Inter-strand crosslinking of DNA by nitrogen mustard

J. Mol. Biol. (1966) 19, 266-288

Inter-strand Crosslinking of DNA by Nitrogen Mustard KURT

W.

KOHN, CARLOS

L.

SPEARS .AND PAUL DOTY

Laboratory of Chemical Pharmacology, National Cancer Institute National Institutes of Health, Bethesda, Maryland and Department of Chemistry Harvard University, Cambridge, Massachusetts, U.S.A. (Received 7 March 1966, and in revised form 4 May 1966) The effect of reaction with nitrogen mustard on the denaturability of DNA was studied in order to clarify the nature of the effect that has been attributed to inter-strand crosslinking. Reaction of bacterial DNA with concentrations of nitrogen mustard as low as 5 X 10- 7 M, corresponding to alkylation of approximately 0'005% of the bases, converted an appreciable fraction of the DNA to a form that did not denature after exposure to dilute sodium hydroxide or to formamide, The hydrogen. bonded structure of the DNA bihelix is broken down by such exposures, but the altered fraction of the DNA reverts to the bihelieal form after removal of the denaturant. (Unreacted DNA remains almost completely denatured after these exposures.) Several physical properties of this recovered bihelical DNA were measured and were found to be similar to those of the original DNA. Measure. menta of the fraction of the DNA rendered resistant to denaturation were performed by CsCl density-gradient equilibrium ultracentrifugation. Studies with density-hybrid DNA showed that the denaturation resistance produced by nitrogen mustard treatment correlates with an inhibition of strand separation. When DNA was subjected to a mild HN2t treatment and then exposed to alkali at various temperatures, the fraction of the DNA denatured was found to be constant over a wide temperature range. This indicates that the denaturation resistance is mediated by covalent bonds. Deneturation-reaistent fractions were produced by the reaction of DNA with the bifunctional nitrogen mustard, HN2, and with bifunctional epoxides. Mono. functional analogues of these agents did not produce this effect. The requirement for sequential reactions of two functional groups was associated with a time-lag in the appearance of denaturation-resistant molecules during reaction with HN2. The formation of denaturation-resistant DNA was initially first-order with respect to HN2 concentration. This indicates that a denaturation-resistant DNA molecule can be produced by the reaction of a single molecule of HN2. The results indicate that nitrogen mustard generates covalent crosslinks between the paired strands of bihelical DNA, and that a single such crosslink per DNA molecule prevents denaturation. Studies with 14C-labeled HN2 showed that only a small fraction, about 4%, of the DNA-bound mustard molecules became effective crosslinks, under the reaction conditions employed.

1. Introduction The possibility that biological alkylating agents might act by forming crosslinks between two sites was first suggested by Goldacre, Loveless & Ross (1949). Their suggestion was based on the observations that the potent tumor inhibition and chromosome breakage effects of alkylating agents occur only with compounds having

t

Abbreviation used: HN2, nitrogen mustard.

266

INTER-STRAND CROSSLINKING OF DNA

267

a.t least two functional groups (Haddow, Kon & Ross, 1948; Loveless, 1951). Alexander & Lett (1960) presented evidence that bifunctional alkylating agents produce crosslinks betwe en different DNA molecules; specifically, treatment of cells or sperm heads with bifunctional nitrogen mustards was found to convert part of the DNA to an insoluble gel. The formation of inter-strand crosslinks within DNA molecules, however, was first suggested by Geiduschek (1961) who discovered that nitrogen mustard-treated DNA does not denature normally. Brookes & Lawley (1961) have isolated from nitrogen mustard-treated DNA a. diguanyl derivative of nitrogen mustard which may be the chemical basis for inter-strand crosslinking. This report concerns observations supporting the picture that bifunctional alkylating agents can form covalent crosslinks between paired DNA strands, that these crosslinks prevent strand separation, and that a single crosslink per DNA molecule can prevent irreversible denaturation]'. A preliminary report of this work has been presented elsewhere (Kohn, Green & Doty, 1963).

2. Materials and Methods (a) Alkylating agentB

N-methyl-bis(2-chloroethyl)amine'HCl (HN2) was donated by Merck, Sharp & Dohme R esearch Laboratories. N,N-dimethyl.2.chloroethylamine·HCl (HNl) was purchased from Ma theson, Coleman & Bell, Inc. and recrystallized from acetone. 1.(2,3-allyloxy.)-2,3. epoxypropane (NSC 631), bis(2,3.epoxypropyl)ether (NSC 54739), and butane-l:2,3 :4. di epoxide (NSC 629) were obtained from the Cancer Chemotherapy National Service Center. N .methyl.bis(2.chloroethyV 4C)amine·HCI, 1· 65 X 10 6 disintegrations/mintpIIlole, was obtained from the Merck Co., Canada, To prepare samples of known composition, this material was dissolved in acetone to a concentration of 0'01 Y. Portions of the solution were evaporated in vials in vacuo and stored at -15°C. (b) DNA isolation

Bacterial DNA was isolated by lysis of the cells with sodium lauryl sulfate, deproteinization with chloroform-isoamyl alcohol or with phenol, dig estion with ribonuclease, and isopropanol precipitation, following procedures similar to those described by Marrnur (1961) and Saito & Miure (1963). (c) Absorbance-temperature profiles A Beckman DU spectrophotometer was equipped with thermal spacers connected to a temperature-controlled circulating bath. DNA solutions of approximately 20 p.g/rnl. concentration were measured in Teflon-stoppered quartz cuvettes, At least 10 min was allowed for equilibration at each temperature. (d) Viscosity

A four-bulb capillary viscometer (Eigner, 1960) was used to measure viscosity at several rates of shear at 24'70 ± 0·005°C. (e) Density.gradient equilibrium centrifugation

Samples were centrifuged in 12.mm analytical cells in the Be ckman Model E ultra. centrifuge, usually at 44,770 rev./min, at 25°C for at least 18 hr (Meselson & Stahl, 1958). The samples were trans-illuminated by a filtered low-pressure mercury-vapor lamp (254 mil) and photographed on Kodak Commercial film. The films were traced with a Joyce-Loebl microdensitometer.

t TefTTIinology. The terms "denaturation" and "renaturation" have been applied to DNA in different ways by different authors. In an attempt to resolve the ambigu ity, we ha.ve outlined the problem and selected specifio definitions (see Appendix).

268

K. W. KOHN, C. L. SPEARS AND P. DOTY

(f) Determination of the relative amounts of helical and denatured DNA A solution containing approximately 3 p.g of DNA was mixed with solid CsCI (99'7%, optical grade, obtained from the Maywood Chemical Co. and from the Harshaw Chemical Co.). The CsCl concentrations were adjusted by measurements of refraction to produce a density of 1·71 g/cm3 (Ifft, Voet & Vinograd, 1961). The solutions were buffered at pH 8 with 0·02 :M-Tris-HCI or at pH 10·3 to 10·7 with 0·2 M-sodium carbonate. The higher pH was useful for increasing the separation between helical and den9tured bands (Vinograd, Morris, Davidson & Dove, 1963). Linearity of film response was checked by comparing tracings from photographs of different exposure times. The areas of the DNA bands were measured from the tracings by means of a polar planimeter. The quantitative measurement of the fraction of DNA denatured was hampered in the early parts of this work by a disproportionate loss of denatured (compared with helical) DNA upon banding in CsCI. This loss was more marked at lower DNA concentrations, suggesting that an adsorption effect might be responsible. It was found that a small amount of sodium lauryl sulfate, added to the CsCl solution, tends to reduce this loss (Table 1). (Sodium lauryl sulfate does not affect the absorbance of DNA.) The hyperchromicity of denatured DNA in CsCI was found to be in the range 5 to 15%, depending on pH and type of DNA. This does not substantially affect the results and tends to oppose any error due to selective adsorption of denatured DNA. Hence, no hyperchromicity corrections were applied. (g) Buoyant den8itie8 Buoyant density differences were calculated from the equation: £02 P2 - Pl = 2fl (r~ - '1).

For CsCI solutions of density 1'7 g/cm3 , centrifuged at 44,770 rev./min, 25°C, the value, £02/2fl = 0·01006 g/cm 5 , was used. This value takes into account compressibility effects (Hearst,Ifft & Vinograd, 1961) and is approximately 9% greater than scales which ignore these effects. The exact value used is not critical in the present work, however, since we will be concerned mainly with relative differences. (h) Binding of [uO]nitrogen mustard to DNA DNA was precipitated from [14C]HN2 reaction mixtures with 2 vol, of 95% ethanol. (The reaction mixtures were adjusted to contain 0·15 M-NaCI-0'015 M-sodium citrate (pH 7).) The precipitated DNA was sedimentated at 14,000 g for 10 min, and the super. natant fraction was decanted. The DNA was dispersed in 1 msr-sodium EDTA, and the procedure was repeated for a total of four ethanol precipitations. The final DNA recovery was approximately 75%. The concentration of the DNA in the final solution (in 1 ml. of 1 rmr-sodium EDTA) was determined by optical density, using a value of E~~ = 200. The sample was then transferred to a glass bottle of the type used in the Packard scinbillation counter. One ml, of 1 rmr-aodium EDTA was used as rinse to assure quantitative transfer of the sample. Concentrated HCI was added to make a final HCl concentration of 3·3 N. The resulting solution was evaporated at 105°C. The residue was dissolved in 0·2 ml. of 0·5 M-KOH, and 18 ml. of the following scintillation solvent was added: 3 gil. 2,5diphcnyloxazole, 0·1 gil. p-bis[2-(5-phenyloxazole)]-benzene in 30% methanol-70% toluene. Radioactivity was measured in a Packard liquid-scintillation spectrometer. The counting efficiency (fraction of disintegrations counted) was determined with each experiment by counting some of the samples before and after the addition of a known 14Cactivity. Counting efficiencies were generally about 60%. This method of internal standardization detects any quenching due to light-absorbing materials in the samples. Quenching was usually found to be negligible.

(a)

3. Results Formation of a denaturation-resistant fraction

Bihelical DNA that has been treated with HN2 does not denature normally. Geiduschek (1961) found that when HN2-treated DNA is heated in 7·2 M-NaCl04 , it

INTER·STRAND CROSSLINKING OF DNA

269

exhibits a normal helix-eoil transition. The midpoint temperature of the transition (T m) and the extent of hyperchromicity were similar to those of normal DNA. Upon rapid cooling, normal DNA does not recover its original low absorbance. The HN2treated DNA, however, did return to the initial hypochromio state. In order to clarify the mechanism of this effect on denaturability, we examined the denaturation of DNA after relatively small extents of reaction with HN2. Mild denaturing agents were used so as to avoid the breakdown of alkylated DNA which may occur at elevated temperatures. The denaturing agents used were sodium hydroxide (Ehrlich & Doty, 1958) and formamide (Marmur & Ts'o, 1961). TAllLE

1

Relative recovery of native and denatured DNA by analytical density-gradient uUracentrifugation

Source of DNA

B. IfUbtili8

M.lyaodeikticu8

CsCI·t buffer

SLSt

Neutr. Neutr. C0 3·A C0 3-A C0 3·A C0 3·A C0 3·B C0 3-B

0 40 0 40 0 10

C0 3·B

(,..g)

Total DNA

Denatured: Native§

(,..g)

expected

found

10

1·7 1·7 0·7 0·7 1·7 1·7 3·0 3·0

1·00 1·00 1·00 1·00 1·00 1·00 3·00 0·333

0·80 1·01 0·72 1-13 0·81 1·04 2·89 0·354

10

2·1

1·00

0·97

10

t Neutr., 0·02 M-Tris-HCI (pH 8); C0 3·A, 0·2 M·sodium carbonate, pH 10·4 (pH and concentration in CaC! solution); C0 3-B, 0·25 )I-sodium carbonate, pH 10·6. t Sodium lauryl sulfate added per 0·8 mI. final solution. § Ratio of areas under denatured and native bands in known mixture of the two types of DNA.

The most useful information was derived from analytical density-gradient equilibrium ultracentrifugation. This procedure, applied to DNA that has been treated with a low concentration of HN2 and then subjected to a transient exposure to formamide or high pH, generally revealed a mixture of two components having buoyant densities close to those ofunreacted helical and denatured DNA's. Unreacted DNA, exposed to these melting conditions, produced almost exclusively denatured DNA. Typical results are illustrated in Fig. 1. Bacterial DNA preparations of approximately 70 p.g/ml. concentration were treated with 2 X 10- 6 M-HN2 at 25°0 (pH 7,2) (0,025 M-triethanolamine-HCl) for two hours. The reaction mixtures were then gently mixed with two volumes of 0·085 N-NaOH, 0·001 M·EDTA and left to stand for four minutes at 35°0. The solutions were neutralized (brought to pH 7 to 8) with two volumes of 0·1 N-oitrio acid, 0·03 M-Tris. The samples were frozen and stored at -15°0. Just prior to ultracentrifugation, 0·4 ml. of sample was mixed with 0·2 ml. of 1 M-NaHOO a-Na 200a (1 : 2), 5 p.!. of 0'2% sodium lauryl sulfate, and 0·5 p.g of a deuteriated Escherichia coli reference DNA. The centrifuge was run at 44,770 rev./min at 25°0 for 20 hours. The bands in Fig. 1, from right to left, are helical component, denatured component and reference DNA, respectively.

270

K. W. KOHN. C. L. SPEARS AND P. DOTY

Some bacterial DNA preparations displayed no material at densities intermediate between the denatured and undenatured bands (Fig. l(a)), whereas other preparations did show significant amounts of such material (Fig. l(c) shows a typical example). Deterioration of DNA after prolonged storage caused a decrease in the denaturationresistant fraction that could be produced by a standard HN2 treatment, and caused a. marked increase in intermediate density material (Fig. 1 (bj], The intermediate

(q)

FIG. 1. Resolution of HN2-treated bacterial DNA into denaturable and undenaturable fractions. The conditions of reaction and alkali exposure are described in the text. The bands, from right to left, are helical component, denatured component and reference DNA. (a) B. subtili« DNA. (b) Same after 3 years of storage. (c) E. coli DNA.

material seemed to be derived from a tailing of the "helical" band towards higher densities. This could be due to single-strand breaks and the consequent loss, during exposure to melting conditions, of single-strand segments from the denaturationresistant molecules. In most of our experiments, we observed DNA peaks only at densities close to "helical" or "denatured". In the case of HN2-treated phage T2 DNA, however, we were able to generate a band at intermediate density by terminating the alkali exposure at high ionic strength. Figure 2 shows the effect of added salt during the neutralization step. With a sodium ion concentration of 0·05 M, only peaks at "helical" and "denatured" densities are visible (a). With 0·45 M-Na+, however, a

INTER-STRAND CROSSLINKING OF

D~A

271

prominent intermediate band was found to appear (b). The position of the intermediate band varied with the experimental conditions.

1·717 1·713

"702

FIG. 2. Effect of ionic strength on helix reformation after exposure to alkali of HN2-treated phage T2 DNA. A 20 p.g/ml. D~A solution was treated with 10 p.~1-HN2 for 100 min at 25°C (pH 7,2) (0,025 M-triethanolamine-HCI). The reaction was terminated with 0-01 M-Na 2S2 0 3 • Three volumes of ~aOH were added to produce a final OH- concentration of 0-03 N. After 2 min at 24°C, the solution was neutralized with 0-5 vol. citric acid with or without added XaCI. (a) Neutralized at low ionic strength (0,05 M). NaCl to make a concentration of 0·45 M was added after neutralization. (b) Same as (a), except that the NaCI was added just prior to neutralization.

Mild HN2-treatment apparently alters a fraction of the DNA molecules so that even after complete helix disruption by a melting agent, the helical structure can quickly re-form. The helix, under some experimental conditions, may be grossly imperfect. Exposures to sodium hydroxide or formamide denaturants at relatively low ionic strengths, however, seemed to permit the recovered DNA to regain a structure very close to that of the original helical DNA. (b) Properties of the denaturation-resistant fraction

In order to determine how close the recovered structure is to the original, comparisons were made in terms of buoyant density, hypochromicity, viscosity and sedimentation coefficient. (i) Buoyant density Banding position in a CsCI gradient is a moderately sensitive index of D~ A secondary structure (Rownd, 1963). Denatured DNA usually bands in neutral CsCI at a position corresponding to approximately 0·016 g/cm 3 higher density than docs helical DNA. One-tenth of this difference can easily be detected. Hence, a 10% deviation of helical DNA towards the denatured form can be measured. The sensitivity of this measure can be increased by carrying out the procedure in CsCI buffered at an elevated pH. Above pH 10, the thymines and guanines of denatured DNA begin to ionize whereas those hydrogen-bonded in helical regions do not ionize. Each negative ion so formed acquires a Cs + counter-ion which increases the effective density of the molecule (Vinograd et al., 1963).

272

K. W. KOHN, C. L. SPEARS AND P. DOTY

Table 2 shows representative data for the density changes resulting from HN2treatment and/or exposure to denaturants (alkali or formamide). The HN2-treatment was such that the two bands produced after exposure to denaturant were of approximately equal size. The densities of these bands were measured with respect to suitable reference DNA's which were added to the ultracentrifuge cells. The first entry in Table 2 shows that the HN2-treatment by itself did not appreciably affect density. TABLE

2

Buoyant density changes of denatured and undenatured components after denaturationexposure of HN2-reacted DNA Denaturant DNA source

Agentt

B. subtilis (prep. 1)

(g/cm a)

(g/cm a)

Neutr. 2 min 25°C Neutr. 4 min 35°C COa-A

-0.0001 0.001 4 0.001 8

0.017 4 0.021 8

Exposure

+ + +

None OHOH-

0

+

OHOHOHfa fa

3 min 34°C 3 min 34°C 10 min 80°C 40 min 35°C 40 min 35°C

COa·B COa-B COa-B C0 3·B COa·B

0 0 + +

OHOHOHOH-

10 min 5°C 4 min 45°C 10 min 5°C 4 min 45°C

COa-B COa-B COa-B COa·B

(prep. 2)

+ + 0 M. lysodeikticus

CsCl.§ buffer

HN2t

pd-Poll

Ph-PO

0.002 6

0'025 a 0.025 7 0'025 3 0.026 8 0.028 4

0.000 2 -0,0002

0.016 3 0.016 2 0.016 0 0.015 6

-0.0001 0.000 5

(0'0005)~

Ph-PO Pd-PO

0·08 0·08 0·00 0·02 0·09

0·01 -0·01

t "+",2 X 10- 6 M-HN2, 2 hr, 25°C, pH 7·2 (0,025 M-triethano1amine-HCI). t "OH", NaOH to produce [OH-] = 0·04 to 0·05 N, followed, after the indicated exposure, by neutralization with citric acid. "fa", Dialysis against 95 to 98 % formamide for 2 to 4 hr at 3°C followed, after the indicated exposure, by dialysis against 0·04 M-NaCI, 0·001 M-sodium EDTA. § "Neutr.", 0·02 M-Tris-HC1 (pH 8); C0 3·A = 0·2 M-sodium carbonate (pH 10,4) (concentration and pH in CsCI solution); COa-B = 0·25 M-sodium carbonate (pH 10,6). II Po, Buoyant density of untreated native DNA. Pd' Ph, Buoyant density of denatured and undenatured fractions, respectively, after HN2 andlor exposure to denaturant. ~ Small residual "helical" band.

The extent of reaction was too small to produce appreciable reduction in buoyant density due to alkylation (Kohn & Spears, 1964). The position of the "denatured" fractions, after HN2-treatment and exposure to denaturant, correspond closely with that of normal denatured DNA. The column farthest to the right in the Table is a measure of the fraction of "denatured" character contained by the helical component. It shows that, as measured by buoyant density, these denaturation-resistant molecules generally recover at least 90% of their "helical" character after exposure to the denaturants. (ii) Hypochromicity

Arrangement of the DNA bases in a bihelix causes a reduction in their molar extinction coefficients. This hypochromicity may be taken as a measure of helical character.

INTER-STRAND CROSSLINKING OF DNA

273

Hypochromicity was determined from absorbance-temperature profiles of Bacillus subtilis DNA which had been treated with various concentrations of HN2 and exposed to 0·04 N-OH-. The reactions were carried out in 0·025 M-triethanolamineHOI (pH 7,2) for 100 minutes at 24°0 and were terminated by a. 35-minute exposure to 0·004 M-sodium thiosulfate. The solutions were made alkaline by adding 0·5 volume of 0·16 N-NaOH--D·002 M-EDTA. After 1·5 minutes at 24°0, the solutions were neutralized with citric acid and dialyzed against 0·15 M-NaOI--D·015 M-sodium citrate. The absorbance-temperature profiles (Fig. 3) show a dose-dependent sequence of curves ranging between those of control native and denatured DNA's. The sharpmelting component of the control denatured DNA (upper curve) was due to partial renaturation during the heating. 'rhis is shown by the presence of a hump at about 50°0 (Doty, Murmur, Eigner & Schildkraut, 1960). Hypochromicity was measured as the ratio of absorbance at 25 or 40°0 to that at 95°0. In Table 3 and Fig. 4, the relative hypochromicity is compared with the fraction of the DNA banding in the density range of helical DNA. There is good agreement throughout the range of HN2 concentration used. (iii) Viscosity

The same comparison was made for viscosity, measured in a capillary viscometer at various rates of shear G (Table 4 and Fig. 5). The intrinsic viscosity is a. measure of tho length and stiffness of the molecules. Denatured DNA is a flexible coil and has a much lower intrinsic viscosity than helical DNA. The fraction of the original viscosity retained after exposure to alkali of DNA treated with low concentrations of HN2 tended to be approximately 15% below the fraction at "helical" density in the ultracentrifuge. At high HN2 concentrations, tho two measurements tended to approach each other. It appears that after a. small extent of reaction with HN2, tho original DNA helical structure is almost, but perhaps not quite, recovered, whereas after longer extents of reaction, recovery is more complete. (iv) Sedimentation velocity A sedimentation velocity study was performed on sample D1 of Tables 3 and 4. This sample had been treated with 5 X 10- 7 M-HN2 and, after exposure to alkali, contained 22% helical DNA, according to density-gradient analysis. Boundary sedimentation revealed two components. The integral sedimentation distribution is shown in Fig. 6. DNA that had not been treated with HN2 showed only the rapid (denatured) component. The slow component appeared to make up 30% of the total DNA. This value may be somewhat too largo because of non-linear film response in the region of high DNA concentrations. The slow component had a median sedimentation coefficient (sgo.w) of 32 s, compared with the value 39 s for the untreated, native DNA. (These values have been corrected to zero concentration according to the relation used by Eigner, Schildkraut & Doty, 1962.) The difference between these figures may not be beyond the experimental errors involved in analyzing the bimodal sedimentation patterns. The values arc relatively close when compared with the much greater value characteristio of denatured DNA. The sedimentation coefficient of the denaturation-resistant component was, if anything, less than that of the untreated DNA. The slight reductions both in viscosity and in sedimentation coefficient suggest that thero may have been a slight reduction in molecular weight. The fact that the sedimentation coefficient

1"0

1- 0 '9 o '0 !:::! Q)

u

~ s

.D

o

Q)

>

";J

o

Qj

~

100 Temper ature (°el

FIG. 3. Absorbance-temperature profiles of HN2-treated B . subtili« DNA after exposure to alkali. HN2 concentration (pM): <>-, 1-0; -0-0-, 2-0; - . - . - , 5-0; -.-t-, 10; -.A.-.A. -,20; -e-e-, 0-0 (not exposed to alkali).

-0-0-, 0-0; -!:;;.- !:;;.-, 0-5; --<)-

INTER-STRAND CROSSLINKING OF DNA TABLE

275

3

Hypochromicity ofB. subtilis DNA after HN2-treatment and exposure to NaOH Hypochromicity

Relative hypochromicity

(260 mfL)

Sample no.

[HN2]

0 0 5 X 10- 7 10- 6 2Xl0- 6 5 X 10- 6 10- 5 2 X 10- 5

NOt DO Dl D2 D3 D4 Do D6

Fraotiont at "helical" density

A 25 • C A S5 ' O

A. o•c A S5 ' O

h 25 • C

h. o•c

0·737 0·835 0·816 0·703 0·782 0·76 1 0·745 0·731

0·732 0·897 0·862 0·834 0·809 0·7 62 0·747 0·7 27

1·00§ 0·00 0·19 0·43 0·54 0·86 0·92 1·06

1·00§ 0·00 0·21 0·38 0·53 0·82 0·91 1·03

1·00 0·04 0·22 0·43 0·60 0·84 0·95 >0·98

t Equilibrium sedimentation in CsCI gradient. t Samplo NO was not exposed to NaOH.

§ h,

At (AAt'E. )_D~5.:'£... . (A~5~JDO - (A:5~JNO

= _

p5

1·0

1'0

,

0

~

.~

E

'>

.s:

> .;;

.,

£

v 0

0 '6

ac

0. o-,

0·8

'0

.~

8 0·4 .;;

0 '4

a "ii

v

~

ex:.

, , " /

0·6

.c:.

.,

./

,,,"""

?;-

.;;;

.~ 0'8

/

u;

/

,

I'

,1',,""

,,""

0·2

0·2

02

0-4

FIG. 4.

06

0'8

1·0 02 Frcct ion at 'helical' density

0-4

06

0'8

1·0

FIG. 5.

FIG. 4. Hypochromieity of denaturation-resistant component of HN2-treatod B. aubtilia DNA. The fraction of normal hypochromicity retained after exposure to alkali of HN2-troated DNA is plotted against the fraction of the DNA remaining undenatured, as determined by density-gradient analysis. See Table 3. FlO . 5. Viscosity of denaturetion-resiatent component of HN2-treated B . BUb/ilia DNA. The fraction of the original viscosity retained after exposure to alkali of HN2·treated DNA is pl otted against fraction undenatured as in Fig. 4. See Table 4. 18

K. W. KOHN, C. L. SPEARS AND P. DOTY

276

TABLE

4

Viscosity of B. subtilis DNA after HN2-treatment and exposure to NaOH

Sample no.

NOt DO Dl D2 D3 Dol D5 D6

(M)

0 0 5xl0- 7 10- 6 2 x 10- 6 5 X 10- 6 10- 5 2 X 10- 6

Fraction of native viscesity

("Isplc) c=O

[HN2]

G=

60 (sec-I)

98·2 5·0 20·7 36·5 51·0 68·7 87·0 93·5

G=

30 (sec-I)

G =0

112 5 23 40 56 80 96·5 105

126 5 26 46 65 90 109 120

G=

60 (sec -1)

1·00 0·05 0·21 0·37 0·52 0·70 0'8!! 0·95

G=

30. (sec t)

G=

0

Fract.ion t at "helical" density

r

1·00 0·04 0·21 0·36 0·50 0·72 0·86 0·94

1·00 0·04 0·21 0·37 0·52 0·71 0·87 0·95

1·00 0·04 0·22 0·43 0·60 0·84 0'!!5 >0·98

t Sample NO was not exposed to :!\aOH. t Donsity-gradient equilibrium ultracentrifugation.

was not increased argues against the presenee of denatured areas in the recovered helices. (e) Inhibition of strand separation Nitrogen mustard-treated DNA, hence, does not readily undergo the changes in physical properties exhibited by normal DNA following exposure to alkali. This inhibition of denaturation implies an inhibition of strand separation. The latter was directly confirmed by studies on density-hybrid DNA. The paired strands ofthis type . of DNA have different buoyant densities; the separated strands form two distinct denatured bands in density-gradient equilibrium experiments. Such hybrid DNA can be prepared from bacteria grown in heavy isotopic medium followed by a period in normal medium. Density-hybrid DNA can also be made in vitro by annealing mixtures of normal and heavy isotopic single strands. Treatment of either of these types of hybrids with HN2 prevented the separation of the components of different density by exposure to alkali or formamide. A similar result was obtained with phage S1>82 DNA, the two strands of which have different buoyant densities (Marmur & Cordes, 1963). Treatment with HN2 again prevented the appearance of two components following exposure to alkali and density-gradient analysis. These results directly confirm that HN2-treatment of bihelical DNA can prevent strand separation. (d) Requirement for two functional groups per alkylating molecule

If the inhibition of strand separation is due to covalent crosslinks formed from the alkylating agent, the effect should be specific for polyfunctional agents. It should not occur with monofunctional alkylating agents even at very high extents of reaction. In order to test this, B. subtilis DNA was treated with mono- and bifunctional representatives of three types of alkylating agents known to attack DNA: 2-chloroethylamines, epoxides and alkylsulfonates. The reactions were carried out at pH 7·4

f·Or-----.----,-----,-----.-----,----,------,,-------,

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t

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120

80

160

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7'0

Distance from centre of rotation (em) (b)

FIG. 6. (a) Integral sedimentation distribution of B. subtilis DNA that has been treated with 5 X 10- 7 M-HN2 and exposed to alkali (sample D1 of Tables 3 and 4). Boundary sedimentation in 0·15 M-NaCI-0'015 M-sodium citrate at 29,500 rev.fmin, 25°C; 30-mm analytical cell; DNA concentration, 18 ftgfml.; ultraviolet optics. The sedimentation patterns were corrected for radial dilution. Sedimentation coefficients were calculated from the slopes of the linear portions of the log r versus time plots for various equi-concentration points on the corrected boundaries. (b) Sedimentation patterns (corrected for radial dilution) used for the calculation of the integral distribution shown in Fig. 6 (a). The patterns are at 4 min intervals during sedimentation.

278

K. W. KOHN, C. L. SPEARS AND P. DOTY

(0,025 M-triethanolamine buffer) at 37°C. Unreacted alkylating agent was removed by dialysis against 0·02 M-NaCl, 0·02 M-Tl"is, 0·002 M-EDTA (pH 8). Sodium hydroxide was then added to produce 0·04 N-OH-. After two minutes at 24°C, the solutions were neutralized with citric acid. Cesium chloride density-gradient analysis was carried out at pH 8. N,N-dimethyl-2-chloroethylamine (H....""1), a mono-functionallanalogue of HN2, was found to be inactive at 1O,OOO-fold higher concentration than that of HN2 required to prevent denaturation of most of the DNA. This is demonstrated in Fig. 7. DNA which had been treated with 4 X 10- 2 M-RN1 for two hou'rs at 37°C (curve C) banded at lower density than untreated helical DNA. This density-reducing effect of alkylation was observed, even more strikingly, with HN2 (Kohn & Spears, 1964). The shift to lower density in curve C indicates that the HN1 did, in fact, react with the DNA under the conditions used. Exposure of DNA treated with HNI DNA to alkali produced a denatured band, also shifted toward lower density (curve D). The broadening of this band compared with control denatured DNA (curve A) is a second indication

r\

I •

I

•

•

I

I•

I•

•

I

I

•

I

I

: 'C

·, : ,

• •I

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]'IG. 7. Denaturability of B. aubtilis DNA treated with mono-functional nitrogen mustard. Curve A, control DNA, incubated 4 hr at 37°C (pH 8), and alkali denatured. Curve B, DNA treated 40 min at 37°C with 2 X 10- 6 l\I·lIN2, and exposed to alkali. Curve C, DNA treated 2 hr at 37°C with 4 X 10- 2 lIl·HNl (N,N-dimethyl-2-chloroethylamino); not exposed to alkali. Curve D, same, exposed to alkali. The right vertical line defines the position of normal helical B. subtilis DNA. Buoyant density increased towards the left.

INTER-STRAND

CROSSLINKI~G

OF DNA

279

that the HNI had a marked effect on the DNA. Despite these effects of HNI on DNA, no denaturation-resistant fraction was produced. Smaller extents of reaction with HNl, which do not have these effects, also do not produce denaturation resistance. On the other hand, DNA treated with high concentrations of HN2 so as to produce marked reductions in density is completely resistant to denaturation. These observations demonstrate that, although HNI reacts extensively with DNA, it docs not produce denaturation resistance. Likewise, the bifunctional epoxides, bis(2,3-epoxypropyl)-ether and butanediepoxide, produced distinct denaturation-resistant fractions (Fig. 8, curves B and C). The monofunctional epoxide, 1-(2,3-allyloxy)-2,3-epoxypropane, had no such effect (Fig. 8, curve A).

A

,

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Ref.

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"

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\\

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.

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FIG. 8. Effect of mono- and bifunctional epoxides on the denaturability of B. subtilis DNA. DNA was treated with 5 X 10- 3 M-epoxide for 4 hr at 37°C, exposed to alkali and ultracentrifuged in neutral CsCl. Curve A, 1-(2,3-allyloxy-)-2,3-epoxypropane; curve B, bis(2,3-epoxypropyl)ether; curve C, butano-L: 2,3: 4.diepoxide.

However, the mono- and bifunctional alkylsulfonates: ethylmethanesulfonate and 1,4-dimcthanesulfonoxybutane ("Myleran"), produced no denaturation resistance under the conditione used (5 X 10- 3 M, 37°C, two hams), although comparable reaction conditions have been reported to yield 7-alkylguanine residues in the DNA (Brookes & Lawley, 1961). These data clearly indicate that the interference with the denaturability of DNA requires simultaneous alkylations at two neighboring sites. (e) Covalent nature of the crosslinks

The inhibition of the denaturation of HN2-treatcd DNA may, in principle, be due (1) to covalent links which hold the strands together, or (2) to a local increase in stability of the bihelix without covalent connections between strands. The second type of effect by small molecules has been reported, for example, for actinomycin (Haselkorn, 1964; Reich, 1964) and aliphatic diamines (Mehrotra & Mahler, 1964). These stabilizing agents 'cause an increase in the 1 'm (midpoint of absorbance-temperature

280

K. W. KOHN, C. L. SPEARS AND P. DOTY

profile) of helical DNA. If HN2-treated DNA were stabilized in this way, one would expect that, with increasing temperature, a critical range would be reached at which melting is complete, permitting strand separation and denaturation. On the other hand, if denaturation requires the rupture of covalent links between paired strands, then the breakdown of the denaturation resistance of HN2-treated DNA should proceed progressively with time at increasing rates as the temperature is raised. To distinguish between these possibilities, the extent of denaturation ofHN2-treated DNA was determined after exposure to alkali at various temperatures. DNA derived from B. subtilis or Micrococcus lysodeikticus, 35 fLgfml. in 0·025 M-triethanolamineHCl (pH 7,3), was treated with 2 fLM-HN2 for two hours at 25°C. Portions of 0·25 ml. were frozen and stored at -15°C. For denaturation, thawed samples were gently mixed at O°C with two volumes of NaOH solution to produce a final OH - concentration of 0'05 N. After exposure to various temperatures for various lengths of time, the samples were cooled and neutralized with citric acid. The samples were ultracentrifuged in CsCI buffered at pH 10·6 with 0·2 M-sodium carbonate and containing 10 fLgfml. sodium lauryl sulfate. The results are given in Table 5. The extent of denaturation of HN2-treated DNA, produced by four- to ten-minute exposures to 0·05 N-OH-, was independent of temperature between 5 and 80°C. The undenatured fraction in the 12 portions of HN2-treated B. subtilis DNA that were exposed to alkali within this temperature range was 0·430 ± 0·008 (mean ± standard deviation). The band profiles were similar over this range except for small increases in material of intermediate density at the

TABLE

5

Alkali stability of crosslinked DNA Alkali exposure DNA

[HX2J (,..M)

Temp. (DC)

Time (min)

Fraction not denatured

B. aubtili8

0 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2

5 45 5 10 14 20 25 30 40 45 64 64 80 80 99 99

10 10 10 10 10 4 4 4 4 4 4 10 4 10 5 15

<0·010 <0·010 0·422 0·438 0·422 0·420 0·427 0·426 0·433 0·434 0·444 0·430 0·439 0·422 0·374 0·21

M. lysodeikticua

0 0 2 2

5 45 5 45

10 10 10 4

<0·020 <0·020 0·626 0·637

INTER - STRAND -CRO SSLINKING OF DNA

2Bl

highest temperatures (Fig . 9(a)}. The elevation above the baseline at intermediate densities is probably du e to some single-strand breaks, producing molecules with both helical and denatured parts. At 99°0 , however, there was progressive disappearance of t he "helical" band (F ig: 9(b)). This was ac companied by an accumulation of molecules with intermediate densities. The loss of denaturation resistance, therefore, probably involved main chain breaks. We conclude that denaturation resistance mu st involve cova lent bonds.

(ol

FIG. 9. Effect of t emperature during exposure to alkali on t he de n at urat ion of HN2-treat ed DNA. (a) HN2-treated B. su bti lie DNA exposed to 0·05 N-OH- for 10 m in at 5°C (solid curve) or BO°C (dashed curve). (b) Same, ex posed at 99°C for 5 min (solid cur ve ) or 15 min (dashed curve). The bands are, from left to right : reference, denatured, helical.

In contrast to this situation it was found that, when neutral formamide was used as denaturant, the fra ction of DNA denatured increased as the t emperature was r aised. Thus, crosslinked DNA apparently is not as stable in neutral formamide as in alka li. Alkali, in fa ct , stabilizes the crosslinks to subsequent heating at neutral pH. Thi s reaction is currentl y under study. (f) Number of alkylations per effe ctive crosslink

Th e inhibition, by reaction with HN2, of the irreversible denaturation of DNA might be explained by the formation of one (or a few) crosslinks which prevent complete st ra nd separat ion, or by the formation of many HN2-bonds which keep the DNA st rands in register at many points. In order to decide between these two general

K. W. KaHN, C. L. SPEARS AND P. DOTY

282

types of possibilities, it was desirable to estimate how much HN2-alkylation is required to affect the denaturation behavior of DNA. To this end, the extent of binding of [14C]HN2 to DNA was compared with the changes produced in denaturability. B. subtilis DNA was allowed to react in 0·025 Mtriethanolamine-HCI (pH 7'2), with various concentrations of [14C]HN2 for two hours at 25°C. (For the higher concentrations of HN2, NaOH was added to the buffer to neutralize the hydrochloride groups of the mustard.) The reactions were stopped by the addition of Na 2S2 0 3 to a final concentration of 0·02 M. The radioactive material bound to DNA was determined as described under Materials and Methods. The result of two independent experiments, covering overlapping ranges of HN2 concentrations, are presented in Table 6. If Na 2S 2 0 3 was added prior to [14C]HN2 (entry 15), a negligible amount of label was retained with the DNA. This shows that the thiosulfate treatment promptly inactivated the mustard, and that the purification procedure removed essentially all of the unbound label. The number of HN2 residues chemically bound per 10 4 nucleotide units (r X 10 4 ) was found to be proportional to the HN2 concentration (c) of the reaction mixture up to c = 40 p.M. At higher concentrations, ric decreased, presumably because of an appreciable reduction in the number of free binding sites. TABLE

6

Binding of [14C]HN2 to B. subtilis DNA

Entry

c

w

A X 10- 2

(I'M)

(fLg)

(ctsjmin)

0·99 1·8 4·3 9·9 18 30 60

265 263 210 260 135 133 147

E

r x 10'

0·53 0·53 0·53 0·53 0·53 0·53 0·53

0·94 1·57 5·18 9·4 16·5 28·0 48·2

r[c »; 10' (fLM- 1)

Experiment 1 1 2 3 4 5 6 7

0·649 1·076 2·83 6·36 5·79 9·66 18·5

0'95 0·87 1·21 0'95 0·92 0'93 0'80

Experiment 2 8 9 10 11 12 13 14 15

8·0 20 40 200 400 667 1000 1000t

67 71

64 65 62 59 58 66

1·50 3·48 6·83 30·9 54·1 75·5 92·9 0·075

0·61 0·61 0·61 0·61 0·61 0·61 0·61 0·61

7·4 16·3 35·1 157 291 426 531 0·4

0'93 0·82 0'88 0·79 0·73 0·64 0·53

Moan r/e, entries 1 to 10: 0·93 ± 0·11 (stand. dev.), Same, excluding entry 3: 0·89 ± 0'06. t Sodium thiosulfate added prior to HN2. r = mol. HN2 bound/mol. nucleotide units.

Am where m is molecular weight per average nucleotide unit = 330, E is counting Ewa efficiency, w is weight of D~A counted (fLg), a is specific activity of [14C]HK2 = 1·65 X 10 8 r

= --,

disintegrations/min/fLIDole.

283

INTER-STRAND CROSSLINKIKG OF DNA

For this particular set of reaction conditions (fixed reaction time of two hours) and for c < 40 p.M, the extent of alkylation (r) may be calculated from the expression: r - X 104 = 0·89 ± 0·06 (p.M)-l. c This figure was then used to determine the extents of alkylation in the lower range of HN2 concentrations at which the change in DNA denaturability is effected. A set of samples covering this range was prepared concurrently with experiment 1 of Table 6. About 30 minutes after the HN2 reactions were stopped with sodium thiosulfate, two volumes of a NaOH solution were added so as to make the final OH- concentration 0·05 N. This exposure to alkali was maintained for two minutes at 24°C, after which the solutions were neutralized with citric acid. The results are plotted in Fig. 10. The H~2 concentration (c) and extent of alkylation (r) scales are both indicated on the horizontal axis. The vertical axis represents the fraction of the DNA which was denatured by the sodium hydroxide treatment as measured by analytical ultracentrifugation in CsCl. Conversion of half the molecules to denaturation resistance occurred when there were only about five HN2 moieties bound per 104 base pairs. It is seen that, except at high HN2 concentrations, the denaturability declines with increasing extent of alkylation according to a first-order relation. This indicates that the rate-determining step of the reaction causing the HN 2 Concentration (j4M)

3

Mol.~4C]

4

5

6

7

8

9

10

4 4 HN2 bound per [0 nucleotides (r x 10 )

FIG. 10. Decline of denaturability of B. eubtili« DNA with increasing extent of alkylation by [ 14C]HN2. Reaction time was fixed at 2 hr at 25°C.

284

K. W. KOHN, C. L. SPEARS AND P. DOTY

abrupt change in denaturability involves a single HN2 molecule. If this reaction is used as the operational definition of crosslinking, then the result clearly implies that a single crosslink prevents irreversible denaturation. The number of crosslinks per molecule should then follow a Poisson distribution (assuming that molecules are crosslinked at random). The fraction of molecules without crosslinks, 10 = exp (-n), where n is the average number of crosslinks per molecule. When there is an average of one crosslink per molecule (one "hit"), the fraction uncrosslinked would be exp (-1) = 0·368. (In the experiment shown in Fig. 10,91 % ofthe control DNA was denaturable. At one hit, the fraction of these molecules that are still denaturable would be 0·369 X 0·91 = 0,335.) One crosslinking hit corresponded to 3·6 molecules of [14C]HN2 bound per 104 nucleotide residues (Fig. 10), or one bound HN2 per 0·9 X 106 molecular weight units. The DNA preparation used in this experiment had a molecular weight of 23 X 106 , determined from sedimentation and viscosity data (Eigner & Doty, 1965). The binding of one molecule of [14C]HN2 per 0·9 X 10 6 molecular weight units, therefore, produced an average of one crosslink per 23 X 10 6 molecular weight units. Hence, to produce one effective crosslink required the binding of approximately 25 molecules of HN2. It can be concluded that, at least under these experimental conditions, only a small fraction of bound HN2 residues form crosslinks. In contrast with the initial first-order kinetics with respect to HN2 concentration, the appearance of crosslinked DNA with reaction time at a fixed HN2 concentration shows a distinct lag. (In these experiments, the HN2 was pre-incubated in buffer for a sufficient time to permit essentially full conversion to the active ethyleneimmonium form.) The observed lag may be due to the time required for the second arm of a bound HN2 molecule to react with the second strand of the DNA. These experiments are described in the accompanying paper (Kohn & Green, 1966).

4. Discussion We have found that reaction of bihelical DNA with nitrogen mustard (HN2) concentrations as low as 5 X 10- 7 M, which correspond to an alkylation of approximately 0'005% of the bases, caused a discrete change in the denaturation behavior of a fraction of the DNA. The altered fraction does not undergo the transformation of physical properties caused in normal DNA by a brief exposure to denaturing conditions. Unlike normal DNA, the HN2-altered fraction, after transient exposure to NaOH, retains buoyant density, hypochromicity, intrinsic viscosity and sedimentation coefficient close to the values for the original native DNA. The change brought about by reaction with HN2 is abrupt; its quality is not significantly altered by increasing the extent of reaction with HN2. The kinetics of this effect were found to be first-order in HN2 concentration. These observations indicate that the reaction of one HN2 molecule per DNA molecule can prevent the denaturation of the entire macromolecule. We may take this first-order, all-or-none conversion of DNA molecules to a denaturation-resistant form as an operational definition of chemical crosslinking. Crosslinking was shown to prevent the complete separation of the paired strands in density-hybrid DNA. We picture that a crosslink consists of a covalent connection between the paired strands which holds the strands together at the point of the crosslink while the DNA molecule as a whole is melted. This picture is supported by the

INTER· STRAND

CROSSLINKI~G

OF

D~A

285

finding that bifunctionality is required for crosslinking by nitrogen mustard and by epoxides. We noted that HN1, a monofunctional analogue of HN2, reacts extensively with DNA but does not crosslink. A bifunctional molecule can be imagined to alkylate a DNA site and then to react, via its second functional group, with a site on the opposite strand. The time interval between these steps may be responsible for the lag we observed in the kinetics of crosslinking. One effective crosslink was produced, under our reaction condition, per approximately 25 molecules of [14C]HN2 chemically bound to the DNA. Hence, the large majority of DNA-alkylations by HN2 did not form crosslinks. Alkylation without crosslinking may occur when binding is at a site from which no second reaction site on the opposite strand can be reached, or when hydrolysis or reaction with other nucleophiles in the solution intervenes. The crosslinking effect was found to be stable in alkali over a very wide temperature range. This indicates that the effect is mediated by covalent bonds. When the denaturation resistance was finally lost at 99°C, the density-gradient patterns showed that many single-strand breaks were produced. It may seem surprising that singly crosslinked melted molecules, upon rapid removal of denaturant ("quenching"), tend to "zipper-up" to form perfect, or nearly perfect, bihelices. However, a crosslink may be considered the kinetic equivalent of a point of nucleation. Extension of helix from this point seems to be the favored reaction, since we find that structures with properties intermediate between helical and denatured do not tend to form (when the ionic strength is kept low). On the other hand, such intermediate structures, containing both bihelical and denatured regions, have been observed after quenching during the early stages of bi-molecular renaturation (Rownd, 1963). The explanation may be that, at the temperature of renaturation (optimal about 20°C below the T m), the non-specific binding between strand regions is strong enough to interfere with helix growth (Wada & Yamagami, 1964). When crosslinked DNA is completely melted and then quenched, however, we would have nucleated molecules which have not yet formed non-specific bonds. Hence, helix growth would be relatively unimpeded. We found that material of intermediate density did occur, however, if crosslinked phage T2 DNA was melted by alkali and then neutralized at high ionic strength (Fig. 2). We interpret this to indicate that high ionic strength favors denaturation of melted DNA, relative to "zippering-up". This might be viewed in terms of competing rate processes leading to helical versus denatured structures during quenching. Once non-specifically bonded structures have formed, helix growth would be inhibited. This is supported by the finding that nucleation of two complementary strands is not followed by helix growth unless annealing conditions are applied (Rownd, 1963). High ionic strength may favor the formation of denatured areas before helix growth is complete and so produce intermediate molecules. Brookes & Lawley (1961) have shown that the major site of alkylation on the bases of bihelical DNA is the N(7) position of guanine. They identified a product consisting of an HN2 moiety bound to two guanine residues and suggested that there may be inter-strand crosslinking between guanines. There was no way, however, to distinguish this possibility from crosslinking between adjacent guanines in the same strand. Examination of a space-filling model indicated that an HN2 crosslink can, with slight distortion ofthe helix, reach between guanine-N(7) positions on opposite strands. The crosslink then lies in the major groove and connects between guanines of adjacent

286

K . W. KOHN, C. L. SPEARS AND P. DOTY

base pairs. The base sequence must be G(3'-+5')0. The sequence 0(3'-+5')G does not permit this type of crosslinking. Although it may seem likely that crosslinking by alkylating agents is between guanine-N(7) positions on opposite strands, this has not yct been established. We have seen that only a small fraction of alkylations by HN2 result in crosslinks. Hence, crosslinking could conceivably be due to reactions at relatively minor alkylation sites, rather than at the major alkylation sit e on guanine. The formation of a denaturation-resistant fraction in chemically altered DNA was first reported by Marmur & Grossman (1961) after ultraviolet irradiation, and by Geiduschek (1961) after treatment with HN0 2 and HN2. Several other agents have now been found to produce this effect, including formaldehyde at neutral pH (Freifelder & Davison, 1963), mitomycin 0 in the presence of reducing agents (Iyer & Szybalski, 1963,1964), and heat at low pH (Freese & Cashel, 1964). Geiduschek (1961) observed that the hyperchromic effect of heating and fastcooling normal bihelical DNA does not occur, or is greatly reduced, if the DNA has been treated with nitrous acid. The intrinsic viscosity of nitrous acid-treated DNA was reported to fall 13% after heating and fast cooling. This is comparable with the viscosity deficit of our HN2-treated DNA samples after exposure to alkali (Fig. 5). Density-gradient analysis revealed discrete denaturation-resistant fractions having a buoyant density close to that of the original native DNA. In these respects, the behavior of HN0 2-treated DNA and HN2-treated DNA are remarkably similar. As will be seen in the following paper (Kohn & Green, 1966), the similarity between these two types of crosslinked DNA is exhibite d also in terms of transforming activity. This work was supported by the National Institutes of H ealth, Grant HD 01229.

REFERENCES Alexander, P. & Lett, J. T. (1960). Bioch em, Pharmacal. 4, 34. Brookes, P. & Lawley, P. D. (1961). Biochem, J. 80, 496. Doty, P., Marrnur, J., Eigner, J . & Schildkraut, C. (1960). P roc. Nat. Acad. Sci., Wash . 46,461. Ehrlich, P. & Doty, P . (1958). J. Amer. Ohern, Soc. 80 , 4251. Eigner, J. (1960). Ph.D. Thesis, Harvard University. Eigner, J. & Doty, P. (1965). J. Mol. B iol. 12, 549. Eigner, J., Schildkraut, C. & Doty, 1>. (1962). Biochim. biophys. Acta, 55, 13. Freese, R. & Cashel, M. (1964). Biochim. biophys. Acta, 91, 67. Froifelder, D. & Davison, P. F. (1963). Biophys. J. 3, 49. Geiduschek, E. P. (1961). Proc. Nat. Acad. Sci., Wash. 47, 950. Goldacre, R. J., Loveless, A. & Ross, W. C. J. (1949). Nature, 163, 667. Haddow, S. A. R., Kon, G. A. R. & Ross, W. C. J. (1948). Nature, 162, 824. Haselkorn, R. (1964). Science, 143, 682. Hearst, J. E., HIt, J. B. & Vinograd, J. (1961). Proc. Nat. Acad. Sci., Wash. 47, 99. Ifft, J. B., Voet, D. H. & Vinograd, J. (1961). J. Phys. Chem, 65, 1138. Iyer, V. N. & Szybalski, W. (1963). Proc, Nat. Acad. Sei., Wash. 50,355. Iyer, V. N. & Szybalski, W. (1964). Scie nce, 145, 55 . Kohn, K. W. & Green, D. M. (1966). J. Mol. Riol. 19, 289. Kahn, K. W., Green, D . M. & Doty, P. (1963). Fed . Proc. 22, 582. Kahn, K. W. & Spears, C. L. (1964). Proc, Amer. Assoc. Cancer Res . 5, 89. Loveless, A. (1951). Nature, 167, 338. Luzzati, V., Mathis, A., Masson, F. & Witz, J. (1964). J. M ol. B iol, 10, 28. Marmur, J. (1961). J. Al ol. R iol . 3, 208. Marmur, J. & Cordes, S. (1963). In Informational 111acromolecules, p. 79. New York: Academic Press.

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287

Marmur, J. & Grossman, L. (1961). Proc. Nat. Acad. Sci., Wash. 47,778. Marmur, J. & Ts'o, P. O. P. (1961). Biochim. biophys. Acta, 51, 32. Mehrotra, B. D. & Mahler, H. R. (1964). Biochim. biophys. Acta, 91, 78. Meselson, M. & Stahl, F. W. (1958). Proc. Nat. Acad. Sci., Wash. 44,670. Reich, E. (1964). Science, 143, 684. Richardson, C. C., Schildkraut, C. L. & Kornberg, A. (1963). Cold Spr, Harb, Symp. Quant. Biol. 28, 9. Rownd, R. H. (1963). Ph.D. Thesis, Harvard University. Saito, H. & Miura, K. (1963). Biochim. biophys. Acta, 72, 619. Vinograd, J., Morris, J., Davidson, N. & Dove, W. F., Jr. (1963). Proc. Nat. Acad. Sci., Wash. 49,12. Wada, A. & Yamagami, H. (1964). Biopolymers, 2,445.

Appendix Terminology of Configurational Changes of DNA in Solution The terminology currently used to describe configurational changes of DNA in solution seems to promote ambiguity and misunderstanding. The term "denatured DNk" is commonly applied to two quite different states: (1) the random-coil which exists when there is little or no interaction between segments of DNA strands; and (2) the irregular compact structure which forms when DNA segments adhere to each other without long-range ordered base pairing. "Denaturation" sometimes refers to the helix-eoil transition and sometimes to conversion to the compact structure. Clear and concise description becomes especially difficult when one is dealing with so-called "reversible" DNA (Geiduschek, 1961). This type of DNA undergoes a normal transition from double-helix to random-coil, but when conditions are then reversed, it does not go on to the compact structure as is the case with ordinary DNA; but, rather it returns to the bihelical state. Thus, when reversible DNA is denatured (definition (1», it does not denature (definition (2»! In order to avoid such confusion, we define "denatured" DNA to be a conformation not having long-range helical order, but existing under conditions in which doublehelical DNA is thermodynamically stable. "Denaturation" then refers to the conversion of bihelical DNA (or some other non-denatured form of DNA) to denatured DNA. By this definition, "denaturation" should not be applied to the helix-coil transition. To designate the helix-coil transition, we may use instead the term "melting", which is already commonly applied to this process. When bihelical DNA is completely melted, in the sense that it has completed a transition, it may go over to a random-coil state. We must keep in mind, however, that the resulting state may occasionally not be a random coil (Luzzati, Mathis, Masson & Witz, 1964). Therefore, it may be wise to retain the non-commital term "melted DNA" for the product resulting from a transition from a relatively ordered to a relatively disordered structure. "Renaturation" is also currently used in two ways: (1) to refer to the slow conversion of denatured DNA to bihelical DNA, usually by annealing at some temperature below the T m; and (2) to refer to a rapid formation of bihelix from melted DNA when conditions are changed so as to favor the stability of bihelix. By the definitions proposed here, the term should be restricted to the first meaning. For process (2), no

288

K . W. KOHN , C. L. SP E A RS AND P. DOTY

completely satisfactory te rm seems to be available. The colloquially used expression "zippering-up" is descriptive of the proc ess, and could perhap s be elevated to the status of a technical term. These relationships are summarized in the following diagram: Bihelix not sta b le~

Bihelix stable

FIG. 11.

To complete the paths between the boxes in the diagram , it only remain s to consider the direct conversion of bihelieal to denatured DNA under condition s in which the bihelix is st able. Thi s occurs , for example, when one st ra nd of the bihelix is enzymically degraded, leaving the complement ary st ra nd intact (Richardson , Schildkraut & Kornberg, 1963). Thi s pro cess is not solely a ph ase transition, but it could be considered to be a kind of " denat uration" . Thi s te rminology can be applied also to parts of DN A molecules. A single molecule can be imagin ed, for example, to cont ain regions that are bihelical, regions that are denatured, and other regions that are melted.