming a linear relation between buoyant density and percent single-stranded. Error bars were determined using repeated measurements of M. lysodeikticus marker DNA present in one of the PM-2 DNA sample cells. u of native PM-2 DNA was found to give a molecular weight of 6 X lo6 from PM-2 DNA using a CsCl effective gradient G = 9.0591 X (0) buffer ratio of 10:5.4; (0 ) buffer ratio of 10:6.3.

diate-state band. At the one-half denatured point, the DNA has the maximum possible number of partially denatured conformations. As the PM-2 DNA is further melted, the DNA strands are held together only by a few (G + C)-rich regions. The bandwidth decreases. When these last stable regions are melted, the bandwidth increases again due to the halving of the molecular weight.

The stability gradient, then, causes the increased variance of the partially denatured DNA peak. In other words, the partially denatured DNA sees a resultant gradient that is the effective gradient17 reduced by the stability gradient. When the DNA is totally native, the resultant gradient is equal to the effective gradient. The DNA is not affected by the stability gradient when all of the DNA molecules are totally native. A t the one-half denaturation point, the resultant gradient is (1/1.7)2 or about one-third of the effective gradient. Here, when the DNA is most affected, the stability gradient is two-thirds as large as the effective gradient. When the DNA strands are held together only by a few (G + C)-rich nuclei, they are less affected by the stability gradient and the resultant gradient increases again. After the strands separate, the stability gradient again has no effect on the


r"" i I

70 t .a iL:T,".

Temperature CC)

Fig. 6. Optical melt of singly nicked PM-2 DNA in alkaline CsCl. Buffer ratio is 10:5.4. The T, is 3OoC and the 1690% width is 3.1OC.

Fig. 7. Analytical tracings of mouse satellite DNA melt. Buffer ratio = 105.4.


(f1 340

(g 30'

1.675 1.743

Fig. 8. Analytical tracings of D. melanogaster 1.687 satellite DNA melt. Buffer ratio = 10:3.6.

DNA and the resultant gradient is again equal to the effective gradient. The final increase in the variance of the DNA peak is not due to the stability gradient, but merely reflects the halving of the DNA molecular weight.

Figure 6 shows an optical melting curve of PM-2 in alkaline CsC1. The optical melting transition has a width of 3.1"C from the point a t which 1Wo of the DNA is denatured to the point a t which 90% of the DNA is denatured. The 10-90% width of the PM-2 melt done in the centrifuge is 2.2OC. This is consistent with the idea of a stability gradient in the centrifuge causing a sharpening of the melting transition. Unsheared T7 DNA has a melting behavior in alkaline CsCl gradients similar to PM-2 DNA.

Mouse Satellite DNA

Mouse satellite DNA is an example of a relatively homogeneous base sequence DNA.lsJg It melts with an all-or-none transition in the alkaline CsCl density gradient. When the normal melting curve procedure of 2°C steps in temperature is applied to mouse satellite DNA, the DNA goes from an entirely native density to an entirely denatured density in one step. If the melting curve is done in 0.5"C steps, part of the DNA remains native


1.701 I 7 5 0

Fig. 9. Analytical tracings of total D. mehogaster DNA melt. Buffer ratio = 10:4.8. At the first temperature of this sequence (23OC) all the low-density satellites have already melted.

while the majority of the DNA shows an all-or-none transition. Figure 7 shows the stages in a melt of mouse satellite.

The small peak that remains at native satellite density (Fig. 7d) is thought to be a stable fraction of mouse satellite DNA because its density is the same as the native density of mouse satellite and nearly all of it melts with an all-or-none transition when the temperature is raised by an additional 0.5OC (Fig. 7f). In a separate experiment, more than two-thirds of the stable material was seen to melt with a rise in temperature of only 0.1oc.

The peak that appears between the separated strands of mouse satellite (Fig. 7d) returns to a density of 1.697 when the temperature is lowered to 26°C (Fig. 7e). The area of this peak corresponds well with the area of the main-band density DNA that is present in the sample (Fig. 7b, 7c). This peak does not appear to be merely bulk main-band mouse DNA, however. It shifts to a density of 1.739 with a temperature increase of only 0.5"C. This is 72.5% of the density increase expected for complete titration, and it occurs in an all-or-none fashion. The small peak stays between the separated mouse satellite DNA strands for at least l0C, and then at 29.5OC


the peak disappears under the heavy strand of mouse satellite. This DNA acts like a covalent linkage of 70% mouse satellite with 30% of a relatively (G + C)-rich region. This peak has been seen in every melt (four times) of isolated mouse satellite DNA. It is about 5% of the DNA in the isolated mouse satellite DNA.

Drosophila 1.687 Satellite DNA

Satellite DNA of buoyant-density 1.687 isolated from D. melanogaster shows two all-or-none transitions (Fig. 8). The first results in two separate denatured peaks at densities of 1.701 g/cm3 and 1.762 g/cm3 (measured native density under these conditions is 1.679 g/cm3). The second transition gives two denatured peaks at densities of 1.734 g/cm3 and 1.742 g/cm3. The difference in T, between the two components is 4" f 2°C. This is in agreement with the work of Peacock et ale3 who separated the 1.687 g/cm3 satellite into two components with native CsCl densities of 1.686 g/cm3 and 1.688 g/cm3, and it agrees with Gall et a1.20 who showed that isolated 1.687 g/cm3 DNA melts with a biphasic transition, with the two components melting 5.7"C apart.

Figure 8 shows the melt of isolated 1.687 g/cm3 satellite. There is no obvious evidence of heterogeneity in either of these components. Neither is there any evidence of (G + C)-rich DNA covalently attached to satellite DNA. This is not surprising because these satellites are known to occur in very long ~ t r e t c h e s . ~ Any molecules consisting of (G + C)-rich regions covalently linked to satellites would also tend to be excluded because the 1.687 g/cm3 satellite was isolated by repeated neutral buoyant-density gradients without antibiotics or heavy metals. The melt shown in Figure 8 is done in 2°C steps, and it is conceivable that a melt done in smaller steps would reveal heterogeneity in one or both of the components.

Drosophila melanogaster DNA

Figure 9 is a melt of total DNA from D. melanogaster. This is a DNA sample that includes a broad distribution of molecular weights and base sequences. The bulk main-band DNA still shows the basic features of the PM-2 DNA melt. The bandwidth first increases, then decreases, and finally increases as the DNA denatures.

Micrococc us lysodeiktic us DNA

M. lysodeikticus DNA was melted a t four different molecular weights. As shown in Table I there is no obvious molecular-weight dependence on the melting temperature of M. lysodeikticus DNA in the single-strand molecular-weight range of 1 X lo6 to 10 X lo6. In these alkaline CsCl melting experiments, a change in the measured pH of a sample of 0.1 pH unit has been found to change the melting temperature of the DNA by


TABLE I The Melting Temperatures of Four Different Molecular Weights

of M. lysodeikticus, Mouse, and T4 DNA

Single-strand molecular Transition

weight Trn width (M,X 10-6) ("C) ("C)

M. lysodeikticus, 10.3 32.8 1.6 t 0.3 71% (G+C) 4.2 32.5 1.6 f 0.3

3.8 32.1 1.8 f 0.3 1 . 2 33.3 1 .9 * 0.3

Mouse, 40% ( G + C ) 15 28.6 3.3 * 0.3 5.2 28.75 3.2 t 0.3 4.9 28.8 3.0 * 0.3 1.9 28.6 3.5 * 0.3

4.7 32.5 f 1 < 2 3.3 33.25 t 0.25 2.2 f 0.3

T4,34% ( G + C ) 30.3 31.9 f 1 <2

about 6.5"C (obtained by melting the same DNA at slightly different pH's and observing the change in Tm). For this reason and because the melt is done in 2OC steps, the observed differences in the melting temperature data are not significant.

Figure 10 shows the stages in the melt of M. lysodeikticus DNA at a molecular weight of 10.2 X lo6 (single strand). The bandwidth shows the familiar characteristics of broadening, then narrowing, and finally broadening again as strand separation occurs. Figure 11 shows the melt of M. Zysodeikticus DNA at a molecular weight of 1.2 X 106 (single strand). The intermediate states have larger bandwidths than at the higher molecular weight.

The standard deviations of each of the density-gradient profiles of M . lysodeikticus DNA at each of four molecular weights have been calculated. These are plotted in Figure 12b as a function of the fraction of the DNA which is titrated. The standard deviation of the native peak CN increases as the molecular weight decreases. The standard deviation of the one-half titrated band al /2 also increases, and it increases roughly in proportion to the increase in the native standard deviation. The ratio of a l /2 to CTN is about 3.1, which is larger than the a112 to UN of 1.7 found for PM-2 DNA. This higher ratio is to be expected since M. lysodeikticus D N A is not a collection of identical molecules.

Mouse DNA

Figure 13 shows the melt of total mouse DNA. The first stage in the denaturation is the sharpening of the main band (Fig. 13b, 13c). Next, a shoulder is developed on the heavy-density side of the main band. Near


32 75" (e)

_ _ _ _ _ _ - - .- ---- i \L I 7 2 1 I 1 0 5

Fig. 10. Analytical tracings of M. lysodeikticus DNA melt. The single-strand molecular weight is 10.2 X lo6. Buffer ratio = 105.8.

the midpoint of the titration, the main band becomes bimodal. The decreased relative amount of satellite suggests that part of the satellite has denatured. The small peak on the heavy side of the main band is a t a density of 1.740 g/cm3. This density is consistent with its being the heavy strand of denatured mouse satellite (Fig. 13e). As the denaturation pro- ceeds, the main band becomes one peak with a shoulder on the light side. The last stage is the broadening of the main band.

Table I shows that there is no detectable molecular-weight dependence to the melt of mouse DNA. Figure 14b shows that there is also no detectable increase in the standard deviation of the one-half denatured states as the molecular weight is decreased. In addition, the shape of the partially denatured bands is relatively constant as a function of molecular weight.

The standard deviation of a DNA band in a neutral density gradient is dependent on both the molecular weight of the DNA and the density heterogeneity of different DNA molecules in the band. The standard devia-


1 (el 32.75'

. _. . _.

33.75' (9)

. . .. __ - A

1.721 1.784

Fig. 11. Analytical tracings of M. lysodeikticus DNA melt. The single-strand molecular weight is 1.2 X lo6. Buffer ratio = 105.8.

tion of a band of partially denatured DNA is dependent on the molecular weight of the DNA and the melting heterogeneity of different DNA molecules in the band. If the standard deviation of a band of partially denatured DNA fails to increase as the DNA molecular weight is decreased, it means that the melting heterogeneity is so large that it is determining the bandwidth. The melting heterogeneity is not large enough in M. Zysodeikticus DNA to obscure the molecular-weight dependence of the partially denatured bandwidth, but in mouse DNA it is. This large melting heterogeneity and the unique shape of the mouse DNA partially denatured bands suggests an analysis of mouse DNA by resolving the DNA band into several components. Thi&ry21 has resolved native mouse DNA into five major and three minor components on the basis of fractionation in preparative Ag+- Cs2SO4 gradients. He also found that analytical ultracentrifuge mouse DNA CsCl bands can be resolved into five major and one minor compo-


25 30 35 Temperature ("C)

0 2 0 P

c v)

10

5

0 2 4 6 8 1 0 Fraction Single- Stranded

Fig. 12. M. lysodeikticus DNA melt. (a) Buoyant density of mean band position as a function of temperature. The single-strand molecular weight is 10.3 X lo6. (b) Standard deviation of M. lysodeikticus DNA as a function of fraction single-stranded: (0) 10.3 X lo6; (X) 4.2 X lo6; (0) 3.8 X lo6; (0 ) 1.15 X lo6 (all single-strand molecular weights). Units of standard deviations are cm + 0.0512.

nents. (We define a minor component as one with composition below 5%.) The buoyant densities and amounts of the major components are similar for the two techniques. We have analyzed the partially denatured bands of mouse DNA in order to determine if their shapes could be explained on the basis of a small number of components.

The mouse DNA band is assumed to consist of several components, each having the shape of a Gaussian curve. For native DNA, the standard deviations of all components are assumed to be identical, and the standard deviation is determined by the molecular weight of the DNA. Attempts were made to fit the native mouse DNA bands starting with one Gaussian component. Additional components were added until the average rms deviation approached what was considered to be the experimental error associated with the data (deviation of about 1% of the peak maximum). The number of Gaussian components found to be necessary is six, one over the satellite peak and five in the main band. These components are not


1.688 1.755

Fig. 13. Analytical tracings of the melt of total mouse DNA. The single-strand molecular weight is 15 X lo6. Buffer ratio = 104.8. The sensitivity of the tracings was sometimes changed to keep the peaks on scale.

identical to those of Thi6ry in buoyant density or amount. The results are, however, in qualitative agreement.

Can the same components used in the native band be used to explain the partially denatured profiles? Figure 15 shows the experimental and calculated data for a six-component fit to an alkaline CsCl melt of mouse DNA. The main-band components melt in order of increasing (G -t C) contents. The bandwidth of each component goes through a maximum a t about its one-half denatured density. The ratio of al/z to a~ for each main-band component is about 2.5. The densities, standard deviations, and amounts

DNA DENATURATION

(a 1 1.760 1.750

/

1609

.- 2 1.730 1.720 1.710 1.700

s 1.690 m 1.680

c3 c

22 24 26 28 30 3 2 34 Temperature ("C)

28 ),fi od\ O

16 14 Q 12 0

10 a2

0 2 4 6 8 1 0 Fraction Single- Stranded

Fig. 14. Mouse DNA melt. (a) Buoyant density of mean band position as a function of temperature. The single-strand molecular weight is 15 X lo6. (b) Standard deviation of mouse DNA as a function of fraction single-stranded: (0) 15 X lo6; ( 0 ) 5.2 X lo6; (X) 4.9 X lo6; (0) 1.9 X lo6 (all are single-strand molecular weights). Units of standard deviations are cm + 0.0512.

of the components at each stage in the melt are shown in Table 11. The calculated data seem to be in reasonable agreement with experiment. The least satisfactory fit occurs when the DNA becomes totally denatured. The most (G + C)-rich main-band component (6) is required by the computer to be on the light side of the 34°C tracing (Fig. 15i). It is possible that this component is not yet totally melted.

An analysis of mouse DNA with single-strand molecular weight of 4.9 X lo6 gives similar results. It should be pointed out that better fits to some of the partially denatured bands could be obtained by varying the relative amounts andlor standard deviations of some of the components. The amount of improvement was typically small and so the somewhat poorer fit with more appropriate parameters was preferred.

T4 DNA

As shown in Table I, T4 DNA also shows no detectable molecular-weight dependence on its T,. The high-molecular-weight sample went from totally native to totally denatured in the span of one 2°C jump. The inter-


0) 22.

I 2 3 4 5 6 2 4'

29. I 34 5 6 2 1)

I 5 4 6 3 2 9) 30.

I 349

1.755 1.688

Fig. 15. Tracings of component melt of mouse DNA (1.9 X 106 Mr) . Vertical tic marks denote the digitized data from analytical ultracentrifuge tracings. Gaussian components determined by least squares analysis are labeled 1-6. 1 is mouse satellite. 2-6 are main-band Gaussian components in order of increasing native density. The line through the vertical tics is the sum of the Gaussian components.


TABLE I1 Densities, Standard Deviations, and Fraction of Total DNA

During the Melting of Six Different Components of Mouse DNA

22°C 24°C 26°C 27°C 28°C 29°C 30°C 31°C 34°C

1) 0

P Fraction of total DNA

2) 0 P

Fraction of total DNA

3) 0 P


4) 0 P


5 ) 0 P


6) 0 P


Rms deviation

Sum of squares

(mm)

(mm’)

0.544a 0.544a

1.674 1.677

0.092 0.084

0.544a 0.544a 1.681 1.681 0.184 0.133

0.544a 0.544a 1.686 1.686 0.282 0.303

0.544a 0.544a 1.690 1.690 0.267 0.272

0.544a 0.5448 1.695 1.695 0.141 0.158

0.544s 0.5448 1.701 1.700 0.034 0.053

0.221 0.226

3.53 3.59

0.544a

1.682

0.088a

0.654 1.686 o . i w

0.544a 1.690 0.293a

0.5442 1.693 0.268s

0.544a 1.698 0.152a

0.544a 1.707 0.044a

0.299

6.08

0.5448

1.680

0.088a

0.916 1.691 0.151a

0.598a 1.691 0.295a

0.5448 1.696 0.270a

0.544a 1.701 0.153a

0.544a 1.707 0.044a

0.322

8.17

0.544a

1.681

0.083

1.28 1.713 0.165

0.962 1.699 0.287

0.584 1.699 0.262

0.544a 1.704 0.156

0.544a 1.7108 0.047a

0.318

10.14

0.544a 0.931a

0.941 0.931a 1.734 1.741 0.162 0.159a

1.48 0.9318 1.722 1.741 0.328 0.274a

1.13 1.22 1.707 1.728 0.228 0.274

0.592 1.26 1.705 1.713 0.160 0.151a

0.544a 0.691 1.711a 1.710 0 . 0 4 9 0.044a

0.343 0.194

15.33 4.27

0.931a 0.939b 1.7302 1.737 1.759a 1.768 0.088a 0.088a

0.931a 0.939b 1.745 1.746 0 . 1 ~ 0 . 1 5 ~

0.931a 0.939b 1.748 1.753 0.294a 0.294a

0.931a 0.939b 1.748 1.757 0.268a 0 269a

1.25 0.939’’ 1.732 1.762 0.150a 0.150a

1.05 0.939b 1.718 1.731 0.044a 0.0448

0.364 0.280

14.58 8.07

a Parameters that were not allowed to be varied by the computer program. b All u’s were assumed to be equal for the 34°C data. The computer program

chose u to give the best least squares fit to the data.

mediate-molecular-weight sample also went from totally native to largely denatured in one 2°C jump. The denatured peak had a partially melted light density tail, however. When the temperature was lowered, the bulk of the DNA stayed at a totally denatured density, while part of the DNA (-30%) returned to a native density. The lowest molecular-weight sample showed an early melting fraction as well as a late melting fraction. When the temperature was lowered during the early part of its denaturation, the bulk of the DNA returned to native density while a small part (-5%) was totally denatured (data not shown). Thus phage DNA can be fractionated by shearing to low molecular weight and separating the early and late melting fractions in alkaline CsC1.

T, as a Function of (G + C) Content

Figure 16 shows a plot of the pH required to melt the DNA samples studied here a t 25°C in alkaline CsCl versus the melting temperature in Cit/NaCl for these same DNA samples. The pH’s are those corresponding


pH I T

1 1 I I

00 d5 90 95 I00

T,,, in S S C (Oc)

Fig. 16. pH needed to melt various DNA's a t 25OC in alkaline CsCl as a function of T, in Cit/NaCl (0.15 M NaCl, 0.015 M sodium citrate): 1-D. melanogaster 1.686 DNA; 2--0. melanogaster 1.688 DNA; 3-T4 DNA; 4--0. melanogaster total DNA; 5-mouse satellite DNA, 6-PM-2 DNA; 7-mouse total DNA &A DNA; 9-T7 DNA; 10-M. lysodeikticus DNA.

to the buffer ratios in Figure 1. Melting experiments with T, at temperatures other than 25OC were corrected to 25OC using the conversion factor of 0.1 pH unit per 6.5"C change in melting temperature. The slope of the line in Figure 16 is 0.01 pH unitPC.

DISCUSSION

DNA melting heterogeneity can be observed in the centrifuge. This leads to the hope of preparative fractionation of DNA on the basis of melting behavior.22 The stable fraction of mouse satellite DNA indicates a longer order heterogeneity than has previously been seen in this DNA. This long-range heterogeneity (2 X lo7 daltons double strand) suggests the possibility that satellites may vary from chromosome to chromosome in the mouse karyotype.

The heterogeneity in mouse main-band DNA is similarly long range. There is no obvious change in the melting heterogeneity of mouse DNA in the molecular-weight range studied here. Even though the mouse DNA melt can be treated formally as the melt of six components, these components obviously do not consist of identical molecules (PM-2 DNA type). Their increase in standard deviation at one-half denaturation is clearly greater than that of PM-2 DNA.

The authors thank D. Wandres for help with data analysis, Dr. I. Tinoco for the use of the PDP-8B computer, and Dr. D. Appleby for helpful discussions. They also thank Mrs. M. Malone, Mrs. E. Rotondo, Mrs. D. Miner, and Mr. M. Tuttle for technical assistance.


This work was supported by United States Public Health Service Grants GM-11180 and GM-15661. T.C. was supported by a National Science Foundation Graduate Fellowship.

References

1. Cech, T. R., Rosenfeld, A. & Hearst, J. E. (1973) J. Mol. Biol. 81,299-325. 2. Corneo, G., Ginelli, E., Soave, C. & Bernardi, G. (1968) Biochemistry 7,4373-4379. 3. Peacock, W. J., Brutlag, D., Goldring, E., Appels, R., Hinton, C. W. & Lindsley, D. L.

4. Wang, J. C. (1971) in Procedures in Nucleic Acid Research, Cantoni, G. L. & Davies,

5. Ifft, J . B., Voet, D. & Vinograd, J. (1961) J. Phys. Chem. 65,1138-1145. 6. Hearst, J. E., Ifft, J. B. & Vinograd, J. (1961). Proc. Nat. Acad. Sci. U S . 47,1015-1025. 7. Schmid, C. W. & Hearst, J. E. (1971) Biopolymers 10,1901-1924. 8. Studier, F. W. (1965) J. Mol. Biol. 11,373-390. 9. Hartley, H. 0. (1961) Technometrics 3,269-280.

(1973) Cold Spring Harbor Symp. Quantitative Biol. 38,405-416.

D. R., Eds., Harper and Row, San Francisco, Vol. 2, pp. 407-416.

10. Baldwin, R. L. & Shooter, E. M. (1963) J. Mol. Biol. 7,511-526. 11. Vinograd, J., Morris, J., Davidson, N. & Dove, Jr., W. F. (1963) Proc. Nat. Acad. Sci.

12. Wang, J. C. (1974) J. Mol. Biol. 89,783-801. 13. Meselson, M., Stahl, F. W. & Vinograd, J. (1957) Proc. Nat. Acad. Sci. U S . 43,581-588. 14. Vinograd, J. & Greenwald, R. (1965) Biopolymers 3,109-114. 15. Hawley, S. A. & Macleod, R. M. (1974) Biopolymers 13,1417-1426. 16. Chapman, Jr., R. E. & Sturtevant, J. M. (1970) Biopolymers 9,445-457. 17. Hearst, J . E. & Vinograd, J. (1961) Proc. Nat. Acad. Sci. U S . 47,999-1004. 18. Southern, E. M. (1975) J. Mol. Biol. 94,51-69. 19. Biro, P. A., Carr-Brown, A., Southern, E. M., & Walker, P. M. B. (1975) J . Mol. Biol.

20. Gall, J . G., Cohen, E. H. & Polan, M. L. (1971) Chromosoma 33,319-344. 21. ThiBry, J.-P. (1974) These de Doctorat d’Etat, UniversitB Paris VII. 22. Cech, T. R., Wiesehahn, G. & Hearst, J. E. (1976) Biochemistry 15,1865-1873.

U.S. 49,12-17.

94,71-86.

Received October 30,1975 Accepted March 3,1976

A study of DNA denaturation in the ultracentrifuge · 2008. 1. 5. · BIOPOLY MERS VOL. 15, 1591-1613 (1976) A Study of DNA Denaturation in the U1 t racent rif uge GARY WIESEHAHN,

Documents