THE JOURNALBIOLOGICAL OF CHEMISTRY 0 1986 by The American Soeiety of Biological Chemists, Inc

Vol. 261,No. 13, Issue of May 5, pp. 60264033,1986 Printed in U.S.A.

Denaturation of Covalently Closed Circular DNA

KINETICS, COMPARISON OF SEVERAL DNAs, MECHANISM AND IONIC EFFECTS*
(Received for publication, August 6, 1985)

Merrill N. Camien and RobertC. Warner$

From the Department Molecular Biology and Biochemistry, Universityof California, Iruine, California92717 of

The rates of the alkaline denaturationof the cova- constraint imposed by covalent closure is chiefly due to the lently closed, circular DNAs (form I) of the replicative work of Vinograd and his colleagues and has been reviewed forms (RF) phages G4,4X174, and fd, of plasmid by Bauer and Vinograd (1). Other aspects of the unique of and pBR322 and phage PM2 have been measuredat 0 "C behavior of form I DNA in alkali have come to our attention and some at higher temperatures. These ratesare or- as a result of our recent study (2) of the kinetics of renaturaders of magnitude slower than the denaturation of tion of form b from 4X174-RF and have motivated an inveslinear DNA because of the increased stability of the tigation of the kinetics of the denaturation reaction. helix to deprotonation that results from the accumuThe principal effect, in this regard, of the treatment form of lating positive superhelicity during denaturation.DeI DNA with alkali was the large dependence of the rate of naturation reactions were initiated by rapid, infrasonic mixing (Camien, M. N., and Warner,R. C. (1984) renaturation on the conditions under which the alkaline denaturation had been carried out. This was demonstrated by Anal. Biochem. 138,329-334), and their progress was the following observations (2); (i) the rate of renaturation is measured by analytical ultracentrifugal analysis for the amounts of form I and denatured (Id) DNA after pseudo-first-order; i.e. it is independent of the DNA concentration, but falls off from an initial first-order rate as the neutralization of the alkaline reaction. The comparativerates of the fiveDNAs varied over reaction proceeds; (ii) the rate of renaturation depends on the a wide range; the fastest, G4-RF, denatured a t 500- temperature at which the denaturation is carried out and on fold the rate of the slowest, fd-RF. The differencesare the supercoil density of the form I DNA used to prepare Id; accounted for by the interaction of positive superhel- and (iii) the molecules of I d are heterogeneous in configuration icity with the sequence-dependent regions of relative and renature at different rates. Point iiigives rise to the helix stability in the various DNAs. Renaturation rates characteristics of the reaction indicated in iand ii. The of I d DNAs varied similarly for hs prepared at 0 "C, differences in configuration among the Id molecules are estabbut onlya few-fold for Ids prepared at50 "C. lished during the denaturation reaction. The rate of denaturation of G4-RF was determined It is also known that there are large differences in therates over a wide range of NaOH and NaCl concentrations of renaturation of Id from the form I of different DNAs. at 0 "C, and the pH, was determined as a function of ionic strength and temperature. The effects of ionic Preliminary work indicated that this is also true of denaturstrength have been analyzed in an application of the ation ratesto a degree not observed for linear or nicked DNA. Manning ion condensation-screening theory (Manning, Differences were noted (2) between the closely related RFs of G. S. (1978) Q. Rev. Biophys. 11, 179-246) which is phages 9x174 and G4. More substantial differences between shown to account for the large destablizing effect of PM2 DNA and other form I DNAs were shown by Ostrander salts on the helix. The pH region of transition at 50 "C and Gray (3). When PM2 DNA was denatured in alkali, a from renaturation to denaturation wasexamined, and fraction renatured to form I simply on neutralization without it was found that the maximum rate of renaturation exposure to annealing conditions such as those required for occurred at a pH about 0.05 units below the pH,. 4X-Id. We havemade similar observations on chloroplast DNA (4) and on PM2 DNA (2). Lau and Gray (5) compared the renaturation of +X-RF, X and PM2 DNAs and found , that XDNA shared this property. Two additional examples, The denaturation of circular form I1 DNA in alkaline solution differs in a number of significant ways from the alkaline fd-RF and pBR322 DNAs, are reported in this study. The denaturation of its linear or nicked counterpart. The under- phenomenon is apparently common and is dependent on the standing of these differences and their origin in the topological conditions of denaturation (2, 5, this study); we refer to it as zero time renaturation (ZTR). During the course of our renaturation studies, preliminary *This research was supported by National Institutes of Health Grant GM28769 from the National Institute of General Medical observations were made indicating that therate of denaturaSciences. The costs of publication of this article were defrayed in part tion of +X-RFI under some conditions of alkalinity and by the payment of page charges. This article musttherefore be hereby temperature canbe slow, 4 in therange of 1-3000 s. A similar marked "aduertisement" in accordance with 18 U.S.C. Section 1734 slow denaturation of PM2 DNA was noted by Lau and Gray solely to indicate this fact. (Fig. 10of Ref. 5). These rates are orders of magnitude slower $ To whom correspondence should be addressed. The abbreviations used are: form I, covalently closed, circular, than those for denaturation of linear DNA, but may still be duplex DNA; RF, replicative form; form 4, denatured form I DNA; too fast to measure by the methodology we used for renatuform I,, relaxed form I DNA; form I,, superhelical form I; form 11, ration rates. We have designed and constructed an infrasonic nicked form I; EtdBr, ethidium bromide; bp, base pairs; ZTR, zerotime renaturability; p, ionic strength, O"-Id, Id DNA prepared by mixing device (6) which permits the rapid initiationand quenching of microliter volumes of a denaturationreaction at denaturation at 0 "C or other indicated temperature. 5,titratable a constant temperatufe and thus measurement of reaction the superhelical density.

6026

Denaturation of Covalently Closed Circular DNA

6027

I
f

Denoturation -Renaturation t, Ii(j) d

1
IItW
-ecId(l)

[Neutral supercoiled, form I , Is, orrelaxed,partially 1 ,circular , titrofed

~~

L1 9, Z T R

Llk

Iti)

I Z G i q

1-

Multiple species varying in configurotion

DNA

Species that retain residual protonoted regions and yield I on neutrolizotion

Species thet an neutralization yield Id except far the formation of I by ZTR

FIG. 5. Minimum scheme to account for the kinetics of denaturation and renaturation and for the properties of the denatured species. Reactions b-d are those of denaturation: accumulation of positive supercoiling and deprotonation of thymine and guanine residues remaining in the duplex configuration. Reactions e and g are neutralizations. Reaction g, ZTR, we have defined as that fraction of Id(j) and I(k) of some DNAs denatured a t low temperatures that assume the form I configuration on neutralization without the exposure to the particular renaturation conditions required for species which is indicated by reaction f.The distinction between I&) is that provided by our assay for the progress of denaturation. The parenthetical intermediate species I(i) and designations, (i), (j), ( k ) , ( l ) , are included to indicate that such species have multiple configurations and therefore different properties with respect both to further denaturation steps and to renaturation. Neither the necessity of exposure of neutral Id to annealing conditions nor the possible formation of intermediates inreaction f are indicated on the diagram.

rates having half-times as low as a few seconds. We report a study of the dependence of the rate of denaturation of G4-RF on temperature, ionic strength, and NaOH concentration and an examination of the dependence of denaturation and renaturation on NaOH concentration in the range in which the transition between the two takes place. Comparative observations on the rates of denaturation and renaturation of G4-, 4X-, and fd-RFs and pBR322 and PM2 DNAs have been correlated with local relative stabilities calculated for the four DNAs for which sequences are available.

measurable rate and the higher pH of denaturation of circular as compared with linear DNA. Random configurations arising early in the reaction do not have time to relax and equilibrate as denaturationproceeds, leading to a heterogeneous range of kinetically trapped configurations. A minimal reaction scheme for both denaturation and renaturationispresentedin Fig. 5. It includes operational definitions of the classes of intermediate species that account for the sigmoid nature of the logarithmic rate curve and other features of both reactions. The first step, reaction a, represents the initial part of the alkaline sedimentation titration of form I (14). The rapidly sedimenting species that accumuEXPERIMENTAL PROCEDURES AND RESULTS late during this titration are designated I& and I(i). A disDISCUSSION tinction between these two species is made in order to explain The Denaturation Reaction-The characteristics of dena- the lag in the initial denaturation rate shown in Fig. 4. The turation of form I DNA shown in Figs. 2-4 and theirrelation distribution among the I(i) species is stochastic; except for to the renaturation of form I shown in Fig. 1 can be under- the heterogeneity in linking number, all I& molecules have d stood by an extension of the analysis of these reactions given the same potentiality, but variations in the initial random in our previous study (2).It was there shown that thenonlin- configurations commit the molecules to different subsequent ear first-order plots of renaturation rate and thedependence configurations at different rates. The distinction between I(i) and Id(j) species isthat of this rateon the conditions of denaturation were a result of the formation during denaturation a range of configurations defined by the reversibility criteria of Rush and Warner (17) of of the denatured species differing in the rates atwhich they and Ostrander and Gray (3). It is the same distinction as could be renatured.These configurations wereviewed as provided by our quenching assay for denaturation; Id species arising during the denaturation reaction by an interaction of are detected after neutralization by their more rapid sedimenthe increasing positive superhelical density with the primary tation. If reaction c is sufficiently slow then a lag phase will helical windings (1, 2, 14-16). Positive supercoiling acts as a be observed. If the configurational changes taking place durbarrier to deprotonation and denaturation of the residual ing the initial stages of the alkaline titration aretime-dependhelical regions and is responsible for the low and easily ent to a degree that contributes to the measured rate of denaturation, it may not be possible to distinguish between I& and I species. A range of rates for the conversion of I Portions of this paper (including Experimental Procedures, Results, parts of Discussion, Figs. 1-4 and 6-10, Tables 1-111, and species to Id species leads to the decrease in the measured additional Footnotes 1 and 2) are presented in miniprint at the end rate of denaturation as the reaction proceeds (Figs. 2-4). The of this paper. Miniprint is easily read with the aid of a standard range of rates results both from the inherent heterogeneity of magnifying glass. Full size photocopies are available from the Journal linking numbers among the molecules of form I andfrom the of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. consequences of the heterogeneity of I(i) species. The Id(j) Request Document No. 85M-2632, cite the authors, and include a check or money order for $8.80 per set of photocopies. Full size species are further maturated configurationally than I(i) in photocopies are also included in the microfilm edition of the Journal the sense that they are committed to denaturation, but still that is available from Waverly Press. differ from Id in configuration. Their existence is indicated by

6028

Denaturation of Covalently Closed Circular DNA

9. Smiley, B. L., and Warner,R.C. (1979) Nucleic Acids Res. 6 , 1979-1991 10. Tinoco, I., Jr., Borer, P. N., Dengler, B., Levine, M. D., Uhlenbeck, 0. C., Crothers, D. M., and Gralla, J. (1973) Nature New Biol. 246,40-41 11. Denhardt, D. T., Dressler, D., and Ray, D. S. (eds) (1978) The Single-Stranded Phages, pp. 139, 659 and 671, Cold Spring Harbor Laboratory, ColdSpring Harbor, NY 12. Sutcliffe, J. G. (1979) Cold Spring Harbor Symp. Quant. Biol. 4 3 , 77-90 13. Funnell, B. E., and Inman, R. B. (1979) J. Mol. Biol. 131, 331340 14. Vinograd, J., Lebowitz, J., Radloff, R., Watson, R., and Lapis, R. (1965) Proc. Natl. Acad. Sci. U. S. A. 5 3 , 1104-1111 15. Vinograd, J., Lebowitz, J., and Watson, R. (1968) J. Mol. Biol. 33,173-197 16. Schmir, M., Revet, B. M. J., and Vinograd, J. (1974) J. Mol. Biol. 83,35-45 17. Rush, M. G., and Warner, R. C. (1970) J. Biol. Chem. 245,27042708 18. Pouwels, P. H., Knijnenburg, C. M., van Rotterdam, J., Cohen, J. A., and Jansz, H. S. (1968) J. Mol. Biol. 3 2 , 169-182 19. Wang, J. C. (1974) J. Mol. Biol. 89, 783-801 20. Bauer, W. R. (1978) Annu. Rev. Biophys. Bioeng. 7,287-313 21. Lyubchenko, Y. L., Frank-Kamenetskii, M. D., Vologodskii, A. V., Lazurkin, Y. S., and Gause, G. G., Jr. (1976) Biopolymers 15,1019-1036 22. Tachibana, H., Wada, A., Gotoh, O., and Takanami, M. (1978) Biochem. Biophys.Acta 517,319-328 23. Lyubchenko, Y.L., Vologodskii, A. V., and Frank-Kamenetskii, M. D. (1978) Nature 2 7 1 , 2&31 24. Borer, P. N., Dengler, B., Tinoco, I., Jr., and Uhlenbeck, 0. C. (1974) J. Mol. Biol. 8 6 , 8 4 3 4 5 3 25. Chapman, R. E., Jr., and Sturtevant, J. M. (1970) Biopolymers 9,445-457 26. Porter, B. W., Kolodner, R., and Warner, R. C. (1973) J. Bacteriol. 116,163-174 27. Record, M. T , Woodbury, C. P., and Lohman, T. M. (1976) . Jr., Biopolymers 15,893-915 28. Manning, G. S. (1978) Q. Rev. Biophys. 1 1 , 179-246 29. Manning, G. S. (1972) Biopolymers 11,937-949 30. Hamaguchi, K., and Geiduschek, E. P. (1962) J. Am. Chem. SOC. 84,1329-1338 31. Levy, M., and Warner, R. C. (1954) J. Phys. Chem. 5 8 , 106-109 32. Warner, R. C., and Levy, M. (1958) J. Am. Chem. SOC. 80,57355744 33. Record, M. T ,Jr., Anderson, C. F., and Lohman, T. M. (1978) . Q. Rev. Biophys. 1 1 , 103-178 34. Record, M. T., Jr. (1967) Biopolymers 5 , 993-1008

the fact that the pH limit above which only I is formed on d neutralization duringan alkaline sedimentation titration of I occursbefore the changesin sedimentation coefficient are complete (2, 3, 17). Two pathways of renaturation, f and g) must be distinguished. The neutral Id is distinguishable from the alkaline form byits lower sedimentationcoefficient and lower buoyant density. It requires exposure to defined annealing conditions of specified alkaline pH and temperature order to renature in (2). This pathway is shown as leading to Ialk because of our previous demonstrationthat the proximate renatured species has the same sedimentation coefficient as I& at the pH and temperature of renaturation (2). The other pathway, g, also termed ZTR, is included because for certain DNAs a fraction of the alkaline 4 renatures on neutralization as discussed above.3
Acknowledgment-We are indebted to Scott B. Mulrooney for the preparation of most of the DNAs used in this study.
REFERENCES
1. Bauer, W., and Vinograd, J. (1974) in Basic Principles of Nucleic

2. 3. 4.
5.

Acid Chemistry (Ts'o, P., ed) Vol. 1, pp. 265-303,Academic Press, New York Strider, W., Camien, M. N., and Warner, R. C. (1981) J. Biol. Chem. 256,7820-7839 Ostrander, D. A., and Gray, H. B., Jr. (1973) Biopolymers 1 2 , 1387-1419 Kolodner, R., Tewari, K. K., and Warner, R. C. (1976) Biochim. Biophys. Acta 4 4 7 , 144-155 Lau, P. P., and Gray, H. B., Jr. (1980) Nucleic Acids Res. 8,673701

6. Camien, M. N., and Warner, R. C. (1984) Anal. Biochem. 1 3 8 , 329-334 7. Harned, H. S., and Owen, B. B. (1958) The Physical Chemistry of Electrolytic Solutions, 3rd Ed., pp. 638 and 697-764, Reinhold, New York 8. Wheeler, F. C., Fishel, R. A., and Warner, R. C. (1977) Anal. Biochem. 78,260-275 A previously unnoted error appears in our earlier paper (2). The stated pH values at which the data shown in Fig. 4 and those in Fig. 5 were obtained were inadvertently reversed. The correct pH for Fig. 4 is 11.35 and that for Fig. 5 is 10.95.

SUPPLEMENT to DENATURATION OF COVALENTLY-CLOSED CIRCULAR DNA: KINETICS. CMPARISON O SEVERAL F DNAs. MECHANIgl A D IONIC EFFECTS N by M e r r i l l N Canien and Robert C. Warner . EXPERIMENTAL PRDCEDURES RF and o t h e r c i r c u l a r DNAs. i n c l u d i n g samples relaxedwithtopoisanerase 1, were Sephacryl c o l m n prepared as previouslydescribed ( 2 ) except f o r t h e m i s s i o n o f t h e I n a l l casesform I was p u r i f i e d by the useof EtdBr-CsCl step i n preparing pBR322 DNA. equilibriumgradients. FU2 DNA was a g i f t of Or. Richard Kolodner; another sample o f PM2 D A ws purchased f r a n Boeringer-Hannheim. N Other preparations, ultracentrifuge methods and denaturation and renaturation procedures were those previously described (2) except f o rt h e use o f rapid. infrasonic mixing. The c o n s t r u c t i o no ft h em i x i n g device and i t s use f o r measuring denaturation rates have been reported (6). Sone of the renaturation rates reported here were measured using the older mixing methods (2). The r e a c t i o n i x t u r e o r i t h e r e n a t u r a t i o n m f e d or renaturation, unless othervise indicated. had a f i n a l v o l u n e o f 45 el and consisted of approximately 33 eg of DNA per m l i n NaOH a t an i n d i c a t e dc o n c e n t r a t i o nw i t h EDTA a t 4.67 nN t om i n i m i z en i c k i n go f t h e DNA a t l k a l i n e a pHs and w i t h NaCl included t o d j u s t h e o n i c t r e n g t h . a i s Each reported NaOH c o w e n t r a t i o n has been c o r r e c t e d o t h e o u n o f f r m t NaOH consmed i n t i t r a t i m jt h e EDTA. The reported ionic strengths, cmputed i nt h e usual manner, are based on concentrations of Na+ OHC1- and EDTA4-. Uhere pHs of these Iolutionr r n aregiven,they were c a l c u l a t e d f a ' the NaOH concentration and i o n i cs t r e n g t hu s i n g (7). the values for pKw and a c t i v i t c o e f f i c i e n t atth e p p r o p r i a t ee m p e r a t u r e y s a t Other pHs were measured as previouslydescribed ( 2 ) . The reactlons were quenched w i t h a 5-10 el drop of from 0.5 t o 4 M T r i s HCI, containing tw e q u i v a l e n t so fT r i s HCI f o r each e q u i v a l e n t f o NaOH i n the reaction mixture. Sane experiments on renaturaThese s o l u t i o s t i o n were done i n h e t NaC1-phosphate buffer previously used (2). contai ed 1 M NaCl. 4.67 nN EDTA and 77 d phosphate w i t hav a r y i n gr a t i o 4 Of HPOjt o P049- depending on the pHand had an ionic strength of approximately 1.3. D e n a t u r a t i o nr e a c t i o nm i x t u r e si n t e n d e dt op r d u c es u f f i c i e n tI df o rr e n a t u r a t i o n studies were prepared as described above except withconcentrationsof OVA increased t o as much as 1 mg per ml. The product was dialysedagainst 2 nN EDTA (pH 8). d i l u t e d M to approximately 50 pg pr m l w i t h 2 r EDTA (pH 8 ) . and s t o r e d a t O'C.

Gel electrophoresis was conducted as previouslydescribed (8) except forthe use ofaHoeferminigel apparatus.Thismethod was used as a means o f l o c a t i n g r e g i o n s o f c r i t i c ailo n i c t r e n g t h s and a l k a l i n i t y o r r a n s i t i o n s f t between the I and Id-DNAs. Forms I and I d g i v e d i s t i n c t , well-separated bands then run i n Ebuffer (8) containing I eg/mI Ether. The cmpleteness o fe i t h e r a d e n a t u r a t i o n r e n a t u r a t i o ne a c t i o n o r can bedetermined by t h i s method t o w i t h i n 11. The r e l a t i v e m o u n t s o f t h e two cmponents a t i n t e r m e d i a t e degreesofconversion. where given, were determined by canparison ofthephotograph o ft h eg e lw i t ht h a to fas e t of D standards and areaccurateto M about t 15%.

Calculated Stabilit Stability those used f o r canparins' etemduplexes Of are based on t h e AGs f o i base s t a c k i n go f on short RNA f r a ne q u i l i b r i u nn a s U r e m t S t h of et h e re phage Mus S u t cThee (121. s l i d i n g l l liff ca

p-

maps were conputed by methods s i m i l a rt o 1 X and G4 w i t ht h e i r sequerces (9) They RNA duplexesderived by Tinoco e t a i (In) duolexes of know sequence. ThFs&nces ' pRR322 was determined by average Of t h e bG
~~~. l r n a t h n f the .. the -.
~

mnlwwlm

as done byFunnell and Inman'il3jfoF A + Tcontent. The values o f AG per bp a t 25-c were taken fm, t h et a b l e ofTincco e ta l . (10) and averaged f o r bp 1 t o n, 2 t o ~ + 1 etc., through 70C4 t o N+6999. T h e s e s l i a i n g averages were plottedagainst bp positio; on the standard sequence using Tektmnix a 4051 Graphics Systan. The c a l c u l a t i o n 7000 bp because t h i s i s l a r g e r t h a n t h e l a r g e s t sequenced was c o n t i n u e d a r b i t r a r i l y t o molecule ve have used. t h a t o f fd-RF. The graphs thus repeat after the nunber o f base p a i r si nt h em o l e c u l ei s reached. O maps displayed i n Fig. 6, N n 300; when N was reduced t o 150 o r 75 the peaks and v a l l e y s became more frequent and the maximum t AG excursions fran the mean were approximately double those f o r N = 300.

I i s ashortcaningthatthe t AGs employed here were derivedfor RNA; however, i t seems reasonable t o use them f o r D A a t l e a s t i n as much as theyprovideaplausible N relative weighting the for stacking of the various conbinations o f base p a i r s The Stabilityprofilesobtainedinthis way a r e v e r y s i m i l a r t o t h o s e ye have Consthcted on thebasis of unveighted A + T frequency. These maps arenot shown b u t our p m f i l e f o r I X may beconpared w i t h t h a t o f F u n n e l l and lrunan (13).

Denaturation of Covalently Closed Circular D N A

RESULTS FURTHER AN0 DISCUSSION Renaturation curves rate are shown i n Fig. 1 Denaturation and Renaturation I ,ionic trength n s (II= 0.15, Fig. 1A) and determined both a t ai x e dr e l a t i v e l y f , u n d e rt h eh i g hi o n i cs t r e n g t hc o n d i t i o n s used previously ( 2 ) (Figs. 1B and IC). Wide d l i f f w e n c e s i n i.. i t.a l r a t e s are evident amma I,+ DNAs DreDared under the sane conditions . n i frm different form I DNAs. The j r e a t er a t e r e n a t u r a t i o n of of o o - I d t h a n f 50'-Id o seen i nt h e 0X curves of Fig. 1 was Roted previously (2) and i s 4 . seen here t oc h a r a c t e r i z et h eo t h e rt h r e e ONAS, although i i s l e s s pronounced i n G4. t The f dc u r v e so fF i g . 1B p l a t e a u ?t r e n a t u r a t i o nl e v e l so fa p p r o x i m a t e l y 4RI. and 79% f o rt h e 5o"Id and o * - i d ,r e s p e c t i v e l y ,a l t h o u g ht h ei n i t i a lr a t ef o rt h ef d0 * 4 d QX O*-Id these under conditions. PM2 o o - l d was i s seen t o b e l o s eoh afto r c tt ONAS f o r whichratesHeredetermined and t h eo n l y t h e most r a p i d l y r e n a t u r a b l e o f t h e one f o r w h i c h e n a t u r a t i o n t r a O T could be demonstrated. I t s treatment at CO ' with I t h e h i g h i o n i c s t r e n g t h p h o s p h a t e b u f f e r o f F i g . C a t e i t h e r pH 11.9 o r pH 12.0 resulted in nearly cmplete renaturation in 20 min (data not shoun).
~ ~ ~~~~

6029

StrinGly

80

160 Time, s

240

1800 3600
and

Fig. 3. F i r s t o r d e rp l o t s of denaturation r a t e so f of PMZ and pBR DNAs a t 2O'C i n 40 nM NaOH a t ~ 1 3 . '.6

fd-RF a t several temperatures

decrease i n the r a t e t o zero. This ZTR i s e v i d e n ti n a denaturation curve by a Time ~I I i s more c l e a r l y seen i n t h e denaturation t i s approximatedby t h e pBR curve i n Fig. 2. Fig. 1 F i r s to r d e rp l o t s . showing r e n a t u r a t i o nr a t e s measured a t 50C f o rv a r i o u sI d offd a t 3OoC i n Fig. 3 Where no f u r t h e rd e n a t u r a t i o n was observed when thetime was DNAs A. Renaturations i n 40 mM NaOH p=O.I5. The I d ' s were prepareddenaturing by extended several-fold beyond t h e minimum r e q u i r e d o r h e l a t e a u t f t p a I n I s -1.62, I, D i n sf o r 1 min a t ooc i n 200 nt(( eaOH, ~ = O . Z Sf o r Do-1d's and a t jooc i n 65 m~ ZTR 20%. ZTR was observed w i t ha l lt h r e e DNAs having rates denaturation, slow of NaOH, y = 0.15 f o rt h e 50"-Id's. Superposition the of 64 OO-Id and t h e CIX 50"-Id but with neither of those having a fast rate. curves i s coincidental. 8. Renaturations i nh i o hi o n i cs t r e n o t h ~ ~ . ~ . . ." . ~ OH . ~ Dhocnhate buffer.. . _ 11.03 (see Experimental Procedures). The 0X r e i a t u i a t i o c u r v e s r e i a shorn as dashed QX- and G4-RFs are shorn i n Fig. 4 on an expanded t i m es c a l et o Ratecurvesfor curve i s based on a i n g l e o i nitn t e r p o l a t e d s p frm the curve l i n e s . The 0X 50'-I d e f i n e more c l e a r l y t h e i n i t i a l l a g b a r e l y seen i n Fig. 2 b e f o r et h er a t e reaches i t s shown i n Fig. 3s. Re?. (2). C. Renaturations i n h i g h o n i c t r e n g t h i s phosphate b u f f e r decrease i n r a t e throughout rest the of maximum value. The maximum is f o l l a a d by a a t pH 11.35 f otrh 5 O 0 - I d ' s e and a t pH 10.65 f o t h e r OO-Id's. The r e n a t u r a t i o n the reaction. In c o n t r a st th r e n a t u r a t i o r a t fea l lo fcfo n t i n u o u s l y e n s fa i t s r n curve shorn f o r P P D*-Id i s an estimate based on the observation of cmplete denaturaM i n i t i a l value(Fig. 1). The r e a c t i o n i s pseudc-firstorder i n b o t hd i r e c t i o n si nt h a t 10 min i n 0.1 U NaOH, t i o n i n 1 minute. The I d ' s used i n B and C weredenaturedfor t h er a t ei s independent o f i n i t i a l ONA concentration, as shown f o rr e n a t u r a t i o n (2) 1.78 t NaCl and n e u t r a l i z e dtth en d i c a t e de m p e r a t u r e . i a i t The h i g ho n i c t r e n g t h i s (data for denaturation not shorn). b u f f e ra t pH 11.35 has t h e pH found (2) t o b eo p t i m mf o rt h er e n a t u r a t i o n of 0 X a t 5VC. The o t h e r pHs used i n R and C wereselected i no r d e rt oa c h i e v es l o w e rr e a c t i o n i m rates.Vertical dashed l i n e si n d i c a t e breaks i n t h e t e scale. Table 1

The phenmenonof ZTR i s shown i n t h e r e n a t u r a t i o n c u r v e s as the zero-time intercept on t h e r d i n a t e o r o f O"-Id o f fd-RF i n Fig. 18. I nI d = -0.69, ZTR = 50%; and f o r ZTR 5 25%. ZTR disappeared when the DNA was O'-Id of PM2 i n Fig. I C , I n I d = -0.28, deoatursd a t S O T a s seen i n t h e 5 0 ' 4 ~ curves f o rt h e same DNAs i n Fios. 1B and IC. .~~ . . The dependence of 0" t h e temperat& d&turati& shown b y ~ f dand-%? i s s i m i l a r tothatdiscussedby Lau and Gray (5) where i t i s shown t h a t ZTR i s presentneither i n PM2 denatured a t 5C 0 ' or h i g h e r (2, 5 ) nor i n A OW denatured a t 2 5 T o r higher ( 5 ) . I n a separate experiment i t wasshown t h a t o'-Id of pBR DNA prepared under the Conditions o f Fig. 18 and 1 C gave a ZTR o f 90%.

Half-times of denaturation of @X- and 64-RFs Heasurenentswere made i n 40 nM NaOH a t t h e i n d i c a t e d t e n p r a t u r e under t h e same c o n d i t i o n s a s g i v e n f o r Figs. 2 and 3. Half-time i n s a t temperature.
C '

Zm

of

ON& @X-RF 1.8 64-RF pBR322

"

2' 1 1

1 0 '

"

5-

00

of t h e s ef i v e DNAs were campared (Figs. 7 and 3 ) a When t h ed e n a t u r a t i o nr a t e s range of denaturation rates about of 500-fold was found. I n t h i s small sample there is a sharp d i v i s i o ni n t ot h ef a s tr a t e s found w i t h 64 and qX DNAs and the very much s l o w e rr a t e st h a tc h a r a c t e r i z e fd. PER and PM2 ONAs ONAs. t h a t were slow t o denature tended t o be among t h ef a s t e rt or e n a t u r e ,a l t h o u g has t r i c t reverse order rates of Relaxation supercoiling of (G4-Ip curve) reduced t h er a t e a change was not found. i nt h e same d i r e c t i o n as i t s effect on t h er a t eo fr e n a t u r a t i o n .(2). The effect f o fd-RF and p 2 DNA i s shown i n Fig. 7 and M . temperature on t h e rate o fd e n a t u r a t i o no f c a p a r a b l e d a t a f o r OX- and G4-RFs and pBR322 DNA are presented i n Table I.

9.1

17.2
4.4

1M
11.7

12.9

1350

Reducing the NaOH c o n c e n t r a t i o n t o 30 nM ( o t h e r c o n d i t i o n s unchanged) i n c r e a s e d t h e h a l f - t i m e measured a t 5'C t o 18.6 s f o r G4-RF and t o 204 s for
LIY-PF.

Time, s (upper curve) 7 1 4

21

-0.6
W

.+
0

5 -1.2

E = -1.8 -2.4

200
Time, s

400

600

30

60

90 Time, s

120

150

rrEl NaOH a t "-1 36

Fig. 2. F i r s t order p l o t s o f d e n a t u r a t i o n r a t e s measured forseveral DNAs a t Oo i n 40 The 64 I c u r v e r e f e r s t o GI-RF relaxedbytreat-nentwithtopoismerase I. The ias(ed l i n e foF fd DNA was e x t r a p o l a t e d r a n h e d a t a n + t fd i Fig. 3. I t c o i n c i d e sw i t h i nt h ep r e c i s i o no ft h ed a t aw i t ht w oe x p e r i m e n t a lp a i n t s( o p e nc i r c l e s ) f o r PH2 ONA.

Fig 4 Rates a fd e n a t u r a t i o n o f RFs o f PX and 64. RateCurvesareshorn a t 0' and S9C'in 40 nM NaOH a t u13. ..6 The t i n es c a l ef o rt h e 5' 64 c u r v e .i n d i c a t e da l o n gt h e i s shifted and expanded w i t h e s p e c t r 10 t h a ti n d i c a t e df o r t o pr i g h t of t h ef i g u r e , t h eo t h e rt w o curves. The t i n es c a l e s were s e l e c t e d t o i l l u s t r a t e t h e s i g n o i d charact e r i s t i c of these curves.

6030

Denaturation of Covalently Closed Circular D N A

Rate of Oenaturation The general discussion o ft h ek i n e t i c s of denaturation and r e n a t u r a t i o n based on Fig. 5 i n t h e f i r s t s e c t i o n of t h i s paper be must further developed i n o r d e r t o i d e n t i f y t h e f a c t o r s d e t e r m i n i n g t h e r a t e l i m i t i n g s t e p i n denatur a t i o n and the reasons for the differences among DNAs i n t h e i r r a t e s o f b o t h d e n a t u r a t i o n and renaturation. The concept O f t r a n s l o c a t i o n (2, 3, 5, 18) applied i n t h e d i s c u s s i o n of t h er e n a t U r a t i o n of t h e !d o f 0 X Wagain be used. Translocation i st h es l i p p a g e l l i o f one s t r a n d w i t h r e s p e c t t o i t s canplement as thedouble-strandedcoilconfiguration replaces native the duplex and the axial length the of double strand increases This increase i n l e n g t h r o more p r e c i s e l y h e n c r e a s e n h e t i it average number of b p per t u r ni nt h ec o i l overhe elix etermineshe xtenopositive upercoiling t h d t e tf s The increase i nt h e average axial nucleotide spacing on denaturation i s predicted'to be roughly 40% (see section on I o n Condensation Theory). W a s s o c i a t eh e r o b a b i l i t y e t p o f n u c l e a t i o n and t h e r e f o r e t h e r a t e - l i m i t i n g s t e p o f r e n a t u r a t i o n w i t h t h e d e g r e e o f t r a n s l o c a t i o n ( 2 ) and we w use t h e r a t e o f r e n a t u r a t i o n o f d i f f e r e n t i l ONAs o r of t h e different conditions as a u a l i t a t i v en d i c a t i o n tf h e q i o degree of same DNA under t r a n s l o c a t i o n imposed i n the preceding denaturation reaction. A large translocation r e s u l t s nl o w a t e ia r as i n 50D-!d whereas a m a ltlr a n s l o c a t i o n i e l d sh ea s t s y t f r a t eo f OO-Id. W now add t h e case o f no t r a n s l o c a t i o nt oy i e l d e ZTR This provides am e c h a n i s t i ci n t e r p r e t a t i o no f ZTR as t h er e s u l to f complete deprotonation without translocation. The primary event that determines t h e e x t e n t and r a t e of any a l k a l i n e denaturation o f DNA i s t h e d e p r o t o n a t i o n o f thymine and guanine residues i n r e l a t i o n t o t h e v a r i a b l e s o f pH. t h en h e r e ns t a b i l i t y a e l i c a l i t oh f segment and i t s c o o p e r a t i v i t yI.nh e t case o f closed, c i r c u l a r DNA t h e a n i n a n t f f e c t f o s i t i v e u p e r h e l i c i t y d e op s must be w i n c r e a s eh e es t a b i l i t y a l i t n t of added. The f r e e n e r g y tfh i s u p e r h e l i c i t y e o l i a pH t o becane deprotonated (15) At h e l i c a l sequence so t h a t it w r e q u i r eh i g h e r a pH above pH,, a t which we measure t h er a t et h ef r e ee n e r g yc o n t r i b u t e st ot h ee n e r g y of a c t i v a t i o n and g i v e st h er e a c t i o nt h ec h a r a c t e ro fas l o wd e p r o t o n a t i o n A direct determination proton of release under equilibrium conditions i s given by ;he buoyant (19). The l a t t e rt i t r a t i o n s d e n s i t yt i t r a t i o n so f Vinograd e t 11. (15) and o fW a g contrast the behavior of a samplFof-PPI2 havinga h i g h u = -0.15 w i t h t h a t of a relaxed u = -0.008. The delay i n deprotonation and t h eg r e a t e rc o o p e r a t i v i t yo ft h e sample. , process, r e s u l t i n g f r a n t h e h i g h e r + B o f the I d f r a n t h e I sample are clearly evident (Fig. 3 of Ref. 19). I n e x a n i n i n gt h er a t el i m i t i n gs t e pi nd e n a t u r a t i o nf o rt h e purpose of canparing d i f f e r e n t DNAs it must be recognized. t h a ti n I DNAs having normal negative values o f 3 most o f t h e d e p r o t o n a t i o n , l o s s o f s t r u c t u r & h e l i x and increase i n sedimentation c o e f f i c i e n t w o c c u rr a p i d l yo v e rt h er e v e r s i b l ep a r to fas e d i m e n t a t i o nt i t r a t i o n . i l The O A deprotonatedduringthis N phase o f t h e r e a c t i o n w i l l i n c l u d e t h e l e a s t - s t a b l e AT-richregions as shown by aconsiderableboiyofevidencefrandenaturation mappin; and high-resolution thermal melting studies. A canparison of the change o f sedimentation c o e f f i c i e n tw i t ht h ec r i t e r i o n of r e v e r s i b i l i t y i n t h e t i t r a t i o n s o f Rush and Warner (171 and of Ostrander and Gray (3) shows t h a t t h e c o n f i g u r a t i o n a l changes as represented i t m by the sedimentation c o e f f i c i e n t a r e 80 t o 90% canplete a t t h e pH l i of r e v e r s i b i l i t y . i t m an pH l i may be used t o make i n d i r e c t estimate of t h e change The p o s i t i o n o f t h i s i n buoyant density and i n A26 a tt h a tp o i n ti nt h et i t r a t i o n . The buoyant density t i t r a t i o n o f Vinograd al-..f!5) of polyoma DNA was done underthe same conditions as t h e i p r e v i o u s e d l m e n t a t l o ni t r a t i o n r t (14) and comparison of each w i t hh e t pH l i i n d i c a t e sh aa p p r o x i m a t e l y i t m t t 80% of t h e e p r o t o n a t i o n d was canpleted a t h a t l i m i t . I n making t h i s comparisonallowancemust bemade f o r t h e f a c t t h a t t h e t i t r a t i o n s s t t o (3. of Rush and Uarner (17) were done a t a lmr i o n i c t r e n g t hh a nh e t h e r s 15. 19) as discussed by Ostrander and Gray (3). A s i m i l a r r e s u l t i s seen i n t h e spect r o p h o t a n e t r i ct i t r a t i o no f 0X-RF shorn i n Fig. 7s of our previous study ( 2 ) where it i s superimposed on a sedimentation titration obtained under the same conditions. Here again! about 90% of t h e A 60 change was canpleted a t t h e pH l i m i t o f r e v e r s i b i l i t y . These ConsTderations suggest % a tt h er a t e of denaturation i n I i s d e t e m i n e dp r i n c i 10-2oX of mol&ule, the corresponding to p a l l yd u r i n gt h ed e p r o t o n a t i o no ft h el a s t t h e r e a c t i o n i n d i c a t e d as I ' A I ' d i n Fig. 5. When amolecule o f agiven DNA reaches t h i s t a g et s i energy of activation, EA, f ocro n p l e t i o o f e n a t u r a t i o w i l l n d n be augmented determined by t h e AG of t h e i n h e r e n t s t a b i l i t y o f t h e l a s t c o o p e r a t i v e u n i t by the AG o fs u p e r h e l i c i t ya tt h ep a r t i c u l a r degree o f conversion of h e l i x t o c o i l t h ac h a r a c t e r i z e sh as t a g e tfh e e a c t i o nT h e r e i l l t t t o r . w be a i s t r i b u t i o n f ' E A d o among themoleculesforthereasonsdiscussed above g i v i n gr i s et ot h eh e t e r o g e n e i t y ofreactionrate.Relaxationofthenegative= as i n 1 w i l l i n c r e a s e t h e p o s i t i v e 3 , a tt h e same degree deprotonation of and g i v er i s et ot h es l o w e rr a t e shown for I , o f 64 i n Fig. 2. The differences among moleculesarethus t o be a t t r i b u t e dp r i m a r i l y t o t h e c a n p a r a t i v e s t a b i l i t y o f t h e most s t a b l e r e g i o n o f each.

Fig. 6. Calculateddenaturation maps forfour sequenced DNAs and a canputer-generated randan sequence. The o r d i n a t e i s AG p e r bp i n kcal calculated as as l i d i n g average f o r each 300 bp overthemolecule as described i n ExperimentalProcedures The abscissa i s t h e nunber bp the of fran conventional zero f o rt h e sequence The' s o l i d l i n e i s and t h e d o t t e d 1in;s show t h e p o s i t i o n s theaverage AG per bp f o r t h e e n t i r e m o l e c u l e of f 0.2 kcal and 0 4 kcal i n AG f r a n h e . t average. The sequences t h a t r e h o r t e r a s end of t h e molecule.which i s i n d i c a t e d b y anarrow than 7 kbp are repeated after the The sign of AG hasbeen changed f r a n t h a t i n t h e t a b l e s f r a n which t h e maps were d e r i v 2 (10)because a l l of t h e d i s c u s s i o n focuseson t h e r e a c t i o n h e l i x - c o i l . These canparirons are made using maps based on stacking energies at 25'C (10) They were i n Part derived frrn thermodynamic measurements on o l i g o n u c l w t i d e s i : the temperature range 0' t o 4O0C by Borer e t a1. (24). Since entropies the obtained forthestackingof GC p a i r sa r e more n e g x i i F t h a n thosefor AT p a i r s o r f o r mixed interactionsthemagnitude of t h ed i f f e r e n c eb e t w e nt h e maxima on a map as w as e l l O'C t h a na t 2 5 O C . W e s t i m a t ef r a nt h e s e e t h e average s t a b i l i t i e s w i l l b eg r e a t e ra t 66 between t h e maxima f o r 64 and f d w be increased by i l d a t at h a tt h ed i f f e r e n c ei n about 0.1 k c a la t D O C . On t h eo t h e r hand. al e v e l i n go ft h e peaks and valleysHould small trend i n AG with temperature may be responsible for be expected a t 50T. This ( 2 ) between t h er a t e o fr e n a t u r a t i o no f0 " I d t h el a r g ed i f f e r e n c ep r e v i o u s l yn o t e d and 5O0-Id of @X and found here t o o l d o r t h e r h f o DNAs (Fig. 1) When t h e a r i o u s v ONAs arecanparedthedifferences i n r a t e a r e seen t o be l a r g e o n i y among t h e OO-Id'S i n which the sequence dependent effects predominate and t o be small amng slowly the r e n a t u r i n g 5n'-Id's. The t r a n S l o C a t i o na t 50'C a tt h et i m eo ft h ef i n a ld e p r o t o n a t i o n i s expected t o be greater bmause of the greater thermal energy, t h e more r a p i d r e a c t i o n and t h el o w e rs t a b i l i t yo ft h e sequence. The change i n r e n a t u r a b i l i t y o f g x - l d w i t h respect to temperature denaturation the of was shown t o be achieved by 25'C and t o ( 2 ) . S i m i l a r l y , ZTR i n t h e DNAs t h a t a r e s l w t o denature a t b ei n t e r m e d i a t ea t1 0 T OC disappears as the denaturation temperature i s raised. For A DNA 25-C was found t o be s u f f i c i e n tb y Lau and Gray ( 5 ) ; PM2 required 50C ( 2 5 and Fig. lC) as d i d fd Thus, t h e consequences o f l o w t o zero t r a n s l o c ~ t i o na r ep r a n i n e n to n l ya t (Fig. 1R). O'C and a r e n o t e v i d e n t u n d e r t h e c o n d i t i o n s of r a p i d d e n a t u r a t i o n a t 50'C. A of these comparisons are l made among DNAs i n which the I forms are characterized by a s i m i l a r and nonnalFOfapproximately -0.06 and do not appTy t o I which y i e l d s I The & a n i n a n e f f e c t t a much more t h e o s i t i v e u p e r h e l i c a I d h a n h a t r a n p s l t t f : of the more p o s i t i v e z m a y be t h a t t h e d e n a t u r a t i o n o f I i mre cooperative (19) as discussed above. This would l e a d og r e a t e r r a n $ o c a t i o n h a n n ta t t i I and ( 0 a l o w er e n a t u r a t i o na t e r Of I d from I as previously found (2). Additiona? evi'dence s t h ahh i g h e ro s i t i vs u p e r h e l i c &o fe n a t u r e d tt e p e d y I a l t e rists t r u c t u r ies shown by i t s higher sedimentation coefficient ( 1 6 ) and ? t s greater buoyant density

I n c a n p a r i n gh e i f f e r e n t t d ONAs we assume t h a t h e form Is ,of each has t h e B and thereforeatacanparablefractionaldeprotonationaboutthe same about t h e same c o n t r i b u t i o no fp o s i t i v e superp o s i t i v e F , and as a r e s u l t a l l have h e l i c a l AG t ot h e EA. The indication ran he ata ollected n auer's eview 20) f t d c i B r ( i st h a tt h ev a r i a t i o ni ni s small and t h a tt h e DNAs i n our sample t h a t are slow t o denature, PPI2 and fd ( e q u i v a l e n t t o M13). have more negative 3 ' s than 0 X , t h a t i s . i n t h e wrong d i r e c t i o n t o e x p l a i n t h e i r s l o w r a t e s o f d e n a t u r a t i o n .

same negative

It i s i m p o s s i b l e t o c a n p a r e d i f f e r e n t DNA molecuCanparison w i t h Stability Haps l e sw i t hr e s p e c tt or e g i o n so f greater and l e s s e r s t a b i l i t y u s i n g e x p e r i m e n t a l d e n a t u are r a t i o n maus exceot i n t h e most q u a l i t a t i v e sense. The conditions of denaturation notstandardized;thedegreesof'denaturation examined arevariable, and t h ed a t aa r e n o ts u f f i c i e n t l ye x t e n s i v e . W have used sequenced DNAs and have c a l c u l a t e ds t a b i l i t y e maps as described i n Experimental Pmcedures and shown i n Fig. 6. A l o f h e l t maps There i s a c o r r e l a t i o n o f t h e magnitudeof havepronounced maxima and minima o f AG. t h e AG values f o r t h e most s t a b l e regions between t h e DNAs i n our small sample t h a t have l o w a t e s d e n a t u r a t i o n r of and show ZTR and those that have h i g h a t e s r and no ZTR. OX and G4 have maximum AG's c l o s et o 2.7 kcal and do not have excursions fran t h e i r mean g r e a t e t h a n h o s e tf h e a n d a n r t o r sequence, whereas fd and PER. t h e two examples o f t h e s l o w l y d e n a t u r i n g c l a s s , have maximum A G ' S t h a t a r e g r e a t e r by 0 1 and . 0.4 kcal. respectively. When t h e maps derived a franrunning average of N = 150 bp and N = 75 bp (see Experimental Procedures) were s i m i l a r l y analyzed, the difference between t h es l o w l y and t h er a p i d l yd e n a t u r i n gc l a s s e s ranged fran 0.2 t o 0.5 k c a l f o r N = 150 and from 03 t o 0.8 k c a l f o r N . 75. The absence o f an e f f e c t of t h e average AG o f a DNA on t h e r a t e , seen i n canparing fd and PUR, i s consistentwiththeproposal t h a tt h er a t ei s determined by t h e most stable 10-20% o f t h e molecule. It does not DNAs w i t h i n t h e s l o w o r f a s t c l a s s t o appear t o be p o s s i b l e t o r e l a t e t h e r a t e s o f t h e t e maps, e.g.. h O X and 64. This may be due t o t h e inadequacy o fo u ra s s m p t i o nt h a t u i s t h e same f o r a l l of the DNAs o r because t h e r a t e i s dependenton the breadth and distributionoftheseveralstabilityregionsofamolecule as well as t h e maximum AG.

(19).
Zero Time R e n a t u r a b i l i t ZTR was associatedmongthefive DNAs s t u d i e dw i t ha slow r a t e 0 denaturation k d f ot r fe a c t i oo f Ie o te n a t u r ea te r o hr nh n r t d d z 1). W have i n t e r p r e t e dt h i s phenanenon e time, w i t h :fast r a t e of renaiuration(Fig. t o i n d i c a t e t h e absence of any s i g n i f i c a n t t r a n s l o c a t i o n h e n t h e t i t r a t i o n i s c a n p l e t e presumably because t h ea l r e a d yt i t r a t e dc o i l DNA i s so t i g h t l y wound i n these .more no slippage s t a h l e DNAs t h a tt h ef i n a lp r o t o nl o s so c c u r si nac o n f o r m a t i o ni nw h i c h i s possible. There i sd i r e c ts u p w r tf o rt h i s view i n experiments on PPI2 near 25OC Lau and Gray (5) determined rate denaturation a of at 2 5 T f o rt h el a s t 14% o f th; 3% ZTR a f t e r 10 min and n of u r t h e r Process and showed a l o w e v e l i n g f f o b o u t s l o ta found 10%ZTR a f t e r change t o 120 rnin (Fig. 10 of Ref. 5 ) . I n otherexperimentsthey 5 t o 120 min exposure. dropping t o 3-4% after19 hours. The a l k a l i n e s o l u t i o n used i n these experiments contained 0.19 H KOH and 2.1 P CsCl and had a pH a t 25' t h a t we c a l c u l a t e t o be 12.7. The removal of protons i n a buoyant density equilibrium experiment on PM2 was measured by Uang (191. He showed t h ad e p r o t o n a t i o n t was canpleted i n I Cooperative Process a t pH 11.8 11.9 a t 20'C S i m i l a r lO s t r a r d e r y and Gray (3) found t h a t t h e t r a n s i t i o n i n a s e d i m e n t a t i o n v e l o c i t y t i t r a t i o n of PM2 a t 25'c Occurred j u s t below pH 12.1. W conclude t h ad e p r o t o n a t i o n e t was canpleted i n about 10 min under c o n d i t i o n s i n which 3 t o 10% 2TR was observed.

No sequence i sa v a i l a b l ef o r PM2. However, it can be c l a s s i f i e dw i t hf d and pBR w i t h r e s p e c t t o t h e AG O f i t s most stable region fran a comparison o f i t s h i g h r e s o l u t i o n FU2 1211 and f d 1 2 2 ) linear OP fann I 1 m e l t i n a r o f i l e i t hh o s e o w t of f d and OX. # DNAs ! shorn t o have d i f f e r e n t i a l m e i t i n g p r o f i i e s i i t h a i&dthinthe range ii 8' t o 12'C and t o have as i g n i f i c a n t peak of s t a b i l i t y ' t o 5-C above t h e . , 4 T In contrast the breadth of a similar profile for WX (23) was about 3'C and had no s t a b i l i t y W , e i n f e rt h a t PM2 must have s t a b i l i t yr e g i o n sw i t h peaks more than 2'C above t he .T A G ' S canparable t o t h e maximun i n fd.

Denaturation of Covalently Closed Circular D N A

A t OC no d i r e c d e t e r m i n a t i o n d e p r o t o n a t i o n t of i s available. Calculations of t h e pHs o ft h er e a c t i o n s shown i n Figs. 2, 3 and 9 f o r fd and PMZ, and e s t i m a t e so f p~, a t O O C frm t h e a l u e o r v f Pn2 a t 20C (19) and CApHm/6Tlu and [A pHm/Alog r j l ~ determined for here 64 (Table 11) i n d i c a t e a substantially g r e a t e v a l u eo r r f (pH p b ) than a t 25OC. These denaturations thus appear be to occurring under c o n d i t i o n s r t h a t would l e a d o m p l e t e e p r o t o n a t i o n . tc d However a t O T t h e e a c t i o n was so slow t h a t t h e I0 minallowed i n t h e experiments shown i n i i g s . 1R and 1 C f o r t h e p r e p a r a t i o n o f fl'-Id from PM2 and fd was p r o b a b l yn s u f f i c i e n f o r m p l e t e e n a t u r a t i o n . i t c d As a r e s u l t s m e u n r e a c t e d form I may beincluded i n t h e c a l c u l a t e d ZlR. I n separateexperimentson PM2 u s i n g t h e s a v e a l k a l i n e b u f f e r as for Fig. 1R and IC and a IO minexposure we found a ZTR o f 15%. 5% and 0% a t O, ' C 2SC and 50C r e s p e c t i v e l y (2). I vould t r e q u i r ea method f o rd e t e r m i n i n gt h er a t e of proton removal under t h ec o n d i t i o n so f kineticexperimentstodisentangletheprocess o f s l w conversion o f I t o I d a t C ' O frm t h a t o f p r o t o n removal. The pH i n d i c a t o r method devised by Chamansand Sturdevant (25) f o rf o l l o w i n gp r o t o nl o s sd u r i n gt h ea l k a l i n ed e n a t u r a t i o no fl i n e a r DNA might beadequate f o r t h i s purpose.

6031

ZTR was noted by Porter, Kolodner An o b s e r v a t i o nt h a t appeared t o b er e l a t e dt o present i n Shi e l l ads e n t e r i a e Y6R on and Warner (26). I n f r a c t i o n a t i n g t h e plasmids a l k a l i n e sucrosegradientsamixtureofthetwosmallest ol*ll&"f Mna in molecular mass. wis obtained frm t h e gradient.^ O n - n e i t r a i i z a t i o n ' t h e 0 . 6 3 ~ M i a - p l a i m i d r e n a t u r e dc w p l e t e l y whereas t h e 1.1 Mna plasmiddidnot.Thispropertypermittedthe s e p a r a t i o no ft h e t w plasmids on aneutralgradient.Subsequently. i was found t h a t t i f t h ea l k a l i n em i x t u r eo ft h e W plasmids ?as c h i l l e d t o C to ' O and thenneutralized, both plasmids retained the I configuration. The n e u t r a l i z e d I frm t h e 0.93 Mna plasmidrenaturedveryraoidlvd a t 2S0t simolv on r a i z i n o t h e oH t o qD-11. W attrihvtp e t h i s behavior,not t ZTk wa o e; have d fe e; d i t -here: b u i io f h e " f o i a t i & ' i f c o n f i g u r a t i o ni n which, perhaps because of i t s smalsize, he ranslocation l t t i s such t h a tab r i e f exposure t o a pH i nt h e range of known r e n a t u r a t i o n o n d i t i o n s ( 2 ) i s c s u f f i c i e n t a t 25-C b u t n o t a t OC t o i n i t i a t e nucleation. Unpublishedobservations, thislaboratory.

Ionic strength
Fig. 8. Effect of i o n i c s t r e n g t h o n t h e r a t e o f d e n a t u r a t i o n o f GI-RF a t O'C a t Constant NaOH concentration. The s i n g l e - p o i n tr a t e s were measured on G4-RF a t 60 s a tv a r y i n g ionicstrengthsproduced by a d j u s t i n gt h e NaCl concentrations. The NaOH COnCentratiOn for each curve i s i n d i c a t e d on t h e graph.
I

.i-ii

E f f e coI o n i c t r e n g t h tf S Our previous measurgnents o r e n a t u r a t i o n f ( 2 ) were u > 1, p r i m a r i l yt o keep t h er a t e s measurable w i t h i n made a t ah i g hi o n i cs t r e n g t h , t h e pH range of buffering alkaline by orthophosphate. Denaturation rates were f i r s t measured (Figs. 7-4) a t a similar ionic strength. Extension lower to ionic strengths and determinationof dependence on NaOH concentration and i o n i c s t r e n g t h hasbeen done Fig. 7 shows sane sample r a t e curves f o r v a r i a t i o n o f t h e using M - R F , c h i e f l y a t 0'. NaOH concentration without added s a l t e x c e p t f o r EDTA. The curveshavethe same charact e r i s t i c s as those i n Figs. 3 and 4 and a t an i n t e n n e d i a t er a t et h es i g n o i d shape i s barely evident. A s y s t e m a t i c a r i a t i o n ifo n i c t r e n g t h c o n s t a n t v o s at NaOH i s shown Fig. 9 These a r es i n g l e - p o i n tr a t ed e t e m i n a . i n Fig. R and t h er e v e r s ev a r i a t i o ni n 60 s The t i o n si n which each p o i n t shows t h ee x t e n to ft h er e a c t i o na tat i m eo f P l o t i s e q u i v a l e n t t o a p l o t Of a first-order rate constant determined at the fraciional I ) . T h i sp l a c e sal i m i t a t i o n extent of r e a c t i o n i n d i c a t e d b y t h e v a l u e o f I n ( f r a c t i o n on analyzing the dependence o r a t e n f o u and (OH-) because o ft h en o n l i n e a r i t y of Plotsof I n ( f r a c t i o n I ) VS. time (Figs. 2. 3 . 4 and 7 ) . The upward curvature the of PlotsinFig. 8 and 9 athighnegativevaluesof I n ( f r a c t i o n I ) i s due t o t h e f a l l i n g Off of t h e r a t e a s t h e r e a c t i o n proceeds, e.g. as i n Fig. 7; w i t h o u tt h i se f f e c tt h e of p l o t s would continue t o i n c r e a s e i n s l o p e i n t h i s region. W have takenthevalues e LI and (OH-) frm each c u r va t p o i n tq u i v a l e n t eh e e t to t i e a It n f r a c t i o n ( [Y (OH')] = 0.027 2 O.%?fo; ;he 8 curves. The 1) = -0.69 and f i n d t h a t t h e p r o d u c t constancy of t h i sp r o d u c ti n d i c a t e st h e same r e l a t i v e dependence o f r a t e on each o f t h e two variables; however. t h e steepnessofthecurvessuggeststhattheactualrate i s af u n c t i o no far a t h e rh i g h exponent of [u(C")l. AP l o to fI n k VS. I n Cu(OH-)l y i e l d ss l o p e a equa1 t o t h i s exponent. Values ranging from 2.7 t o 7.8 (mean. 5.9 t 1.8) wmeobtained frm t h i s t y p e of p l o t o f t h e p o i n t s i n t h e c e n t r a l r e g i o n o f each o f t h e R curves. The l o g a r i t h i c p l o t s a r e r o u g h l y l i n e a r b u t t h e r e s u l t s a r e n e v e r t h e less crude estimates becauseof t h e problemsmentioned above. Another, p r o b a b l y b e t t e r , 7 i n whichthedenaturation estimate be can made frm t h et h r e er a t ec u r v e si nF i g . has proceeded beyond 50%. The r a t e t a ti 2 f otrh ea t t etrh r e e u r v e ss i v e n l c ig bytheexpression,k = Cr(OH-)ln k', where i s theestimatedrate, n i s t h e exponent r e f e r r e dt o above and k * i s aConstantforthethree curves. When n and k'aretaken t o be 35 and 7225. r e s p e c t i v e l y , h e . t average absolute difference between k and t h e measured r a t e i s 14%.

" 2

0.2

-3

20 40 60 80 10 0 (NoOH) , mM
120
140

1 s

135 PH

IS7

160

180

200

Fig. 9. E f f e c to f NaOH concentration on t h er a t eo fd e n a t u r a t i o no f constantionicstrength. The r a t e s were measured as described inthe 8. The i o n i cs t r e n g t hf o r each curve i si n d i c a t e d on t h e graph. Two shown f o rt h ed e n a t u r a t i o no ff d DNA a t 180s a t O'C. The i n s e t shows curve for 64 a t y = 0.49 on c o o r d i n a t e s o f pH vs. f r a c t i o n I d .

64-RF a t OC a t legend for Fig. p o i n t sa r ea l s o ar e p l o to ft h e

I1

120mM

o f a DNA which i s much slower todenaturethan For canparisonwiththebehavior t w NaOH concentrations f o r fd denaturation a t O'C, Y = 0.5 and a r e a c t i o n t i m eo f 3 min ape shown i n Fig. 9. These p o i n t si n d i c a t et h a tt oo b t a i nar a t ea b o u t o n e - t h i r dt h a to f 64 a t u = 0.49 it was necessary t o increasethe NaOH concentration more than 3-fold. The s l o p e f h e i n e h r o u g h h e o i n t s l s o ot l t t p a shows a much lower dependence o f t h e r a t e on NaOH c o n c e n t r a t i o n f o r f d t h a n f o r 64. With a further increase i n NaOH t o 0.4 M and p t o 2.0 t h er a t eo fd e n a t u r a t i o no ff d became f a s t ( t l 2 s). b u tt h er e a c t i o ns t i l lf a i l e dt o go t oc w p l e t i o n . i.e. a ZTR o f about (5%=wZ: found. R systematic examinationofthedenaturationof f d i n t h i s range of s a l t and NaOH concentrations was not made.
64. points for

the dewcan also be used t o estimate The data from Figs. 8 and 9 a tt i l ? w i t hh e n a l y s i s t a by Record et for canparison (27) Of t h e dence o f p , on H . 1 4 r l i n a t i t r n t i n n o f l i n e a r DNA. The t r a n s i t i o n oH. D, H . cannot be d i r e c t l v de-

A.

d i f f e r e n c e between pHm and pH112 i s independent of U . The appearance Of One of thecurvesonthe more conventTonal p l o t of pH vs f r a c t i o n I d i s i l l u s t r a t e d i n t h e i n s e t on Fig. 9. W f i n dt h a tf o rt h es i xp a i r s e of adjacent curves on Figs 8 and 9, (ApH,/nlog u ) = -0.9 t 0.15. ~ This value i s entered i n Table 11. I i s negative as t expectedfrom t h e r e s u l t s o n t h e a l k a l i n e d e n a t u r a t i o n of l i n e a r DNA (27) and i n d i c a t e s a d e s t a b i l i z a t i o n of t h e h e l i x by s a l t as discussedsubsequently.

30

60 Time, s

90

I20

Fig. 7. Denaturation of G4-RF a t 0 atth en d i c a t e d o n c e n t r a t i o n s ' i c of NaOH. The reactionmixturescontained EOTA asdescribed i n ExperimentalProcedureswithoutaddit i o n s of NaCl. R e s u l t i n gi o n i cs t r e n g t h sf o rt h em i x t u r e sw i t h NaOH a t 120, 140, 160 and 2011 "N_ e r e ~=0.17, 0.14, 0.21 and 0.25, respectively.

-~

Oob 400b

Fran Figs. F r m F. ; g

R and 9 2. Ref. (2)

-0.9 f 0.16

for for

u
Y

= 0.2

t o 1.3

-0.6

= 0.1 t o 1 M

-1.0

for e =

1t o 2 M

pH frm Fig. 10 and from a V a l u e sd e r i v e df r o md i r e c td e t e r m i n a t i o n so f similardataatothertemperatures and i o n i c stren!ths (datanot shown). bValuesobtained as described i n thetext.

6032

Denaturation of Covalently Closed Circular DNA

T r a n s i t i o n Reqion between Denaturation and Renaturation The sharp maximum p r e v i ously obsewed ( 2 ) i n t h e r a t e o f r e n a t u r a t i o n w i t h r e s p e c t t o pH at a given temperature becanes slow a~ t h e nH i s defined on t h e i o h h DH s i d e v b a d e c r e a r i n o a t eh a t r t appmaches t h e p.; H i n &de; t o eiamyne ~the-rel;tion"ofdenat&ationto&tu;&& in this transition region a series of experiments were done w i t h 64-RF a t temperatures s u f f i c i e n t l yh i g ht h a tr e n a t u r a t i o na sw e l l as denaturation could followed. a be At l o w Y o f 0;15 o r 0.25 r e n a t u r a t i o nr a t e sc o u l db e measured a t 40' o r 50'C a t NanH concentrations i n t h e range o f 30 t o 60 M; t h er a t e s became very slow a t 30T. A t t h e s et e m p e r a t u r e st h er a t eo fd e n a t u r a t i o n i s t o of a s tt o measure b yo u r methods. and r e ... . a.i -.n . a.u r t .n . n t An e x w r i m e n ta t 50" i s shorn i n Fia. 10 whichdisolavsdenaturation curie$~for-G4-RF f i r arangeofNafHconcentrauoni.~"The-&nts on t h ed e n a t u r a t i o n curvearestationaryvalues o f thefractionofdenaturation measured a f t e r one min and do not change f u r t h ew i t hi m e . r t The t r a n s i t i o n o m p l e t e e n a t u r a t i o ns e r y tc d iv rharp; a change i n NaMl c o n c e n t r a t i o no f 1 M i s e q u i v a l e n tt oa pH change of about ) 0 units. .1 As a e s u l t p o i n t s r . on t h e e n a t u r a t i o n u w e n h e r a n s i t i o n e g i o n d c i t t r Are d i f f i c u l t t o reproduce. The curve has am i d p o i n ta t 56 M (OH-) which defines a p , = 11.88. H

4min

\
*

I.

Effect of Ouenchins on K i n e t i c s Our standard assay forhe rogress f oth t p ob denaturation and r e n a t u r a t i o n i s a nonambient.quenchingassayemplovinq neutralization reaction. Ouenchino assavs are known t o chsnae t h e .anoarant C i n r t i r c n f t o St00 the denaturation of l i n e a r DNA ( 3 0 ) - a n d ~ c f s&e p&i& f3i';-$i;- y e zue concerned t h a tt h ec o n f i g u r a t i o n a l changes t h a t we i n f e r t o occurduringdenaturation and w h i c h a n i n a t eh ee n a t u r a t i o n i n e t i c s i g hb e o d i f i e d y e u t r a l i z a t i o n . d t r k m tm bn assay, t h i sp a r t i c u l a rp o i n tc o u l d be Although we do not have an a l t e r n a t i v e ambient i n v e s t i g a t e d s i n g h en f r a s o n i c i x i n g p p a r a t u s n d e r o n d i t i o n s f u t i m a u c o Fig. 1 . The 0 procedure n a s an extension o f t h e usualthree-dropletdenaturationexperiment (6) to a four-droplet. stage three denaturation and r e n a t u r a t i o n sequence. The neutral DNA d r o p l e t was merged w i t h an NaOH d r o p l e tt oe s t a b l i s hd e n a t u r a t i o nc o n d i t i o n sa t 50-C u = 0.15. (OH-) > 60 After 2 m i n a t h i r d d r o p was merged t h a t i n i t i a t e d r e n a t u r a t i o ; M m t i s a b y i l u t i n gh e d t (OH-) t o 40 mM o r 50 m w h i l e a i n t a i n i n g h eo n i c t r e n g t h t 0.15. A f t e rat i m e di n t e r v a l ,r e n a t u r a t i o n was stoppedasusualwith 2 M T r i s HCl and the olution s was analyzed f o r forms I and id. An experiment a t 40 rrU i s shown i n Fig. 10 w i t hc m p l e t ea n a l y s i s and o t h e r p o i n t s a t 30 M and 50 rrU determined gel by e l e c t r o p h o r e s i s .F o rc a n p a r i s o nw i t ht h er a t eo fr e n a t u r a t i o no fan e u t r a l i z e d 50'-Id on the4-nincurve on Fig. 10 a t 40 M NaOH and aratecurveunder t h e r e i s ap o i n t t h e same c o n d i t i o n s n i g . i F 1A. The i n i t i a r a t e o n s t a n t s o r h e e u t r a l i z e d l c f t n and t h u n n e u t r a l i z e Id e d were w i t h i n 3.3 t 0.3 ks; t h ea t etfh e e u t r a l i z e dd r o n I decreased more at longer intervals. experiments time Other employing analysis gel showed the same r a t e f o r t h e tw k i n d so fI dt o 70% r e a c t i o nw i t h i nt h ep r e c i s i o no f t h e method. W c o n c l u d e t h a t n e u t r a l i z a t i o n o f a d e n a t u r a t i o n r e a c t i o n e does n o t r e s u l t i n a s u b s t a n t i a l change i n t h e conformationofthe I d moleculesfranthoseestablished a t anbienttemperature. W cannot make a s i m i l a r c a n p a r i s o n o f t h e e f f e c t o f n e u t r a l i e z a t i o i i ' b nt h er e n a t u r a t i o nr e a c t i o n . However, ne havefound i n a number of cmparisons (data not shown) t h a tt h er a t eo far e n a t u r a t i o nr e a c t i o ni st h e same whether i t i s n e u t r a l i z e d a t ambient temperature o r l m r e d t o C ' simultaneously with neutralization. O

~~r't~&i

n. M

2m

30min

\j
50
I

30

40 50 (NaOH) ,mM

60

I o n Condensation Theor The a p p l i c a t i o no f Manning t h e o r yt ot h ed e n a t u r a t i o n u : ideas o ft h en a t u r e Of ionic effects the on process by of ONA (29) has revised o distinguishingthecontributionsofioncondensation and Debye-Huckel Screening and by providingasimple means f o r t h e d i r e c t c a l c u l a t i o n of each. Record and h i s c a o r k e r s (27, 33) have r e c o n s i d e r e d t h e a p p l i c a t i o n o f t h e t h e o r y t o t h e d e n a t u r a t i o n of l i n e a r DNA i nn e u t r a ls o l u t i o n and have extended i t ot h ea l k a l i n ed e n a t u r a t i o no fl i n e a r t DNA. I n b o t h cases an i m p o r t a n t c o n t r i b u t i o n t o t h e change i n chargedensityondenatur a t i o ni st h ea c c m p a n y i n g change i n l i n e a r dimension, measured by t h e a x i a l phosphate spacing. I n o u r case o f a l k a l i n e I thisspacingincreaseswiththedegree of a l k a l i n e t i t r a t i o n as found by Record ( 2 7f)olr n e a r i IINA. F otrh i s reason w have e found it convenient t oi n t r o d u c eap a r a m e t e ri n t ot h ei n i t i a le q u a t i o nf o rt h ea x i a l phosphate spacing i n t h e denatured. c o i lf o r mr a t h e rt h a na d j u s t i n g i a t t h e end of t the alculation. C The effectthe of increased negative charge resulting frm alkal i n et i t r a t i o ni si n c l u d e di n a small modification Of the treatment O f Record e t al. (27). The developnent o f equations i s o u t l i n e d b e l o w f o r t h e h e l i x - c o i l t r a n s i t i o n , character and i s t i t r a t e d helix(1) -coil(ld), in which t h e c o i l r e t a i n s i t s d u p l e x t o a maximm of k = 0.5. b ya l k a l it ot h ee x t e n t of k equivalents nucleotide per

9.

by t h e l i n e a r d e n s i t y o f f i x e d Bothioncondensation and screeningaredetennined negativechargeson DNA. T h i s d e n s i t y was defined by Manning ( 2 9 ) as

E. 11 .4
b
by . n e u t r a l i z a t i o n a y i e s c r ; b e d - f o r F i g . - i i . b - . i . (points on the curve) and o t h e r s i n g l e . t i m e p o i n t s f o r t h e i n d i c a t e d n$ber o f m i n a t 40 nM and 50 rrU NaOH; t h e 5Oo-Id used i n these experiments prepared vas without n e u t r a l i z a t i o n as described i n t h e t e x t . The f i l l e ds y n b o l si n d i c a t e . samples analyzed by t h e t a n d a r d e n t r i f u g a l s c method; t h e open s p b o l si n d i c a t e samples analyzed only by gel electrophoresis as described i n Procedures. where b = average l i n e a rs p a c i n gi n of f i x e d charges. Ue d e f i n ed = average l i n e a r any DNA configuration. Thus f o rn e u t r a l spacing o f phosphates on as i n g l es t r a n do f DNA, b = df o ras i n g l es t r a n d and b = d l 2 f o ra double-stranded configuration. For

w be the i fran l B c o n f i g u r a t i o nar e l a t i v e Since a l l changes i n b denaturation on axialphosphatespacing 1 = dcfdh w i l l be used. If therearecharges due t o t i t r a t i o n i na d d i t i o nt ot h ep h o i p h a t e c ag , h;e t h et o t a lc h a r g ep e rn u c l e o t i d ew i l lb e (1 + k ) for double-stranded coil such and the value of b w i l l be b t c = d t c / 2 ( 1 + kc) a The e x t e n o r e n a t u r a t i o n f tf o 5Oo-Id i s shornFig. on 10 a f t e r 4 min and 30 min as I d and b t c = d t c l ( l + kc) a for Single-stranded A coil. factor of I1 + kh) over range a o f NaOH concentrations; additional points shorn time are at 40 and 50 rrU for artiatitration p l of t h e e l i x i l l h w be included in reliminary quations ut p e b NaOH f o r 50'-Id t h a t was n o n e u t r a l i z e d t between denaturation and r e n a t u r a t i o n s q u a r e i ltl h e n e g l l e c t e d ( w bn because kh"kC (27). These d e f i n i t i o n sa l l o wt h ef o n u l a t i o n p i n t s ; discussed below). The curves show a maximun r a t ew i t hr e s p e c tt o pH as previof &c. t h ev a l u eo f& f o rt h et i t r a t e dc o i l formed i n ad e n a t u r a t i o nt r a n s i t i o n , as ously found a th i g hi o n i cs t r e n g t h (2); under these conditions i t occurred a t 50 rrU (OH-). e q u i v a l e n t o pH = 11.83. The maximun r a t ei s 0.05 pH u n i t s below pH,,and (3) renaturation still took place at 55 rrU NaOH. o n l y 0.01 pH u n i t s b e l w p ,The maximum r a ta t e 50 mM was confirmed bv s i m i l a r n a t u r a t i oo f a re n 0 I* for mrn o v eh e tr same range o f NaOH concentrations(datanot shown). As expected"frmpreviousresults (2). OO-Id r e n a t u r e dt oag r e a t e re x t e n t i n 2 min 50-Id than i n 4 min; t h e i n i t i a l F o ra l k a l i n el i n e a r DNA for which coil a the is separated single strand. 1 must have a rates renaturation these 1d'S cmpared of of two are at 40 IrM NaOH i n Fig. 1A. c o e f f i c i e n t Of 2; f o r e u t r a l i n e a r n DNA. kc and k h r e l s o e t a a s = 0.
~

Similar denaturation experiments i n theransition egion t r have been done a t = 0.75 300, 400 and 5ooc. At 40'. r e n a t u r a t i o n of o o - I d w s also The measured. p a t t e r no fc u r v e si nb o t hd i r e c t i o n s was s i m i l a r t o t h o s e i n Fig. 10 and t h e p s i t i o n of t h e maximum r a t e o f r e n a t u r a t i o n was a l s o 0.05 PH u n i t s below p ~ m (data notshorn). values for pbderivedfranthese curves a r eg i v e ni n Table 1 1 along u l a t e d w i t hc a l c coefficients Of (Ap+/Alog and (ApHm/AT) The former is in agreement with the Of -0.a derived frm ~ i and s u i~ . These 8 ~ t ooc. have been used below as t h o r r e s p o n d i n gf f e r e n t ica l e f f i c i e ni t s ce di o n an a p p l i c a tti o fn he i o n o n d e n s a t i o nh e o r o f c t y Manning (78, 29) t o h e t dependence of denaturation on ionic strength. Coefficients have a l s o been d e r i v e d r a n h e f f e c o s a l t f t e tf on t h e 2 of Ref. 7) assuming by t h a t p , bears H Constant a relation r a t e of r e n a t u r a t i oF i g . (n t o t h e pH o f maximum r a t e o f r e n a t u r a t i o n and aregiven i n Table 11. F o r t h e i n t e r v a l 0.1 1 M PaCl. (APHm/Alog u ) = -0.6 and for 1 2 M NaCl. = -1.0. T h i sc a l c u l a t i o n does n o t r e f e r t o t h e r e n a t u r a t i o n r e a c t i o n ; t h e r e n a t u r a t i o n r a t e was used only as a means of l o c a t i n g p b .

Eq (3) i s s i m p l yd e f i n i t i o n a of i n t e n s of t h e change i nl e n g t h and i n charge from i t s v a l u ef o rt h eh e l i x . nett?ow apply ion the condensati n c r i t e r i o no f 1 forpolYeleCtrolYtessuch as DNA forwhich 1, i n t h e Manning (28). [ ( n e t ) The f r a c t i o n of t o t 1chargeneutralizedbycondensationonapartialusual way (27) l y t i t r a t e d s p k i s i s t h e n (1 (1 + k ) and t h ef r a c t i o n a lr e s i d u a lu n n e u t r a l i z ed charge i s + kT.a k i ntgd i f f e r e n cb e t m etn e sv a l u e s hc o i l ) he e h e for e t and fto re ltih e r m e to fr h pa x e Record et ( 2 7 ) . i. equals i h i c = fractional amount O f Na* discharged per nucleotide On Therefore*

{>

t-f~

- t-')

d. l

i =(l-~')(i+k~)-(l-;~)(l+k~)- k

= &:(I

+ k) ,

- Ehl(l+kh)] - k

(4 )

I n Fig. 1(1 i t i s shown t h a t a t (OH-) concentrations 0 0 IFM o r more below t h e .3 In early t r a n s i t i o n no Id i s formed. experiments employing our standard 3-droplet procedurewiththeinfrasonicmixingdevice ( 6 ) I d was always formed up t o a f r a c t i o n o f 0.2 a t 50 m . decreasing t o an e g l i g i b l ef r a c t i o na t H 25 M. ThisformationofId was l a t e r shown t o be t h e r e s u l t ofthemixingprocesswhich was t o o slow r e l a t i v e t o theveryhighrateofdenaturationat 5O'C t o p r e v e n t t h e t r a n s i e n t f o n a t i o n o f l o c a l regionsofhigh (OH-) and Conseauent p a r t i a ld e n a t u r a t i o n . If such ar e a c t i o nm i x t u r e containing 2oi. I da t 50 mw was. a l l & t o remain a t 50-C f o r d d i t i o n a t i m e a l the 1d r a t e o f r e n a t u r a t i o n Shown i n Fig. 10 disappeared i n about 30 min, c o n s i s t e n t w i t h t h e IA was seen a t b o o r . but wac h a r o l v a t 50 rrU NaOH. The same t r a n s i e n tf o r m a t i o no f p e r c e p t i b l e a t 30*C and undetectdble a t l o w e r t e m p ~ r a t u - r e i ~ - r t c o ; l d ' b e - i v o ; d e i ' i ~ N p l e t e l y b y a floc mixing of D A and NanH followedbyraisingthetemperaturetothe under these conditions is desired value. That satisfactory ixing an m c even be slow e v i d e n tf r a nt h ed a t ai nF i g .9f o rt h ed e n a t u r a t i o n of 64-RF a t O'C and al o wi o n i c strength.Thisprwedureis.ofcourse,notSatisfactoryfor measurimJ ratesof denat u r a t i o n .b u t we have attempted not t o do t h i sa th i g h e r temperatures the than 30C used f o r F"2 DNA i n Fig. 3. The p o i n t s on Fig. 10 between 20 and 53 mM were obtained using OC m i x i n g t o e s t a b l i s h t h e i n d i c a t e d NanH concentration.

kh. The q u a n t i t yi nb r a c k e t s = q as defined by Manning (29) t o g i v e where k = k t h e d i f f e r e k e i n screening between c and h because the screening depends on t h e r e s i d u a l charge a f t e r condensation. The q u a n t i t i e s i and 7) a r e i d e n t i f i e d i n r e l a t i o n t o thennodynamic q u a n t i t i e s b y They show t h a t h ec o n t r i b u t i o n of i o n condenthereatmenof t t Record St (27) of h c ' i s RI n (Na*) and t h a t t h e c o n t r i b u t i o n o f s c r e e n i n g i i s a t i o n t o t h e AG Thus t h e c o n d e n s a t i o n c o n t r i b u t i o n s t a b i l i z e s , w h i l e t h e i s (- 71 1 2 ) RT ?kS(Na*). s c r e e n i n g o n t r i b u t i o n e s t a b i l i z e s t h e e l i x i t h e s p e c t o h e o i fl o r i n e a r c d . h w r t c l 7)1 2 ) RT I n (Na'). Experimentalvaluesfor DNA. The n e te f f e c to f (Na+) b e c m s ( i differentiacoefficients erived ran he l d f t thennodynamlc treatment (27) y i e l d i ,and C m b i n i n gt h ed e f i n i t i o n o f q frm eqs. (4) and ( 5 ) w i t h eq. (21 and n g l l e c t r n g we o b t a i n

'

9 .

' ?Eh+

(6 1

Denaturation of Covalently Closed Circular DNA

A value of 1 fran eq. t h ep a r m e t e r sd t cb t c behavior. Eq.*(6) 'applies providedacoefficient b are employed.
(6) ca be used w i t ht h ed e f i n i t i o n sp r e s e n t e d above t oo b t a i n and tc, t h a tc h a r a c t e r i z et h e COY1 and I t sp O ~ y e l e c t r o l Y t e to oth eutral b n and a l k a l i n e e n a t u r a t i o n lfi n e a r d o ONA and t h ea p p r o p r i a t ed e f i n i t i o n so fd and of 2 i s a p p l i e dt o1
t be seen i n Table 111 t h a t b t c and are approximately l i n e a r ONA (27). I can Constant btc Since = ( l l l + k ) Zbh. the increase i n 1 i si n e a w i t h i r (1 + kc). The same'result was noted 6ecord by & (27) f o rt i t r a t e dl i n e a r DNA. A direct

& i1

a.

canparison n i t h I d can be canbe made using

made b yc a l c u l a t i o n

Of

if frm t h e i r data.

I n both cases

The a p p l i c a t i o n o f t h i s r e s u l t t o t h e I - I d t r a n s i t i o n

derived by Record e ta l . (27). T h i sd i f f e r e n t i a lc o e f f i c i e n t has been taken t o equal ApHmlAlog L f o r uh??hThe value -0.91 was derived frm ourdata(Table 11). Combining t h i s r e s u l t w i t h eqs ( 5 ) (6) and (7) we o b t a i n ? = 0.045 and 1 = 1.19 a t p b where T h i s r e s u i t ha; been extended i n Table 111 t o t h e c a l c u l a t i o n Of 7) and a l l k = 0.25. r e l a t e d parameters f o r r b i t r a r y a l u e s k a v of on the assumption t h a t eqs. (4). ( 5 ) and (7) a r e v a l i d o v e r t h e e n t i r e t i t r a t i o n .

Table I11 I o n condensation and screeninq parameters f o r t h e a l k a l i n e d e n a t u r a t i o n ofform

i n d i c a t e s t h a t t h e same fraction of the total charge on t h e c o i l the constancy o f remains unneutralized condensation. since total by but the charge i s increasing. the actual net charge a l s on c r e a s e s u r i n gh ei t r a t i o nT h i s e s u l t i d t t . r may provide an electrostatic basis the for molecular extension of the single-stranded DNA. I n the case of I d we a t t r i b u t e i i n p a r t t o t h e t magnitudes Of t h e average nunber of base pairs per turn (the equivalent of a heIical repeat) Of the noninteracting single strands about each other a tt h et i g h t n e s s o f winding forces formation that the Of p a s i t i v e supercoils. the Since increase i n l e n g t h i s s i m i l a rf o rl i n e a r DNA and f o rI dt h e differences i n t h e f f e c t e of s a l t on the alkaline denaturation the derive of tno mainly frm thedifferences i n c o n f i g u r a t i o n o f t h e r e s p e c t i v e c o i l species. The fact t b a t I d r e t a i n s i t s duplex character results i n i t s r e t e n t i o n of a high charge density. = 4.3 4.6, s l i g h t l y g r e a t e r t h a n t h a t OF the double stranded helix. AS a r e s u l t but increases the residual as net charge i s increased by t i t r a t i o n . The 7 1 2 . thus Stabilizes the coil form as i n other DNA transitions. However. the increased charge density and the increase i n t o t a l charge before condensaso t h a t i i s negative the over t i o n ccubine to reverse ion the condensation effect are up during denaturation rather being than e n t i r et i t r a t i o n and Na+ ions taken released. The canbinedparameter (i- 7 1 2 ) is. o f course. s t i l l more negative because both condensation and screening cmbine to Stabilize the coil. I n contrast it r m a i n e d o s i t i v e o r h e l k a l i n ei n e a r p f t a l DNA t r a n s i t i o n and only becme negitive toward t h e end of t h e t i t r a t i o n (Fig. 1 of Ref. p 7 , 34) where 7) i s large.

L &i

I DNA

Quantities havebeen c a l c u l a t e d f o r a r b i t r a r y v a l u e s o f t h e f r a c t i o n a l t i t r a t i o n , k . frm theexperimental[Ap&/Alog(Na+)lT as explained i n t h e t e x t ,

40% increase i n l e n g t hf o r The value Of 1 f o rt h ef u l l yt i t r a t e dI di m p l i e sa the double-stranded This coil. is c o n s i s t e nw i t hh e i g h o s i t i v e u p e r c o i l i n g . t t h p S An actualvalueforthesuperhelicaldensitycannotbecalculatedwithout an estimate of the effective helical repeat. Q u a l i t a t i v e l y t h e i o n condensationtheory accounts f o r theeffect of s a l t on the denaturation Of form I ONA. The differences between t h e a l k a l i n e denaturation of of t h e two c o i l l i n e a r and c i r c u l a r ONd t h a t depend on t h ed i f f e r e n c ei ns t r u c t u r e f o m s r e a r t i c u l a r l y e ld e f i n e d a P w l by the theory. There i s as y e t no independent confirmation Of the quantitative predictions dimensional of change. I i s assmed i n t t h e above CalCulationSthattheaxialchargedensity s t i l l determines i o n condensation according t o t h e Manning theory i n s p i t e o f t h e h i g h l y compact, supercoiled arrangment of the double-stranded c o io f l Id. Rough c a l c u l a t i o n s of the averaae s m a r a t i o n between charges on d i f f e r e n t segments of a coiled-updoublestrand i n i n eqhivalent hydrodynanic spherethe of size o f I i n d i c a t e sh a t t it i s much greater the than axial phosphatespacing and probably w not modify ion condensation. i l The value of t h e coefficient (ApH /AT) given i n Table I I predicts a A H O f o r denaturation of about 8 kcal bp et&loyfng the r e l a t i o n o f Record & per (27).

k 0.018 0.1 0.2 0.25 0.045 0.3 0.072 0.4

0.5

7
0.036

1 1.08 1.15 1.19

dtc 3.7 3.9 4.0 4.2 4.4 4.7

btc 1.7
1.6

ii
0.23

i-712

-.8 00
-0.16
-0.20

-0.09
-0.18 -0.23 -0.27 -0.36 -0.45

0.23
0.23 0.22 0.22 0.22

1.6 1.6 1.6 1.5

0.054

1.23 1.30

-0.25
-0.33 -0.41

0.090

1.38

fi.

2 I n Table 1 of Record e t a l . (27) thevaluesgivenfortheaxial phosphate spacing are identical with those cTccuTated frm t h e i r dataemployingour eq ( 6 ) and t h e r e l a t ed definitions. However. i n h e a b l e h i s u a n t i t y t t t q i s designated.as which bps in (1 + k ) for single-stranded DA N: Ue have Freferred to our terminology equals btA n m b e r of conclusions can be drawn frm Table 111 and frm acmparison of t h er e t a i nt h eo r i g i n a ld e f i n i k i o n of b 129) as an average spacing charge per and t o include r e s u l t sw i t ht h o s e Of Record e t a1 (27) f o rt ha l k a l i n ed e n a t u r a t i o n of l i n e a r DNA e x p l i c i t l yt h eq u a n t i t y , (1 + k c ) ,t oa d j u s t i t o a per t nucleotide basis where necessThe axial phosphate separation of as described i n Table I and F G . 7 ' o f Ref. (27)t. i se s s e n t i a l l yt h e same as l~',)"defined by Record St. (27). I d ,d t c .i n c r e a s e sw i t hi n c r e a s i n gt i t r a t i o n Only a l i t t l e l e s s t h a nt h a tf o rt i t r a t e d ary.

E;: