j. biol. chem.-1986-camien-6026-33

8/3/2019 J. Biol. Chem.-1986-Camien-6026-33

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THE JOURNAL0 1986 by The American Soeiety of Biological Chemists,OF BIOLOGICALHEMISTRY Inc Vol. 261,No. 13, Issue of May 5, pp. 60264033,1986Printed in U.S.A.

Denaturation of Covalently Closed Circular DNAKINETICS, COMPARISON OF SEVERAL DNAs, MECHANISM AND IONIC EFFECTS*

(Received for publication, August 6, 1985)

Merri ll N. Camien and RobertC. Warner$Fro m the Depa rtme nt f Molecular Biology and Biochemistry, Universityof California, Iruine, California92717

The rates of the alkaline denaturationof the cova-lently closed, circu lar DNAs (form I) of the replicativeforms (RF) f phages G4,4X174, and fd, andf plasmidpBR322 and phage PM2 have been measuredat 0 "Cand some at higher temperatures. These rates re or-de rs of magnitude slower than the denaturation oflinear DNA because of the increased stability of thehelix to d eprotonation that resu lts from the accumu-lating positive superhelic ity during denaturation.De-naturation reactions were initiated by rapid, infra-sonic mixing (Camien, M. N., and Warner,R. C. (1984)Anal. Biochem. 138,329-334), and their progress wasmeasured by analytical ultracentrifugal analysis forthe amounts of form I and denatured (Id) DNA afterneutralization of the alkaline eaction.The comparative ates of the fiveDNAs varied overa wide range; the fastest, G4-RF, denatured a t 500-fold the rate of th e slowest, fd-RF. The differencesareaccounted for by the interaction of positive superhel-icity with the sequence-dependent regions of relativehelix stability in the variousNAs. Rena turation ratesof I d DNAs varied similarly for hs prepared at 0 "C,but onlya few-fold for Ids prepared at50 "C.The rate of denaturation of G4-RF was determinedover a wide range of NaOH and NaCl concentrationsa t 0 "C, and the pH, was determined as a function ofionic strength and temperature. The effects of ionicstrength have been analyzed in a n application of theManning ion condensation-screening th eory (Manning,G. S . (1978)Q.Rev. Biophys. 11, 179-246) which isshown to account for the large destablizing effect ofsalts on the helix. The pH region of transition at 50 "Cfrom renaturation to denaturation wasexamined, andit was found that the maximum rate of renaturationoccurred at a pH about 0.05 units below th e pH,.

The denaturation of circular form I1 DNA in alkaline so-lution differs in a umber of significant ways from the alkalinedenaturation of its linear or nicked counterpart. The under-standing of these differences and their rigin in theopological*This research was supported by National Institutes of HealthGran t GM28769 from the National Inst itu te of General MedicalSciences. The costs of publication of this article were defrayed in par tby the payment of page charges. This article must herefore be herebymarked "aduertisement" in accordance with 18U.S.C. Section 1734solely to indicate this fact.$ To whom correspondence should be addressed.The abbreviations used are: form I, covalently closed, circular,duplex DNA; RF, replicative form; form 4, denatured form I DNA;form I,, relaxed form I DNA; form I,, superhelical form I; form 11,nicked form I; EtdBr, ethidium bromide; bp, base pairs; ZTR, zero-time renaturability; p, ionic strength, O"-Id, Id DNA prepared bydenaturation at 0 "C or other indicated temperature. 5, itratablesuperhelical density.

constraint imposed by covalent closure is chiefly due to thework of Vinograd and his colleagues and has been reviewedby Bauer and Vinograd (1). Other aspects of the uniquebehavior of form I DNA in alkali have come to our attentionas a result of our recent study (2 ) of the kinetics of renatura-tion of form b rom 4X174-RF and have motivated an inves-tigation of the kinetics of the denaturation reaction.The principal effect, in this egard, of the treatment f formI DNA with alkali was the large dependence of the rate ofrenaturation on the conditions under which the alkaline de-naturation had been carried out. This was demonstrated bythe following observations (2); (i) the rate of renaturation ispseudo-first-order; i.e. it is independent of the DNA concen-tration, but falls off from an initial first-order rate as thereaction proceeds; (ii) the rate of renaturation depends on thetemperature at which the denaturation is carried out and onthe supercoil density of the form I DNA used to prepare Id;and (iii) the molecules of I d are heterogeneous in configurationand renature at different rates. Point iiigives rise to thecharacteristics of the reaction indicated in iand ii. Thedifferences in configuration among the Id molecules are estab-lished during the denaturation reaction.It is also known that there are arge differences in the atesof renaturation of Id from the form I of different DNAs.Preliminary work indicated that this is also true of denatur-ation rates o a degree not observed for linear or nicked DNA.Differences were noted (2 ) between the closely related RFs ofphages 9x174 and G4. More substantial differences betweenPM2 DNA and other form I DNAs were shown by Ostranderand Gray (3). When PM2 DNA was denatured in alkali, afraction renatured to form I simply on neutralization withoutexposure to annealing conditions such as those required for4X-Id. We havemade similar observations on chloroplastDNA (4) and on PM2 DNA (2). Lau and Gray (5) comparedthe renaturation of +X-RF, X and PM2 DNAs and foundthat XDNA shared this property. Two additional examples,fd-RF and pBR322 DNAs, are reported in this study. Thephenomenon is apparently common and is dependent on theconditions of denaturation (2 , 5, this study); we refer to it aszero time renaturation (ZTR).During the course of our renaturation studies, preliminaryobservations were made indicating that the ate of denatura-tion of +X-RFI under some conditions of alkalinity andtemperature canbe slow,4 in the ange of 1-3000 s. A similarslow denaturation of PM2 DNA was noted by Lau and Gray(Fig. 10of Ref. 5). These rates are rders of magnitude slowerthan those for denaturation of linear DNA, but may still betoo fast to measure by the methodology we used for renatu-ration rates. We have designed and constructed an infrasonicmixing device (6) which permits the rapid initiationandquenching of microliter volumes of a denaturation eaction a ta constant temperatufe and thus theeasurement of reaction

6026


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Denaturation of Covalently Closed Circular D N AI Denoturation 1

-Renaturation-,f9, Z T RL-l k-Iti) I i ( j )-IItW - e c I d ( l )[Neutral 1 ~ I Z G i q -supercoiled, form I , I1, , ircularitrofedIs, rrelaxed,partiallyD N A

Multiple species varying in configurotion

6027

S p e c i e s th atretain residual S p e c ie s th e t a nneutra l izat ionprotonoted regionsand yield I on y ie ld I d exceptfar the formationneutrolizotion of I by Z T RFIG.5 . Minimum scheme to account for the kinetics of denaturation and renaturation and for theproperties of the denatured species. Reactions b-d are those of denaturation: accumulation of positivesupercoiling and deprotonation of thymine and guanine residues remaining in the duplex configuration. Reactionse and g are neutralizations. Reaction g, ZTR, we have defined as that fraction of Id(j) and I(k) of some DNAsdenatured a t low temperatures that assume the form I configuration on neutralization without the exposure to theparticular renaturation conditions required for species which is indicated by reaction f.The distinction betweenintermediate species I(i) and &) is th at provided by our assay for the progress of denaturation. The parentheticaldesignations, (i), (j), ( k ) , l ) , are included to indicate that such species have multiple configurations and thereforedifferent properties with respect both to further denaturation steps and to enaturation. Neither the necessity ofexposure of neutral Id to annealing conditions nor the possible formation of intermediates in eaction f are indicatedon the diagram.

rates having half-times as low as a few seconds.We report a study of the dependence of the rate of dena-turation of G4-RF on temperature, onic strength, and NaOHconcentration and an examination of the dependence of de-naturation and renaturation on NaOH concentration in therange in which the transition between the two takes place.Comparative observations on the rates of denaturation andrenaturation of G4-, 4X-, and fd-RFs and pBR322 and PM 2DNAs have been correlated with local relative stabilitiescalculated for the four DNAs for which sequences are avail-able.

EXPERIMENTAL PROCEDURES A N D RESULTSDISCUSSION

The Denaturation Reaction-The characteristics of dena-turation of form I DNA shown in Figs. 2-4 and their elationto the renaturation of form Id shown in Fig. 1 can be under-stood by an extension of the analysis of these reactions givenin our previous study (2). t was there shown tha t the onlin-ear first-order plots of renaturation rate and thedependenceof this rateon the conditions of denaturation were a result ofthe formation during denaturationf a range of configurationsof the denatured species differing in the rates atwhich theycould be renatured.These configurations wereviewed asarising during the denaturation reaction by an interaction ofthe increasing positive superhelical density with the primaryhelical windings (1, 2, 14-16). Positive supercoiling acts as abarrier to deprotonation and denaturation of the residualhelical regions and is responsible for the low and easily

Portions of this paper (including Experimental Procedures,Results, parts of Discussion, Figs. 1-4 and 6-10, Tables 1-111, andadditional Footnotes 1 and 2) are presented in miniprint at the endof this paper. Miniprint is easily read with the aid of a standardmagnifying glass. Full size photocopies are available from the Journalof Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814.Request Document No. 85M-2632, cite the authors, and include acheck or money order for $8.80 per set of photocopies. Full sizephotocopies are also included in the microfilm edition of the Journalthat is available from Waverly Press.

measurable rate and theigher pH of denaturation of circularas compared with linear DNA. Random configurations arisingearly in the eaction do not have time to relax and equilibrateas denaturation proceeds, leading to a heterogeneous range ofkinetically trapped configurations.A minimal reaction scheme for both denaturation and re-naturation spresented n Fig. 5 . It includes operationaldefinitions of the classes of intermediate species that accountfor the sigmoid nature of the logarithmic rate curve and otherfeatures of both reactions. The first step, reaction a, repre-sents the initial part of the alkaline sedimentation titrationof form I (14). The rapidly sedimenting species that accumu-late during this titration are designated I& and I(i). A dis-tinction between these two species is made in order to explainthe lag in the initial denaturation rate shown in Fig. 4. Thedistribution among the I(i) species is stochastic; except forthe heterogeneity in linking number, all I& molecules havethe same potentiality, but variations in the initial randomconfigurations commit the molecules to different subsequentconfigurations at different rates.The distinction between I(i) and Id(j) species is hatdefined by the reversibility criteria of Rush and Warner (17)and Ostrander and Gray (3). It is the same distinction asprovided by our quenching assay for denaturation; Id speciesare detected after neutralization by their more rapid sedimen-tation. If reaction c is sufficiently slow then a lag phase willbe observed. If the configurational changes taking place dur-ing the initial stages of the alkaline titration are ime-depend-ent o a degree tha t contributes to he measured rate ofdenaturation, it may not be possible to distinguish betweenI& and I species. A range of rates for the conversion of Ispecies to Id species leads to the decrease in the measuredrate of denaturation as the reaction proceeds (Figs. 2-4). Therange of rates results both rom the inherent heterogeneity oflinking numbers among the molecules of form I andfrom theconsequences of the heterogeneity of I(i) species. Th e Id(j)species are further maturated configurationally than I(i) inthe sense that they are committed to denaturation, but stilldiffer from Id in configuration. Their existence is indicated by


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6028 Denaturat ion of Covalently Closed Circular D N Athe fact that the pH limit above which only Id is formed onneutralization duringan alkaline sedimentation titration of Ioccursbefore the changes n sedimentation coefficient arecomplete (2 , 3, 17).Two pathways of renaturation, f and g)must be distin-guished. The neutral Id is distinguishable from the alkalineform by ts lower sedimentationcoefficient and lower buoyantdensity. It requires exposure to defined annealing conditionsof specified alkaline pH and temperaturen order to renature(2). This pathway is shown as leading to Ialk because of ourprevious demonstration that the proximate renatured specieshas the same sedimentation coefficient as I& at the pH andtemperature of renaturation (2). The other pathway, g, alsotermed ZTR, is included because for certainDNAs a fractionof the alkaline 4 renatures on neutralization as discussedabove.3

Acknowledgment-We are indebted to Scott B . M ulrooney for thepreparation of most of the D NA s used in this study.REFERENCES

1. Bauer, W., and Vinograd, J. (1974) in Basic PrinciplesofNucleicAcid Chemistry (Ts'o, P., ed) Vol. 1, pp. 265-303,AcademicPress, New York2. Strider, W., Camien, M. N., and Warner, R. C. (1981) J. Biol.Chem. 256,7820-78393. Ostrander, D. A., and Gray, H. B., Jr. (1973) Biopolymers 1 2 ,

4. Kolodner, R., Tewari, K. K ., and Warner, R. C. (1976) Biochim.5. Lau, P. P., and Gray, H. B.,Jr. (1980) Nucleic Acids Res. 8 , 673-6. Camien, M. N., and Warner, R. C. (1984) Anal. Biochem. 1 3 8 ,7. Harned, H. S., and Owen, B. B. (1958) The Physical Chemistry

of Electrolytic Solutions, 3rd Ed., pp. 638 and 697-764, R ein-hold, New York8. Wheeler, F. C., Fishel, R. A., and Warner, R. C. (1977) Anal.Biochem. 78,260-275

1387-1419Biophys. Acta 44 7, 144-15570 1329-334

A previously unnoted error appears in our earlier paper (2) . Thestated pH values at which the data shown in Fig. 4 and those in Fig.5 were obtained were inadvertently reversed. Th e correct pH for Fig.4 is 11.35 and that for Fig. 5 is 10.95.

SUPPLEMENTt oDNAs. MECH ANIgl AND IONIC EFFECTS

M e r r i l l N C an ie n an d R o be rt C WarnerbyEXPERIMENTALPRDCEDURES

DENATURATIONFOVALENTLY-CLOSEDIRCULARNA: KIN ETIC S. CMPARISON OF SEVERAL

RF and otherc i r cu la r DNAs. in c lud ing samples re laxedwi th opo isanerase 1, wereprepared as p rev io us ly escr ibed ( 2 ) e x c ep t f o r h em i s s i o no f h e S e ph a cr yl c o l m ne q u i l i b r i u mg r a d i e n t s . FU2 DNA was ag i f t o f Or . Richar d Kolodner; nother sample o fs t e p i n p r e p a r in g pBR322 DNA I n a l l c a se s o r m I was pu r i f ie d by he useof E tdBr -CsClmethods and denat u ra t io n and rena tu ra t ion rocedures were those rev ious ly escr ibedPM2 DNA ws purchased fr an Boeringer-Hannheim. Oth er r e p a r a t i o n s ,l t r a c e n t r i f u g e(2 ) e x c ep t f o r h e u s e o f r a p i d .n f rason icm i x i n g . T he c o n s t r u c t i o n f h em i x i n gdev ice and i t s use fo r measuring denatu ra t ion a tes have een repor ted (6 ) . Sone ofthe ena tu ra t ion a tes epor t ed here were measured us ing he o l d e r m ix ing methods (2).

The reac t ionm i x t u r e o r i t h e r e n a t u r a t i o n or r e n a t u r a t i o n , n l e s s t h e r v i s em l i n NaOH a t a n i n d i c a t e d o n c e n t r a t i o nwi th EDTA a t 4.67 nN to m i n i m i z en i c k i n go fi n d i ca t e d . h ad a i n a l v o l u n e o f 45 el and cons is ted of appro x ima te ly 33 eg o f DNA pert h e DNA a t l k a l i n e pH snd w i th NaCl inc luded to d ju s t h eo n i c t r e n g t h . E ac hrepor ted NaOH co we nt ra t io n has been cor r ec te do rh em o u n t f NaOH c o n sm e d i nt i t r a t im j he EDTA. The repor ted on ic t reng th s , mpute d in he usua l manner , a rebased on concentrat ions of Na+H- C1 - and EDTA4-. Uhe re pHs ofh e s e I o l u t i o n rar eg i v e n , h e y w e re c a l c u l a t e d fran' the NaOH conc ent rat ion and ion ics t r e n g t hu s i n gth ea l u e sor pKw and ac tiv i ty o e f f i c i e n t s th e p p r o p r i a t ee m p e r a t u r e ( 7 ) .Other pHs were measured as previouslyd e s c r i b e d ( 2 ) . The rea ctlo ns were quenched wi tha 5-10 el drop of from 0.5 t o 4 M T r i s H C I, c o n t a i n i n g t w e q u i v a l e n t so fTr is HCIfo r each equ iv a len t f NaOH i n the e a c t i o nm i x t u r e . Sane experiments on renatura-c o n t a i e d 1 M NaCl. 4.67 nN EDTA and 77 d4 phosphate witha a r y i n g a t i o Of H P O j -t i on were done i n h e NaC1-phosphate buff er rev ious ly used (2). These solut io sto P049- depending on the pHand had an ion ic s t ren g th o f appro x ima te ly 1.3.

D e n a t u r a t i o n e a c t i o n m i x t u r e s n t e n d e d op r d u c es u f f i c i e n t d o r e n a t u r a t i o ns tud ie s were repared as descr ibed above excep t w i th oncent ra t ionsof OV A i nc reasedt o as much as 1 mg pe r m l. The product was d i a l y s e da g a i n s t 2 nNDTA (pH 8 ). d i l u t e dt o a p p r o x i m a t e l y 50 pg pr m l w i t h 2 rM EDTA (pH 8 ) . a nd s t o r e d a t O'C.

9. Smiley, B. L., and Warner, R. C. (1979) Nucleic Acids Res. 6 ,1979-199110. Tinoco, I., Jr., Borer, P. N., Dengler, B., Levine, M. D., Uhlen-beck, 0.C., C rothers, D. M., and Gralla, J. (1973) Nature NewBiol. 246 , 40-4111. Denhardt, D. T., Dressler, D., and Ra y, D. S. (eds) (1978) TheSingle-Stranded Phages, pp. 139, 659 and 671, Cold SpringHarbor Laboratory, Cold Spring Harbor, N Y12. Sutcliffe, J. G. (1979) Cold Spring Harbor Symp. Quant. Biol.4 3 ,77-9013. Funnell, B. E., and Inman, R. B. (1979) J. Mol. Biol. 131, 331-34014. Vinograd, J., Lebowitz, J., Radloff, R., Watson, R., and Lapis, R.(1965) Proc. Natl. Acad. Sci. U. S. A. 5 3 , 1104-111115. Vinograd, J., Lebowitz, J., and Watson, R. (1968 ) J. Mol. Biol.33,173-19716. Schmir, M., Revet, B. M. J., and Vinograd, J. (1974) J. Mol. Biol.

17. Rush, M. G., and Warner, R. C. (1970) J. Biol. Chem. 2 4 5 , 2 7 0 4 -270818. Pouwels, P. H., Knijnenburg, C. M., van Rotterdam, J., Cohen,J. A., and Jansz, H. S. (1968) J . Mol. Biol. 3 2 , 169-18219. Wang, J. C. (1974) J. Mol. Biol. 89, 783-80120. Bauer, W. R. (1978) Annu. Rev. Biophys. Bioeng. 7, 87-31321. Lyubchenko, Y. L., Frank-Kamenetskii, M. D., Vologodskii, A.V. , Lazurkin, Y. S., and G ause, G. G., Jr. (1976) Biopolymers22. Tachibana, H., Wada, A., Gotoh, O ., and Takanami, M. (1978)23. Lyubchenko, Y.L., Vologodskii, A. V., and Fran k-Kamen etskii,24. Borer, P. N., Dengler, B., Tinoco, I., Jr., and Uhlenbeck, 0. C.

83 , 35-45

15,1019-1036Biochem. Biophys.Acta 517,319-328M. D. (1978) Nature 27 1 , 2& 31(1974) J. Mol. Biol. 8 6 , 8 4 3 4 5 325. Chapman, R. E., Jr., and Sturtevant, J. M. (1970) Biopolymers9,445-45726. Porter, B. W., Kolodner,R., and Warner, R. C. (1973) J. Bacteriol.

27. Record, M. T., r., Woodbury, C. P. , and Lohman, T. M. (1976)116,163-174Biopolymers 15,893-91528. Manning, G. S. (1978) Q. Rev. Biophys. 1 1 , 179-24629. Manning, G. S. (1972) Biopolymers 11,937-94930. Hamagu chi, K., and Geiduschek, E. P. (1962) J. Am. Chem. SOC.31. Levy, M., and W arner, R. C. (1954) J. Phys. Chem. 5 8 , 106-10932. Warner, R. C., and Levy, M. (1958) J. Am. Chem. SOC.0 , 5735-33. Record, M. T., r., Anderson, C. F., and Lohman, T. M. (1978)34. Record, M. T., Jr. (1967 ) Biopolymers 5 , 993-1008

84,1329-1338

5744Q. Rev. Biophys. 11 , 103-178

Gel e lec t roph ores is was conduc ted as p rev iou s ly escr ibed (8) except for he useofaHoeferminigel apparatus.Thismethod was used as a means of oca tin g eg ion so fc r i t i c a lo n i c t r e n g t h a nd a l k a l i n i t y o r r a n s i t i o n s b et we en t h e I and Id-DNAs.Forms I a nd I d g i v e d i s t i n c t , w e l l- s e pa r a te d b an ds then r un i n Ebuf fe r (8 ) c o n t a i n i n gI eg/mI Ether . The cmple teness o f i t he r a d e n a t u r a t i o n re n a t u r a t i o ne a c t i o ncan bed e te r m in e d b y t h i s m et ho d t o w i t h i n 11. T h e r e l a t i v e m o u n t s o f he two cmpo-n e n t s a t n t e r m e d i a t e d e g r e e sofconversion. where given, were determined by canparisono f hep h o t o g ra p h o f h e g e lw i t h h a to fa s e t o f DM standards and area c c u r a t e oabout t 15%.

those used for canparins'C a l c u l a t e d S t a b i l i tp St ab i l i t y maps were onputed y methods s im i la r oare based on th e AGs f o i b a se s t a c k i n go f RNA dup lexesder ived by T inoco e t a i (In)etemdup lexes Of 1 X and G4 wi th he ir sequerces9) Theyf r a n e q u i l i b r i u n n a s U r e m t S on shor t RNA duo lexes of kn ow sequence. ThFs&nceso f thehree phage Mus ' pRR322 was determinedySutc l i f fe (121 .he ca l ll i d i n g a v e r a g e Of th e bGthe lrnath n f th e m n l w w l mas done byFunne l l and Inm an ' i l3 j oF A + T ontent. The values o f AG p e r b p a t 25-cwere taken fm , t h e a b l e o fTincco eta l . (10) and averaged fo r bp 1 t o n, 2 t o ~ + 1etc., through 70C4 t o N+6999. The ses l ia i ng averages were p lo t te daga in s t bp pos i t io ;on th etandard sequence usi ng e k t m n i x 4051 Graphics Systan. The ca lcu lat ion

molecu le ve have used. t ha t of fd-RF. The graphs thus epeata f te r he nunber o f basew as c o n t i n u e d a r b i t r a r i l y o 7000 bp because th i s s a rg er han he a rg es t sequencedreduced t o 150 o r 75 the peaks and val l ey s became more frequent and the maximum t AGp a i r s n h em o l e c u l e s r ea ch ed . On maps d isp layed i n F ig . 6, N - 300; when N wasexcurs ions ran he mean were approx imate ly doub le hose fo r N = 300.

~~~. -.

seems reasonab le to use them fo r DNA a t e a s t n as much as theyp r o v i d eap l a u s i b l eIt i s as h o r t c a n i n g h a t h e AG s employed here were deri ved or RNA; however, itr e l a t i v ew e i g h t i n gor he tack in g o f the a r ious onb ina t ions o f base pa i r s TheS t a b i l i t y p r o f i l e s o b t a i n e d n h i s way a rev e r ys i m i l a r o h o s e ye have Cons thc tedon th ebasi s of unveighted A + T frequency. These maps ar enot shown bu t ou r p m f i l ef o r I X may beconpared w i t h ha t o f Fu nn e l l and l runan (13) .


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Denaturation of Covalently Closed Circular D N A 6029RESULTSAN0 URTHERDISCUSSION

d e t er m i n e d o t h a t a i x e d ,e l a t i v e l y In, o n i c t r e n g t h (II 0.15, Fig. 1A) andDenatura t ion and Renatu ra t ion - R e n a t u r a t i o nat eurves re shown i n F ig . 1u n d e r h e i g h o n i c t r e n g t h o n d i t i o n s u se d p r e v i o u s l y ( 2) (F igs . 1B and IC). Widedl i f fwences i n i n i t i a l r a t e s are ev iden t amma I,+DNAs DreDared under he sane c o n d i t i o n s. .. . ~~~~t h a n f 5 0' -I d seen i n h e 0X curves o f F ig. 14 was Roted previously (2) and i sfrm d i f f e r e n tor m I DNAs. The StrinGly j r e a t e ra t e f e n a t u r a t i o n o f o o - I dThe fd u rvesofF i g . 1B p l a t e a u ?t r e n a t u r a t i o n e v e l so fa p p r o x i m a t e l y 4RI. and 79%seen here toc h a r a c t e r i z e h eo t h e r h r e e ONAS, a l t h ou g h it i s es s p ronounced i n G4.f o r h e 5 o"I d a nd o * - i d , e s p e c t i v e l y , l t h o u g h h e n i t i a l a t e o r h e d * 4 di s se en t o b e l o s eoh a to r QX O*- Idnderheseond i t ions . PM2 oo- ld wasth e mo s t r a p i d l y e n a t u r a b l eo f h e ONAS f o r w h i c h a t e sHeredeterm ined and the o n l yone f o r w h i c h e n a t u r a t i o n t O T cou ld be demonstra ted . I t s t rea tm ent t O w i t ht h e h i g h 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 . ICa t e i t h e r pH 11.9 or pH 12.0 res ulte di n n e a r l y c m p l e t e e n a t u r a t i o n n 2 0 m i n d a t a n o t s ho un ).

Time ~IFig . 1. F i r s t r d e r l o t s s ho wi ng r e n a t u r a t i o n a t e s m ea su re d a t 50C f o r a r i o u s dDNAs A. Renatura t ions i n 40 mM NaOH p=O.I5. The Id's were preparedyenaturingI, D i n s o r 1 m i n a t ooc i n 200 nt(( eaOH, ~= O. ZS or Do-1d's and a t j o o c i n 65 m~NaOH, y = 0.15 f o r h e 5 0 " - I d ' s . u p e r p o s i t i o n fhe 64 OO-Id and th e CIX 50"-Idc u r v es i s c o i n c i d e n t a l . 8 R e n a tu r a ti o n s i n i o h o n i c t r e n o t h D h oc nh at e b u f fe r . OH11.03 (see Exper imenta l rocedures) . The 0X re i a t u ia t io i u r ves re shorn as dashedlin es . The 0X 50'-I c u r v e i s b a se d on a i n g l e o i n tn t e r p o l a t e d frm th eurvea t pH 11.35 fo rh eO 0 - I d ' s a nd a t pH 10.65 f o r he OO-Id 's . The ren a tu ra t i onshown i n F ig . 3s. Re?. (2). C. R e n a tu r a ti o n s i n h i g hon ic t ren g th phosphate bu f f e rcurve shorn f o r PMP D*- Id i s an estimate based on theobserva t ion of c m p l e t ed e n a t u r a -1.78 ti NaCl and neu t r a l i zed then d i c a t e de m p e r a t u r e . T he h i g ho n i c t r e n g t ht i o n n 1 minute . The Id 's used i n B and C were ena tu red o r 10 m i n i n 0.1 U NaOH,b u f f e rat pH 11.35 has the pH found (2) o b eo p t i m m o r h e e n a t u r a t i o n o f 0 X a t5 V C . The othe r pHs used i n R and C were e lec ted ino r d e r oa c h i e v es l o w e r e a c t i o nra tes .V e r t i c a l d as he d l i n e s n d i c a t e b r ea k s i n h e tme scale.

~ ~ . ~ . " ~ . . ~. _ . .

o n t h e r d i n a t e or O" -Id o f fd-RF in F ig . 18 . I n d = -0.69, ZTR = 50%; and forThe phenmenonof ZTR i s shown in th e r e n a t u r a t i o n c u r v e s a s t h e z e r o - t i m e n t e r c e p tO'-Id of PM2 i n Fig. IC , I n d = -0.28, ZTR 5 25%. ZTR disappeared when the DNA wasdeoatu rsd a t S O T a s seen i n h e 5 0 ' 4 ~ urves f o r h e same DNAs i n Fios. 1B and IC.The dependence of Zm 0" the tempera t& o f d & t u r a t i & s ho wn b y ~ f d n d-% ? i s s i m i l a rt o h a td i s c u s s e dby Lau and Gray (5 ) where i t s shown tha t ZTR i s p resen tn e i t he r i nPM2 denatured a t 50'C or h i g h e r (2 , 5 ) no r i n A OW denatured a t 2 5 T o r h i g h e r ( 5 ) .o f F ig . 18 and 1 C gave a ZTR o f 90%.I n a separate experiment i t was hown th a t o'-Id of pBRDNA prepared under the Cond i t ions

.~~.

range o f ena tu ra t ion a tes f bou t 00- fo ld was found. In h is sma l l sample thereWhen the e na tu r a t ion a tes of th es e ive DNAs were campared (Figs. 7 and 3 ) ai s a h ar p d i v i s i o n n t o h e a s t a t e s f ou nd w i t h 64 and qX DNAs and the ery mucht en de d t o b e a m ong t h e a s t e r o e n a t u r e , l t h o u g h t r i c t reverse o r d e r f a t e sslo wer ate s hat har act eri ze fd. PER and PM2NAs.NAs th a t were s low to denaturewas no t found . Re laxa t i on f uperco i l i ng G4- Ip u rve) reduced th e a t e - a changei n h e sam e d i r e c t i o n a s i t s e ff e c t on t h e a t e f e n a t u r a t i o n .(2).he e f fec t ftempera tu re on t h e rate o fd e n a t u r a t i o nof fd-RF and pM2 DN A i s shown i n F i g. 7. andc a p a r a b l e d a t a f o r OX- and G4-RFs and pBR322 DN A are p r e s en t e d i n T a b l e I.

0

-0.6W

.- -1.2E+0=- -1.8-2.4

0 2 0 400 600T i m e , sFig. 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 a t e s m ea su re d f o rsev eral DNAs a t Oo i n 4 0rrEl NaOH a t "-1 36 The 64 I c u r v e e f e r s o G I- RF r e l a xe dby rea t -nen tw i th opo isme-r a s e I. The ias(ed l i n e foF fd DNA was ex tra pol ate d ran he d ata n Fig. 3. Itc o i n c i d e sw i t h i n h ep r e c i s i o no f h ed a t aw i t h w oe x p e r i m e n t a l p a i n t s o p e nc i r c l e s )for PH2ONA.

0 80 160 240 1800 3600Time, sof PMZ and pBR DNAs a t 2OC i n 40 nM NaOH a t ~'1.36.Fig . 3. F i r s t o r d e rp l o t s o f dena tu ra t ion r a t e sof fd-RF a t several emperatures and

i s a p pr o xi m at e dby the pBR c u rv e i n F i g. 2. It i s m ore c l e a r l y seen in he d e n a t u r a t i o nZTR i s e v i d e n t n a d en a t u ra t i o n curve by a e c re a se i n th er a t e to zero.hi s

o f d a t 3OoC i n F ig . 3 Where no f u r t h e rde nat ura tio n was observed when the ime wasex tended evera l - fo ld beyond the m inimum requ i red o r he la tea u t I n I s -1.62,ZTR - 20%. ZTR was observed w i t h l l h r ee DNAs hav ing lowates f ena tu ra t ion ,b u t w i t h n e i t h e r o f h o s e h a v in g a a s t a t e .Rate u rves o r QX - and G4-RFs are horn i n Fig. 4 on an expanded t im e ca le od e f i n e mo re c l e a r l y h e n i t i a l a gb a r e l y s ee n i n F ig . 2 b e f o r e h e a t e r ea ch es i t smaximum value. The maximum is f o l l a a d b y a e cr ea se i n a t e t h r ou g h ou t h e e s t fth eeac t ion . In c o n t r a s th ee n a t u r a t i o na t ea l l s f fo n t i n u o u s l y fran i t s

t h e a t e s i nd ep en d en t o f n i t i a l ONA concent ra t ion , as shown fo r en a tu ra t ion (2 )i n i t i a l v a lu e F ig . 1). T he r e a c t i o n s p s e u dc - f ir s t r d e r i n b o t h d i r e c t i o n s n h a t( d a t a o r d e n a t u r a t i o n n o t s h o r n ) .Tab le 1

H a l f - t i m e s of denatu ra t ion o f @X- and 64-RFsHeasurenentswere made i n 40 nM NaOH a t h e n d i c a t e d e n p r a t u r e u n d e rth e same con d i t ion s as g iven f o r Figs. 2 and 3.

H a l f- t i m e i n s a t tempera tu re. 'CON& 211' 10' 5 - 00@X-RF 9.1 17.2 1M64-RF.8 4. 4 11.7pBR322 12.9 1350

- ""i nc rea sed he ha l f - t ime measured a t 5C t o 18 .6 s fo r G4-RF and t o 204 s fo rReducing he NaOH co nc en tra t io n o 30 nM (o th er cond i t ion s unchanged)LIY-PF.

0 7 14 21Time, s (upper curve)

I I I I0 30 60 90 120 150T ime , sF i g 4 Rates a fd e n a t u r a t i o n o f RFs o f PX and 64. Rat eCurves re horn a t 0' andS9C'in 40 nM NaOH a t u.1.36. T he t i n e c a l e o r h e 5' 6 4 c u r v e . n d i c a t e da l o n g h et o p i g h t o f t h e i g u r e , i s shif ted and expanded wi th esp ect 10 t h a t n d i c a t e d o rth eo the r wo curves. The t ine s c a l e s w ere s e l e c t e d o l l u s t r a t e h es i g n o i d c h a r a c -t e r i s t i c o f h e securves.


5/8

6030 Denaturat ion of Covalently Closed Circular D N Aand rena tu ra t ion based on Fig. 5 i n h e i r s ts e c t i o n o f t h i s p ap ermust eurtherRate of Oenatura tion - T he g en e r a l i s c u s s io n o f h e i n e t i c s o f d e n a t u r a t i o nd ev el op ed i n o r d e r o d e n t i f y h e a c t o r s d e t e r m i n i n g h e a t e i m i t i n g s t e p n d en at u-ra t i on and the easons o r he d i f fe rences amongDNAs i n 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 nand rena tura tion . The concept O f t r a n s l o c a t i o n (2 , 3, 5 18) a p p l i e d i n h e d i s c u s s i o no f o ne s t r a n d wi t h esp ec t o ts canp lement as thedoub le -s t randed o i l on f igura t iono f the ena tU ra t ion o f the !d o f 0 X Waga in be used . T rans lo ca t ion is he s l i p p a g er e p l a c e she a t ive up lex and the x ia le n g t h fh e o u b l e t r a n dncreases h ist u r n n h e o i l o v e rh e e l i x e t e r m i n e sh e x t e n t f o s i t i v e u p e r c o i l i n g Th ei n c re a s e i n l e n g t h r m or e p r e c i s e l y h e n c r e a s e n he average number of b p perinc rease in he average ax ia l uc leo t ide pac ing on d e n a t u r a t i o n i s p re d i c t e d ' t o b eo f n u c l e a t i o n a nd t h e r e f o r e h e a t e - l im i t i n gs t e po f e n a t u r a t i o nw i t h h ed e g r e eo frough ly 40% ( s e e e c t i o n on Ion Condensa tion heory) . We asso c ia t eh e r o b a b i l i t yt r a n s l o c a t i o n ( 2 ) and we wi u se t h e a t e o f e n a t u r a t i o n o f d i f f e r e n t ONAs o r o f t h et r a n s l o c a t i o n i mp os ed i n t h e r e c e d i n g e n a t u r a t i o ne a c t i o n .a r g er a n s l o c a t i o nsame DNA u n d e r i f f e r e n to n d i t i o n s a s a u a l i t a t i v en d i c a t i o n fhe degreefr e s u l t sn ow a te as i n 50D-!d whereas a ma l lr a n s l o c a t i o n i e l d sh ea str a t eo f OO-Id. We now add th e case o f no t r a n s l o c a t i o n o i e l d ZTR T h i s r o v i d esam e c h a n i s t i c n t e r p r e t a t i o n f ZTR a s t h e e s u l t o f comple te epro tona t ionw i thou tt r a n s l o c a t i o n .o f DNA i s t h e d e p r o t o n a t i o n o f t h y m i n e and guanine res idues i n r e l a t i o n o t h e v a r i a b l e sThe p r i m a r y e v e n t h a t d e t e rm i n e s t h e e x t e n t and ra te o f a n y a l k a l i n e d e n a t u r a t i o no f pH. then h e r e n t t a b i l i t y f e l i c a l s eg me nt a nd i t s c o o p e r a t i v i t y .nhec a se o f c l os e d, 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 m u st b eadded.he f ree nergy fh i s u p e r h e l i c i t y wi i n c r e a s eh e e t t a b i l i t y fhe l i ca l sequence so t h a t it w requ i re igher pH t o becane depro tona ted (15) A ta pH a bo ve pH a t w h ic h we measure the a te he r eee n e r g yc o n t r i b u t e s o h ee n e r g yde te rm ina t ion f ro ton e lease nder qu i l ib r ium ond i t ions i s g iven y ;he buoyanto f a c t i v a t i o n a nd g i v e s h e e a c t i o n h e h a r a c t e r f l o w e p r o t o n a t i o n A d i r e c tc o n t r a s t h e b e h a v i o rof a samplFof-PPI2 ha vin ga h i g h u = -0.15 w i t h h a t of a e laxedd e n s i t y i t r a t i o n s f V in og ra d e t 11. (15) and ofW a g (19). The l a t t e r i t r a t i o n ssample. u = -0.008. T he d e l a y i n d e p r o t o n a t i o n a nd t h eg r e a t e r o o p e r a t i v i t yo f h ep ro ce ss , r e s u l t i n g r a n h e h i g h e r + B o f th e I d fr a n h e I, sample arec l e a r l ye v i d e n t(Fig. 3 of Ref. 19) .d i f fe ren t DNAs it must be recognized . th a t n I DNAs having normal nega tive alue sI n e x a n i n i n g h e a t e i m i t i n gs t e p nd e n a t u r a t i o n o r h e p ur po se of canpar ingo f 3 m ost o f h ed e p r o t o n a t i o n , o s s o f s t r u c t u r &he l ix and inc rease i n sed imenta t ionThe ONA depro to na tedd u r i n g h i s p ha se o f h e e a c t i o nw i l l n c l u d e h e e a s t - s t a b l ec o e f f i c i e n t wi o c c u r a p i d l yo v e r h e e v e r s i b l ep a r tofa e d i m e n t a t i o n i t r at i o n .AT-rich egion s as shown by a ons ider abl eb o i yofe v i d e n c e r a ndenaturation mappin;and h igh- reso lu t ion herma l me l t ing s tud ies . A canpar ison o f he change o f sed imenta t ionc o e f f i c i e n tw i t h h ec r i t e r i o n o f r e v e r s i b i l i t y n h e i t r a t i o n s o f Rush and Warner(171 and of Ost rand er and Gray (3 ) shows tha t he con f igur a t io na l changes as represen tedT h e p o s i t i o n o f h i s pH lmit may be used t o make n i n d i r e c t e s t i m a t e o f t h e c ha ng eby the sed imenta t ion c o e f f i c i e n t a r e 80 t o 9 0% c a n p l e t e a t he pH lmit of r e v e r s i b i l i t y .i n b u o y a n t e n s i t y a nd i n A 26 a t h a t o i n t n h e i t r a t i o n . T he b u o y an t e n s i tyt i t r a t i o n o f V in og ra d 9 l-..f!5) of olyoma DNA was done u nder he same c o n d i t i o n sa s t h e i r 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 (14) and comparisonfach w i thhe pHlmit i n d i c a t e sha t pprox imat e ly 80% o f the epro t ona t io n was canp le ted a t ha tl i m i t . I n making th is compar isona l lo w a nc e m u st b em a de f o r h e a c t h a t h e i t r a t i o n s15 . 19) as discussed by Ostrander and Gray (3). A s i m i l a r e s u l t s s ee n i n h e s pe c-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 ( 3 .t r o p h o t a n e t r i c i t r a t i o n f 0X-RF sh or n i n F ig . 7s of our rev ious tudy ( 2 ) whereit s superimposed on a e d i m e n t a t i o ni t r a t i o n b t a i n e d n d e rhe same cond i t ions .TheseConsTderat ions uggest %a t he a te o f dena tu r a t ion i n I i s d e t e m i n edp r i n c i -Here again!about 90% of the A 60 h an ge w as c a n p le t e d a t h e pH l i m i t o f r e v e r s i b i l i t y .p a l l yd u r i n g h ed e p r o t o n a t i o no f h e a s t 10-2oX of hemol&u le , o r respond ing t ot h i s t a g et s e n e r g yfct iva tion, EA, for o n p l e t i o nfe n a t u r a t i o n i l l b et h e e a c t i o n n d i c a t e d a s I ' A I ' d n F ig . 5. When am o l e c ul e o f agiv en DNA reac hesdetermined y th e AG o f t h e n h e r e n t s t a b i l i t yo f h e a s t c o o p e r a t i v e unit augmentedb y h e AG o f u p e r h e l i c i t ya t h ep a r t i c u l a r d eg re e o f c o nv e rs io n o f h e l i x o o i lt h a t h a r a c t e r i z e sh a t t a g e fh ee a c t i o n . h e r ew i l l b e a i s t r i b u t i o n f ' E Aamong them o l e c u l e s o r h e e a s o n s i s c u s s e d a bo ve g i v i n g i s e o h eh e t e r o g e n e i t ya t he same degree f epro tona t ion and g iv e ise o he low er a te shown fo r I,o f e a c t i o n a t e .R e l a x a t i o no f h en eg at iv e= as i n 1 w i l l i n c re a s e h ep o s i t i v e 3o f 64 i n F i g. 2. The d i f fe rences among mo lecu les re hus t o be a t t r ib u te dp r i m a r i l yt o h e c a n p a r a t i v e s t a b i l i t y o f h e m os t s t a b l e e g i o n o f e ac h.

I n c a n p a r i n ghe i f fe ren t ONAs we assume thathe form Is ,of each has thesame n e g a t i v e B and there fo reat ac a n p a r a b l e r a c t i o n a ld e p r o t o n a t i o nabo ut he sameh e l i c a l AG t o h e E A.he i n d i c a t i o n r a n h e a t a o l l e c t e d nB a u e r 's e v i e w 2 0 )p o s i t i v e F , a n d a s a e s u l t a l l have bou t the same con t r ib u t io no fpos i t i ve super -denature, PPI2 and fd (eq u iv a le n t o M13) . have more negative 3 ' s ha n 0 X, t h a t s . ni s h a t h e a r i a t i o n n s s m a ll a nd t h a t h e DNAs i n o u r s am pl e t h a t a re l o w t ot h e w ro ng d i r e c t i o n o e x p l a i n h e i r s l o w a t e s o f d e n a t u r a t i o n .l e sw i t h e s p e c t o e g i o n so f g rea te r and l e s s e rs t a b i l i t yu s i n ge x p e r i m e n t a l d e n a t u -Canpar ison w i th Stability Haps - It s m p o s s i b l e o c a n p a r e d i f f e r e n t DNA m o le cu -r a t i o n m aus e x c eo t i n h e m o st q u a l i t a t i v e sense. The cond i t ion s o f den a tu ra t ion reno t tandard ize d ; hed e g r e e so f 'dena tu ra t ion examined a re a r iab le , and thed a t aar eno t u f f i c ie n t ly x tens ive . We have used sequenced DNAs and have ca lcu la t ed tab i l i t ymaps as described i n Experimental mcedures and shown i n Fig. 6. A l l f h e m ap sth e AG va lues f o r h e most s tab le r e g i o n s between th e DNAs i n our small sample thatavepronounced maxima and minima o f AG. T he re i s a c o r r e l a t i o n o f th e magn i tude fhave low a tes f ena t u ra t ion andhow ZTR and th os ehat have h ig h a te s and noZTR. OX and G4 have maximum G's cl o se o 2.7 kca l and do no t have excurs ionsra nt h e i r mean g r e a t e rha nh o s e fhe an da n sequence, whereas fd and PER. th e twoexamples of hes l o w l y d e n a t u r i n gcla ss, have maximum AG ' S t h a ta re g r e a t e r b y 0.1 and0.4 kca l. esp ecti vely . When th e maps der ived ran unn ing average o fN = 150 bpand N = 75 bp (see Exper imenta l rocedures) were s i m i la r l y analyzed , the i f fe renceN = 150 and from 0.3 t o 0.8 k c a l o r N - 75. The absence of an ef fe ct of t he averagebetween the low ly and the ap id lyd e n a t u r i n gc las ses ranged f ran 0.2 t o 0.5 k c a l o rAG o f a DNA on the a te , seen i n canpar ing fd and PUR, i s cons is ten tw i t h h ep r o p o s a lt h a t h e a t e s d e te r m in e d y t h e m o st s t a b l e 10-20% o f h e m o l ec u le . It does notthe maps, e.g.. OX and 64. Th is may be due to h e inadequacy ofo u ra s s m p t i o n h a tappear t o be p o s s i b l e o e l a t e h e a t e s o f h e DNAs w i t h i n h e s l o w o r a s t c l a s s ou i s the same fo r a l l o f he DNAs or because th e a t e s dependenton thebreadth andd i s t r i b u t i o n o f h e s e v e r a l s t a b i l i t y e g i o n s o f a mo lec u le as we l l as the max imum AG.

No sequence isa v a i l a b l e o r PM2. However, it c a n b e c l a s s i f i e dwi th d and pBRw i t h r e s p e c t o h e AG O f i t s m os t s t a b le e g i o n r a n a c om pa ri so n o f t s h i g h r e s o l u t i o nm e l t i n a r o f i l e i t hhose o f fd and OX. FU2 1211 and fd 1 2 2 ) linear OP fann I 1DNAs ! s ho rn t o have d i f f e r e n t i a lm e i t i n gp r o f i i e s i t h a i & d t h n t h e r an ge ii8' t o 12'C a nd t o ha ve a i g n i f i c a n t p ea k o f s t a b i l i t y 4 t o 5- C above the T I nc o n t r a s t h e b r e a d t h o f a s i m i l a r p ro f i l e o r WX (23)was abo ut 3C and had no s t a b i l i t yAG ' S canparable to the max imun i n f d.peaks more t h a n 2'C above t h e T i n f e r h a t PM2 m u st a ve s t a b i l i t y e g i o n sw i t h

F ig . 6. C a l c u l a t e dde na tur at ion maps for ou r sequenced DNAs and a anpute r-gener atedrandan sequence . The o rd ina te i s AG p e r bp i n k c a l a l c u l a t e d a s a l i d i n g a v er a gefo r each 300 bp over hemolecu le as descr ibed in Exper imenta lProcedures The abscissai s h e n u nb e r f pra nhe onvent iona l zero f o r h e s eq ue nc e T he ' s o l i d i n e sth eaverage AG per bp fo r he e n t i r em o l e c u l e a nd t h edo tt ed 1in;s show th e p o s i t i o n sof f 0.2 kca l and 0.4 k c al i n AG f r a n he average. The sequences th at re ho rt ert h a n 7 kb pa r e e p e a t e da f te r he end o f th e mo lecu le .w h ic h i s n d i c a t e d b y a narrowThe sign of AG hasbeen changed f ra n ha t n he a b le s ran wh ich th e maps were d e r iv 2(10)because a l l of the d iscu ss ion focuseso n t h e e a c t i o n h e l i x - c o i l .These canpa rirons re made usin g maps based on stac king nerg ies t 25C ( 1 0 )They were i n P ar te r i v e d frrn thermodynamic measurements on o l i g o n u c l w t i d e s i:the empera tu re ange 0' t o 4O0C byBorer e t a1. ( 2 4 ) . Since he n t rop ies b ta ined

i n t e r a c t i o n s h em a g n i t u d e o f t h ed i f f e r e n c eb e t w e n h e m ax im a on a map as weasfo r he tack ing f GC p a i r sa r e m or e n e g x i i F t h a n t h os e o r AT p a i r so r o r m i xe dd a t a h a t h ed i f f e r e n c e n 66 between the maxima for 64 and fd wi be increased byt h e av er ag e s t a b i l i t i e sw i l l b e r e a t e r t OC t h a n t 2 5 O C . We e s t i m a t e r a n h e s eabout 0.1 k c a la t DOC. On t h e t h e r h an d. a e v e l i n go f he peaks and va l leysH o u l dt h e a r g e i f f e r e n c e r e v i o u s l y o t e d ( 2 ) b et we en t h e a t e o f e n a t u r a t i o n f " I dbe xpec ted a t 50T. Th is ma l lr e nd i n AG wi th empe ra tu re may be espons ib le o rand 5O0-Idf @X and found here to o ld o r the r DNAs (Fig. 1) When the ario usONAs ar e an par ed hed i f f e r e nc e s i n a t e a r e s ee n t o b e l a r g e on iy among th e OO-Id'Sren a tu r ing 5n '- Id 's. The t r a n S l o C a t i o nat 50C at he im eo f h e i n a ld e p r o t o n a t i o ni n wh ich he sequence dependent e f fec ts redomina te and t o be ma l l mng he low lyi s e x pe c te d t o b e g r e a t e r b m a u s e o f h e g r e a t e r he r m a l e ne rg y, t h e more r a p i d e a c t i o nrespec t to he emper a tu re f ena tu ra t ion washown t o be chieved y 25C and t oa nd t h e o w e r t a b i l i t y o f he sequence . The change in en a t u r ab i l i t yo fg x - l dw i t hOC d isappears as the ena tu ra t ion e m p e r a t u r e i s r a i se d . o r A DNA 25-C was foundb e n t e r m e d i a t ea t1 0 T ( 2 ) . S i m i l a r l y , ZTR i n h e D NAs t h a t a r e s l w o d e na t ur e a tt o be su f f i c ie n t y Lau and Gray ( 5 ) ; PM2 re qu ir ed 50C (2 5 and Fig. lC) as di d fdOC and ar e not e v i d e n t u n d e r h e c o n d i t i o n s o f r a p i d d e n a t u r a t i o n a t 50 C.(Fig. 1R). Thus, th e consequences of ow 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

by a s i m i l a r a nd n o n n a l F O fa p p r o x i m a t e l y -0.06 and do not appTy to I w h i c h y i e l d sA of hesecompar isonsar e made mongDNAs i n wh ich he I fo rms a re charac te r izeda m uc h m o re t h e o s i t i v e u p e r h e l i c a l d h a n h a t r a n I . T he & a n i n a n t f f e c to f h e m or e p o s i t i v e z m a y b e t h a t h e d e n a t u r a t i o n o f I i: mre c o o p e r a t i v e (19)as discussed above. This would le ad o r e a t e r r a n $ o c a t i o nh a nn I and (0 at h a thei g h e ro s i t i v eu p e r h e l i c & yfe n a t u r e d I a l t e r st st r u c t u r esl o w e re n a t u r a t i o na te Of I d f ro m I as p r e v i o u s l yo u n d (2). Addit iona? evi 'dences ho wn b y i t s h i g h e r e d i m e n t a t i o n o e f f i c i e n t ( 1 6 ) and ?ts grea te r uoyant ens i ty(19).

Z e ro i m e R e n a t u r a b i l i t - ZTR was associatedmon g he ive DNAs stu die dw i t has low ra te 0 d en at ur at io n k d f orh er a c t i o n fh edo te n a t u r e dte rot o n d i c a t e h e a bs en ce o f a ny s i g n i f i c a n t r a n s l o c a t i o n h e n t h e i t r a t i o n s c a n p l e t et ime, w i th : fast ra te o f rena iu ra t ion F ig . 1). W have in t e rp re t ed h is phenanenonp re su m ab ly e ca us e t h e l r e a d y i t r a t e d o i l DNA i s so t i g h t l y w ou nd i n t h e s e . mo rei s p o s si b le . h e r e i s d i r e c ts u p w r t o r h i s v ie w i n e xp er im e nt s on PPI2 near 25OCsta h le DNAs tha t he ina lp r o t o n o s so c c u r s na o n f o r m a t i o n nw h i c h no s l i p p a g eLau and Gray (5) d e t e r m i n e d a t e f e n a t u r a t i o n t 25T f o r h e a s t 1 4% o f th ;change t o 120 rnin (Fig. 10 of Ref. 5 ) . I n other xper iments hey found 10%ZTR a f t e rProcess and showed a lo w e v e l i n g f f o b o u t 3% ZTR af te r 10 min and no u r the r5 t o 120 min xposure. ropping t o 3-4% a f t e r19 hours . The a lk a l i ne s o l u t i o n u se d i nthese xper iments on ta ined 0.19 H KOH and 2.1 P CsCl and had a pH a t 25' t h a t wec a l c u l a t e o b e 12.7. The removal of protons i n abuoyantdens i tyequ i l ib r iumexper imenton PM2 was measured by Uang (191. He showed tha t epr oto nat ion was canpleted i n ICooperativerocess at pH 11.8 - 11.9 a t 20 'C S im i l a r ly s t ra rd er and Gray (3 )f ou nd t h a t h e r a n s i t i o n n a s e d i m e n t a t i o n v e l o c i t y i t r a t i o n o f PM2 a t 25'c O c cu r re dju s t be low pH2.1. We conc lude tha t epro to na t ion wa s canp le ted i n about 10 m inu nd er c o n d i t i o n s n w h ic h 3 t o 10%2TR was observed.


6/8

Denaturat ion of Covalently Closed Circular D N A 6031the pHs o f he eac t ions shown in F igs . 2 , 3 and 9 f o r fd and PMZ, and es tim ate so fA t OC n o d i r e c t e t e r m i n a t i o n f e p r o t o n a t i o n i s a v a il a b le .Ca lcu la t ions fp~ , a t OO C fr m t h e a l u e o r Pn2 a t 20C (19) and CApHm/6Tlu and [A pHm/Alog r j l ~dete rm ined e reor 64 (Tab le 11) i n d i c a t e substantially g r e a t e r a l u eor (pH -p b ) than a t 25OC. These denatu ra t ion sh u s p p e a r o e c c u r r i n g u nd er c o n d i t i o n st h a t h e I0 mi na l lo w e d i n h e e x p e r im e n t s s ho wn i n i g s . 1R and 1 C f o r h e p r e p a r a t i o ntha t wou ld lead o m p l e t e e p r o t o n a t i o n . H ow ev er a t O T h e e a c t i o n w as so slowo f f l ' -Id from PM2 and fd was pro bab lyn s u f f i c i e n to r mp le te ena tu ra t ion . As amentson PM 2 u s i n g h e s a v e a l k a l i n e b u f f e r a s f o r F ig . 1R and IC and a IO mi nexposurer e s u l t s m eunreac ted fo rm I m ay b e n c l u d e d i n h e c a l c u l a t e d Z l R . I n s e p a r at eexper i -we found a ZTR o f 1 5 %. 5% and 0% a t O'C, 2SC and 50C r e s p e c t i v e l y (2). It v o u l dr e q u i r e m et ho d f o r e t e r m i n i n g h e a t e o f p r ot o n r em o va l n d er t h e o n d i t i o n s fk i n e t i c x p e r i m e n t s o i s e n t a n g l e h e r o c e s s o f s l w c on ve rs io n o f I t o d a t Ofrm t h a t of pr ot on removal. The pH in di ca to r method devised by Chamansand Sturdevantbeadequate fo r h is purpose.(25) f o r o l l o w i n g r o t o n o s s u r i n g h e l k a l i n e e n a t u r a t i o n f i n e a r DNA m i g h t

An o b s e r v a t i o n h a t a pp ea re d t o b e e l a t e d o ZTR was noted by Porter,Ko lodnerand Warner (26). I n r a c t i o n a t i n g h e p la s mi ds r es e nt i n S hi e l lad e n t e r i a e Y6R ona l k a l i n e s u c ro s egrad ien ts am i x t u r eo f he wosmallest ol*ll&"fMna i nmo lecu la r mass. wi s obta ined fr m 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 dr e n a t u r e d w p l e t e l y w he re as t h e 1.1 Mna plasmiddi dn o t .T h i sp r o p e r t yp e r m i t t e d h es e p a r a t i o no f h e t w p lasmids on a eu t ra l rad ien t .Subsequent ly . it was found th ati f h ea l k a l i n em i x t u r eo f h e tWo p l a s m id s ?a s c h i l l e d o O and thenneut ra l i zed ,b o t hl a s m i d se t a i n e dhe I c o n f i g u r a t i o n . T he n e u t r a l i z e d I frm th e 0.93 Mnap lasmid ena tu redvery ao id lvd a t 2S0t s i m o l v on r a i z i n o h e oH t o qD - 1 1 . W a t t r i h v t pt h i s b e h av i or ,no t to ZTk ae have deed i t here: b u i i o h e " f o i a t i & ' i f .i-iic o n f i g u r a t i o n n w h ic h, e rh a ps e ca u se f i t s s m a l l i z e , h e r a n s l o c a t i o n i s s uc ht h a t r i e f e x po s ur e t o a pH i n h e r a n ge f k no wn r e n a t u r a t i o n o n d i t i o n s ( 2 ) i ss u f f i c i e n t a t 25-C b ut n o t a t OC t o n i t i a t e n u c l e a t i o n .Unpublishedo b s er v a ti o ns , t h i s a b o r a t o r y .

made a t a igh on ic t reng t h , u > 1, p r i m a r i l y o k ee p t h e a t e s m e as ur ab le w i t h i nE f f e c t fo n i c t r e n g t h - Our p rev ious measurgnents o f ena tu r a t i on ( 2 ) weremeasuredFigs. 7-4) a t a i m i l aro n i c t r e n g t h .E x t e n s i o noo w e ro n i c t r e n g t h sthe pH range o f u f fe r ing y lka l ine r thophospha te .D e n a t u r a t i o n a t e s w er e f i r s tand de te rm ina t ionof dependence on NaOH concentrat ion and ionic s t r e n g t h h a sbeen doneus ing M - R F , c h i e f l y a t 0. Fig. 7 shows sane sample ra te curves f o rv a r i a t i o no f h eNaOH con cen trat ion with out added sal t exc ept or EDTA. he curves have he same charac -t e r i s t i c s as those i n F igs . and 4 and a t an in ten ned ia te a te he igno id shape i sbare ly v iden t . A s y s t e m a t i c a r i a t i o n fon ic t reng th t ons ta n t NaOH i s showni n F ig . R a nd t h e e v e r s e v a r i a t i o n n F ig . 9. These a re ing l e -po in t a ted e t e m i n a -t i o n s n w hi ch a ch p o i n t sh ow s t h e x t e n t f h e e a c t i o n t i m e f 60 s TheP l o t s e q u i v a l e n t o a p l o t Of a i r s t - o r d e r a t e c o n s t a n t d e t e r m i n e d a t h e r a c i i o n a le x t e n t of r e a c t i o n n d i c a t e d b y h ev a l u e o f n f r a c t i o n I ) . T h i sp l a c e sa i m i t a t i o non ana lyz inghe dependence of ate n u and (OH-) because o f he on l in ea r i t y o fP l ot s f I n f r a c t i o n I ) VS. t ime F igs . 2. 3 . 4 and 7 ) . The upward cu rvatu re f heP l o t s n F i g . 8 and 9 ath i g hn e g a t i v ev a l u e so f I n f r a c t i o n I ) i s due t o h e a l l i n gOff o f t h e a t eas he ea c t i on p roceeds , e.g . as i n F ig. 7; w i t h o u t h i se f f e c t h ep l o t s w ou ld c o n t in u e t o n c r e a s e ns l o p e n h i s r e gi o n. We h av e t a k e n h e a l u e s ofLI andOH-) fr m each curvethe o i n tq u i v a l e n to t i a tn f r a c t i o ncons tancy f th is rodu c t nd ic a tes he same re la t i ve dependence o f a t e on each o f1) = -0.69 and f i nd ha t he p r o d u c t [Y (OH')] = 0.027 2 O.%?fo; ;he 8 curves. Thethe two var ia b les ; however. the s teepness f he u rves ugges ts ha t he c tua l a tey ie lds lope equa1 to h is exponent . a luesangingrom 2.7 t o 7.8 (mean. 5.9 ti s a u n ct io nofa a t h e rhig h exponent f [u(C")l. AP l o to f n k VS. I n Cu(OH-) l1.8) wm eobta ined frm t h i s y p e o f p l o t o f h ep o i n t s n h ec e n t r a l e g i o no f eacho f h e R c ur ve s. T he l o g a r i t h i c p l o t s a r e o u g h l y i n e a r b u t h e e s u l t s a r e n e v e r t h e -less c r u d eestimates becauseof the p roblemsmentioned above. Another, pro bab ly bet ter ,estimat e an e made fr m t h e h r e e a t e u r v e s nF i g . 7 i n w h i c h h e e n a t u r a t io nhas proceeded beyond 50%. T he r a t e t ti 2 f o rh ea t t e rh r e e u r v e ss i v e nb y h ee x p r e s s i o n , = Cr(OH-)ln k', where k i s thee s t i m a t e d a t e , n i s h e e x p o n e n tr e f e r r e d o a bo ve a nd k * s aCons tant or he hree curves. When n and k ' re akenmeasured ra te s 14%.t o be 3.5 and 7225. respe c t ive l y , he average abso lu te i f fe rence be tween k and the

I I I1-120mM

0 30 60 90 I20T i m e , sFig. 7. Denatura t ion f G4-RF a t 0 ath en d i c a t e do n c e n t r a t i o n s o f NaOH. T her e a c t i o nm i x t u r e sco nta ine d EOTA asdescr ibed i n Exper imenta lProcedureswithoutaddi-t io ns o f NaCl. Resu l t ing on ic t reng ths o r hem i x t u r e swi th NaOH a t 120, 140, 160and 2011 "N_ e r e ~= 0 .1 7 , 0 .1 4, 0.21 and 0.25, r e s p e c t i v e l y .

Ionic strengthFig. 8. E f f e c t o f i o n i c s t r e n g t h o n h e r a t e o f d e n a t u r a t i o n o f G I- RF a t OC a t C o n st a ntNaOH conc entr atio n. The si ng le -p oi nt at es were measured on G4-RF a t 60 s a t a r y i n gioni c tren gths rod uce d by adj us tin g he NaCl concentra t ions. The NaOH COnCentrat iOnf o r e ac h c u r v e i s i n d i c a t e d o n t h e g ra ph .

I

r

"2 -0.2

-3 1s 1 3 5 IS7PH2 0 40 60 80 100 120 140 160 180 200(NoOH) ,mM

Fig. 9. Eff ect f NaOH con cen trat ion on the a te f ena t u ra t ion f 64-RF a t OC a tcons tan t on ic t reng th . The ra t es were measured as escr ibed in he legend o r F ig.8. T he i o n i c t r e n g t h o r e ac h c u r ve i s n d i c a t e d on th e graph. Two poi nts re lsoshown f or hed e n a t u r a t i o nof d DNA a t 180s a t OC. The in se t shows a ep lo to f h ecurve o r 64 a t y = 0.49 on coo rd in a te s o f pH vs. f ra c t i on d .

64 . p o i n t s o r t w NaOH conc ent rat i ons f o r f d d e n a t u r a t i o n a t OC, Y = 0.5 and a eac t ionFor c a n p a r i s o nw i t h h eb e h a v i o r o f a DNA w h ic h i s much slower tod e n a t u r e h a nt i m eo f 3 min ape shown in F ig. 9. T he se p o i n t s n d i c a t e h a t oo b t a i na a t ea b o u to n e - t h i r d h a tof 64 a t u = 0.49 it was necessary t o incre ase he NaOH con cent rat i onmore than - fo ld . The s lop e f he ine h roug h he o in ts l so shows a much lowerdependence o f he ra t e on NaOH con cen tra t io n or d ha n or 64. Wi t h a u r t h e r n c r e a s es). b u t h e e a c t i o n t i l l a i l e d o go t o w p l e t i o n . i .e . ZTR o f a b o ut (5%=wZ:i n NaOH t o 0. 4 M and p t o 2.0 t h e a t eo fd e n a t u r a t i o no f d b ec am e f a s t t l 2found. R s y s t e m a t i c x a m i n a t i o n f h e e n a t u r a t i o n f f d i n h i s ra ng e of s a l t a n dNaOH concentrations was not made.dence o f pH on f o r a n p a r i s o n w i t hh e n a l y s i s b y Record et A. 27) Of t h eThe data from Figs . 8 and 9 a t i l ? can a lso be used to es t imatehe d e w -. 14r l i na t i t r n t i n n o f l i n e a r DNA. The t ra n s i t i o n oH DH c an no t be d i r e c t l v de-

the u rves n he more conventTona l p l o t o f pH vs f r a c t i o n d s l l u s t r a t e d n h edif fer en ce between pHm and pH112 i s independentf U. The appearance Of One ofi n s e t on Fig . 9. We f i n d h a t o r h e i xpa i rs o f d jacen t u rves on F igs 8 and 9,(A pH ,/n lo g u ) ~ -0.9 t 0.15. T h i s a l u e i s en t e r ed i n T a b le 11. It i s n e g a t i ve a sa d e s t a b i l i z a t i o n o f t h e h e l i x b y s a l t a s di s cu s s edsubsequently.expec ted rom the e su l ts on he a lka l i ne denat u ra t io n of l i n e a r DNA (27) a n d i n d i c a t e s

-~Oob Franigs. R and 9 -0.9 f 0.16 f o r u = 0.2 t o 1.3 M

-0.6 f o r Y = 0.1 t o 1-1.0 f o r e = 1 o 2 M4 0 0 b F r m Fg 2. Ref. (2)

a V a l u e sd e r i v e d r o md i r e c td e t e r m i n a t i o n sof pH frm Fig. 10 and froms i m i l a rd a t aat o the r emp era t u res and ion ic s tren !ths (da tanot shown).bVa lues b ta ined as descr ibed i n the ex t .


7/8

6032 Denaturation of Covalently Closed Circular DN Aous lyobsewed (2 ) i n t h e r a t e o f e n a t u r a t i o n w i t h e s p e c t o pH a t a g iven empera tu reTrans i t ion Req ion be tween Denatu ra t ion and Renatu ra t ion - The sharp maximum previ-i s d e fi n ed on the ioh DH s ide v a d e c r e a r i n oa t eh a t becanes slow a~ t h e nHappmaches th e pH i n &de; t o eiamyne ~there l ; t ion"o fdenat&at ion o - &tu&&s u f f i c i e n t ly i g h h a t e n a tu r a t i o n swe l l as denatu ra t ion ou ld eo l lowed.Ati n h i s r a n s i t i o n e g i o n a s e r i e s o f e x p e r i m e n t s w er e d on e w i t h 6 4- RF a t t e m p e ra t ur e slo w Y o f 0;15 o r 0.25 rena tu ra t ion a tes ou ld e measured a t 40 o r 50C a t NanHc o n c en t r a ti o n s i n h e r an ge o f 30 t o 60 M; the a te s became very low a t 30T. A tt h e s e e m p e r a t u r e s h e a t e f e n a t u r a t i o n i s t o o a s t o m e as ur e b y u r m et ho ds .An exwr imenta t 50 " i s s ho rn i n F ia . 10 w h i c h i s o l a v s e n a t u r a ti o n a nd r e n a t u r a t i n nc u r i e $ ~ f o r - G 4 - R F f i r a angeofNafH oncentrauoni.~"The-&nts on th ed e n a t u r a t i o nc u r v e a re s t a t i o n a r y v a l u e s o f the rac t ion o f dena tu ra t ion measured a f te r one m in anddo not change f urth er i thi m e . T he 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 yrharp ; change i n NaMl con cen t ra t iono f 1 M i s e q ui v al e nt oa pH change of bou t).01 un i ts . As a esu l t . o in ts 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 nA re d i f f i c u l t o r e pr o du c e. T he c u r v e h as am i d p o i n t t 56 M (OH-) which efine spH = 11.88.

.. . .

4min I.\ 2m4min* 30min

5 0

- ,30 40 50 60~\

I ,30 40 50 60(NaOH) ,m Mb y . 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 .(po in ts on thecurve) and o ther s i n g l e . i m ep o i n t s o r h e n d i c a t e d n $b er o f m i n a t40 nM and 50 rrU NaOH; th e 5Oo- Ids ed i n t h es experimentsasrepared i thoutn e u t r a l i z a t i o n a s d e s c ri b e d i n h e e x t . The f i l l e d y n b o l s n d i c a t e . s am pl es a n a ly z e dby the tanda rd en t r i f uga l method ; th e open sp bo ls nd ic a t e samples ana lyzed n lyby ge le lec t rop hores is as descr ibed i n Procedures .

d e n a t u r a t i o n a nd r e n a t u r a t i o n s a n o na m b ie n t.quenchingassaye m p l o v i n q n e u t r a l i z a t i o nEf fec t o f Ouenchins on K in e t i cs - Our s tandard ssay fo r he rogre ss f o tht o St00 the eact ion. Ouenchino ssavs are known t o chsnae th e .anoarant C i n r t i r c n fd e n a t u r a t i o n o f l i n e a r DNA ( 3 0 ) - a n d ~ c f s&e p & i & f3i';-$i;-u e y e - ~~r't~&c o nc e rn e d t h a t h e o n f i g u r a t i o n a l c ha ng es t h a t we i n f e r o o c c u rd u r i n gd e n a t u r a t i o nA l though we do no t have an a l t e rn a t i ve ambien t ssay, th i sp a r t i c u l a rp o i n t o u l d bea nd 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 sm i g h t em o d i f i e d y e u t r a l i z a ti o n .i n v e s t i g a t e d s i n g he n f r a s o n i cm 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 F ig . 10. Thef o u r - d r o p l e t .h r e et a g eena tura tion and ren at ur ati on sequence. The neu tral DNAprocedure n a s an ex tens ion o f h e usua l h ree-drop le tdenatu ra t ionexper iment ( 6 ) t o adr op le t was merged wi th an NaOH dro ple t 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 sat 50-Cu = 0.15. (OH-) > 60 nM A f t e r 2 m i n a h i r d d r o p w as m er ge d t h a t n i t i a t e d e n a t u r a t i o ;0.15. Aftera imed n te rva l , ena tu ra t ion was s topped s sua lw i th 2 M T r i s H C l andb y i l u t i n g he (OH-) t o 40 mM o r 50 mM w h i l em a i n t a i n i n gh eo n i c t r e n g t h tthe o lu t io n was ana lyzed fo r forms I and id. An experiment a t 40 rrU i s s ho wn i nF ig . 10 w i t hc m p l e t ea n a l y s i s a nd o t h e rp o i n t sa t 30 M and 50 rrU determined y elt he re i s ap o i n t on t h e - n i n u r v e on Fig. 10 a t 40 M NaOH and a ate u rve nde re l e c t r o p h o r e s i s .F o r a n p a r i s o nw i t h h e a t eo f e n a t u r a t i o no faneut ra l i zed 50 ' - Idt h e sam e c o n d i t i o n sn i g . 1A. Th e i n i t i a l 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 a ndth en n e u t r a l i z e dd were w i th i n 3.3 t 0.3 k s ; t h ea t e fh e e u t r a l i z e dddecreased more a to n g e rim enterva ls . therxper iments m p l o y i n gel na lys isshowed the same ra te o r he tw k i n d so f d o 70% r e a c t i o nw i t h i n h ep r e c i s i o no fthe method. We conc lu de ha t n e u t r a l i z a t i o n of ad e n a t u r a t i o n e a c t i o n d oe s n o t e s u l ti n as u b s t a n t i a l c ha ng e i n h e c o n f o r m a ti o no f h e I d m o l e c u l e s r a n h o s ee s t a b l i s h e da t a n b ie n t e m p e ra t ur e . We c a n n ot m a ke a s i m i l a r c a n p a r i s o n o f h e e f f e c t o f n e u t r a l i -za t io i i ' bn he ena tu ra t ion eac t io n . However, ne have ound i n a number o f c mpar is ons(da ta o t shown) tha t he a t eo fa e n a t u r a t i o n e a c t i o n s h e same whether it i sn e u t r a l i z e d a t a m bi en t e m pe r at u re o r l m r e d t o O s i m u l t a n e o u s l y w i t h n e u t r a l i z a t i o n .

I o n Condensation heor - T he a p p l i c a t i o n f M a nn in g t h e o r y o h e e n a t u r a t i o nd i s t i n g u i s h i n g h e c o n t r i b u t i o n so f o n co nd en sa tio n and Debye-Huckel Screening and byof ONA ( 2 9) h as r e v i s e d ou i de a s o f h e a t u r e Of i o n i c f f e c t s n h e r o c e s s b yprov id inga imp le means fo r he d i r e c tca lcu la t ion o f each. Record and h i s c a o r k e r s(27, 33 ) h av e r e c o n s i d e r e d h e a p p l i c a t i o n o f h e h e o r y o h e d e n a t u r a t i o n o f l i n e a rDNA i nn e u t r a l o l u t i o n a nd h a ve x t en d ed it t o h e l k a l i n e e n a t u r a t i o n f i n e a rDNA. I n b o t h c a se s a n i m p o r t a n tc o n t r i b u t i o n o h e c ha ng e i n c h a rg edens i tyon denatu -r a t i o n s h e acc mp any ing change in in e ar d imension, measured by theaxial phosphatespacing. Ino u r c a se o f a l k a l i n e I t h i s p a c i n g n c r e a s e sw i t h h ed e g r e e o f a l k a l i n et i t r a t i o n asound by Record 9. ( 2 7 )o rin ea r IINA. Fo rh is reason we havefound it c o n v en i e n t t o n t r o d u c e a r a m e t e r n t o h e n i t i a l q u a t i o n o r h e x i a lp h o sp h a te p a ci n g i n h e d e na t ur e d. c o i l o r m a t h e r h a n d j u s t i n g it a t h e e nd o fthe a lcu l a t ion . The e f fec t fhen c r e a s e d e g a t i v e h a r g ee s u l t i n g fr m a lka-l i n e i t r a t i o n s n c l u d e d n am a l lm o d i f i c a t i o n Of th er e a t m e n t Of Record e t-l. (27) . T he d e v el o pn e nt o f e q u a t i on s i s o u t l i n e d b e l o w o r h e h e l i x - c o i l r a n s i t i o n ,b y l k a l i o h e x t e n t o f k e q u i v a l e n t s e r u c l e o t i d e t o a max imm o f k = 0.5.h e l i x ( 1 ) c o i l ( l d ) , n w hi ch t h e c o i l e t a i n s t s d u p l e x c h ar a ct e r a nd i s t i t r a t e d

Both oncondensa t ion and screen ingar ede tenn ined by t h e i n e a r d e n s i t y o f i x e dnegat ive hargeso n DNA. T h i sden sit y was defined byManning ( 2 9 ) asE . 1.14bwhere b = average l in ear pa c in g n o f f i x ed charges . Ue de f i ne = average l inearspac ing o f phosphates on a i n g l e t r a n d f a ny DNA con f igura t ion. Thus fo r eu t ra lDNA, b = d o r ing le t rand and b = d l 2 f o ra doub le -s t randed on f igura t ion . o r

S ince a l l changes i n b n ena tu ra t ion wi be ra nhe B c o n f i g u r a t i o n e l a t i v eax ia lphosphate pac ing 1 = dc fdh w i l l be used. If t h e r ea r e h a rg e s d ue t o i t r a t i o ni na d d i t i o n o h ep h o ip h a t e chage t h e o t al h a rg ep e rn u c l e o t i d ew i l lb e (1 + k )The ex ten t f e n a t u r a t i o n f 5 O o- Id i s s h or nnig . 10 a f t e r 4 min and 30 mi ns I d a nd b t c = d t c l ( l + kc )o r i n g l e - s t r a n d e d o il .a c t o r f I1 + kh )and thea l u e o f b w i l l b e b t c = d t c / 2 ( 1 + kc )or oub le -s t randed oil suchoverange o f NaOH concent ra t ions ; dd i t iona lime o in ts re horn t 40 and 50 rrU f o r a r t i a l i t r a t i o n o f t h e e l i xw i l l b en c l ud e d i n r e l i m i n a r y q u a t i o n s u tNaOH fo r 50 ' - Id tha t was no t eu t ra l i zed be tween denatu r a t ion and rena tu ra t ion s q u a r ew i l lhen e gl l ect ed because kh"kC ( 2 7 ) . T he se d e f i n i t i o n s l l o w h e o n u l a t i o n

o u s l y f ou nd a t i g h o n i c t r e n g t h ( 2 ) ; u n d e r h e s e o n d i t i o n s it occur red a t 50 rrUp i n t s ; discussed below). The curves show a maximun rat ew i t h e s p e c t o p H a s p r e v i - f & c. t h e a l u eo f& f o r h e i t r a t e d o i l f or me d i n a e n a t u r a t i o n r a n s i t i o n , a sr e n a t u r a t i o n s t i l l o o k p l a c e a t 55 rrU NaOH. on ly 0.01 pH u n i t s b e l w p(OH-). e q u i v a l e n t o pH = 11.83. The max imun ra te s 0.05 pH un it s be low pH,,and

~ I* for ,The maximumr a t et 50 mM was confirmed bv ai m i l a re n a t u r a t i o nf 0 - mrn overhe ( 3 )same range o f NaOH conc entr at ion s data ot shown). As expe cted" frm revio us esult s(2) . O O- Id r e n a t u r e d o r e a t e r x t e n t i n 2 m i nh a n 0 - I d i n 4 m in ; t h e n i t i a l F o ra lk a l i ne ine ar DNA fo rw h i c h h e o i l s epara ted ing le t rand . must ave ara tes fena tu ra t ion fhesewo d 'S re mpared t 40 IrM NaOH i n Fi g. 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 DNA. k c a nd k h r e l s o e t = 0.0.75 300, 400 and 5ooc. At 40'. r e n a t u r a t i o n o f oo - Id ws alsoeasured.he charge f rom i t s va lu e o r he e l i x . net t?ow app ly heo n o n d e n s a t i n c r i t e r i o no fS i m i l a r e n a t u r a t io n x p e r i m e n t s i n t h e ran s i t ion eg i on have been done a t = Eq (3 ) i s s i m p l y e f i n i t i on o f i n e n s o f t h e c ha ng e i n e n g t h an d i no f the maximum ra t e o f ena tu ra t ion was a ls o 0.05 PH u n i t s b el ow p ~ md a t a not horn). usual way (27 ) T he f r a c t i o n o f t o t 1 h a r g eneut ra l i zedb y o n d e n s a t i o nonapar t ia l -p a t t e r no fc u r v e s nb o t hd i r e c t i o n s was s i m i l a r o h o s e n F ig . 10 and t h e p s i t i o n Manning (28). [ ( n e t ) - 1 for olYe leCt rolY tes uch as DNA forwh ich { > , i n h evalues for pb er ived ran hese curves ar eg i v e n n T a bl e 1 1 alongi t h a l c u l a t e d l y t i t r a t e d s p k i s i s h e n (1 - t-') (1 + k ) a nd t h e r a c t i o n a l e s i d u a l n n e u t r a l i z -coefficientsf (Ap+/Alo g and (ApHm/AT) The former is ingreementi t h e d c h ar g e i s t - f ~k) .a k i n ghei f f e r e n c ee t m e nh e s ea l u e sorheoil andt h e Of -0.a derived frm ~ i ~ ~ .and s u i t ooc. These have been fo rh ee l i xh ea r m e t e rf Record et dl. ( 2 7 ) . i. q u al s i h - i c = f r a c t i o n a lused belows t h eo r r e s p o n d i n gi f f e r e n t i a lo e f f i c i e n t sn a n a p p l i c a t i o nfhe amount O f Na* dischargeder nucleotide On Therefore*io no n d e n s a t i o nheory f Manning (78, 29) t o he dependence of ena tu ra t ion oni o n i c t r e n g t h .Coef f i c ie n ts have a lso been der ive d ra n h e f f e c t f a l t on t h er a t e o f r e n a t u r a t i o nF i g . 2 of Ref. 7) bys su m in g t h a t pH b e a rso n s t a n t r e l a t i o n i = ( l - ~ ' ) ( i + k ~ ) - ( l - ; ~ ) ( l + k ~ ) - kto h e pH o f maximum ra t eo f e n a t u r a t i o n a nd a r eg i v e n i n T a bl e 11. F o r h e n t e r v a l0.1 - 1 M PaCl. (APHm/Alog u ) = -0.6nd for 1 - 2 M NaCl. = -1.0. T h i s a l c u l a t i o nd oe s n o t e f e r o h e e n a t u r a t i o n e a c t i o n ; h e e n a t u r a t i o n a t e was u se d o n l y a s am ea ns of l o c a t i n g p b . = &:(I + k,) - E h l ( l + k h ) ] - k (4 )t r a n s i t i o n no Id i s f or me d. In ear ly xper iments mp loy ing u r s tandard 3 - d r o p l e tI n Fig. 1(1 i t i s s ho wn t h a t a t (OH-) co ncent ra t ions 0.03 IFM or more below thep r o c e d u r ew i t h h e n f r a s o n i cm i x i n gd e v i c e ( 6 ) I d was a lways formed up t o a rac t io no f 0.2 a t 50 mH d ec re as in g t o an e g l i g i b l e r a c t i o na t 25 M. T h i s o r m a t i o n f dw as l a t e r s ho wn t o b e t h e e s u l t o f h emix ingprocessw h i ch w as t o o s l ow r e l a t i v e ot h e v e r y h i g h a t e o f d e n a t u r a t i o n a t 5O'C t o p r e v e n t h e r a n s i e n t o n a t i o n o f o c a lreg ions f igh (OH-) and Conseauent par t ia l ena tur ati on. If such a eac t i onm i x t u r ed isappeared i n about 30 min , cons is ten tw i t h h e r a t eo f ren a tu r a t io n Shown i n F ig. 10c o n t a i n i n g 2oi. I d t 50 mw w as. a l l & o r e m ai n a t 50-C f o r d d i t i o n a l i m e t h e 1da t 50 rrU NaOH. Th e same t r a n s i e n t o r m a t i o n f IA was seen a t boo r . bu t wac harolvp l e t e l y b y a floc mix ing o f DNA and NanH fo l lowed y a is ing he empe ra tu re o hep e r c e p t i b l e a t 3 0* C an d u n de t ec t db l e a t o w e r e m p ~ r a t u - r e i ~ - t c o ; l d ' b e - i v o ; d e i ' i ~des i red va lue . ha t sa t is fac to r ymix in g an even be s low nderh e s e o n d i t io n s i se v i d e n t r a n h ed a t a nF i g .9 o r h eden atu ra t i on o f 64-RF a t OC and a ow on ics t reng th .Th is rwedu re s . f ourse , o tSa t is fac to ry o r measur imJ ra tesof dena-tu ra t ion . u t we have o t t tempted t o do th isa th i g h e r t e m p e r a t u r e sha nhe 30Cused fo r F"2 DN A i n F ig . 3 The po in ts on Fig . 10 between 20 and 53 mM were obtainedus ing OC m i x i n g o e s t a b l i s h h e n d i c a t e d NanH c o n c en t r at i o n.

where k = k - kh. he qua nt i ty nb r a c k e t s = q as defined byManning (29) t og i v eth e d i f f e re k e i n c reen ing be tween c and h because the sc reen ing depends on t h e r e s i d u a lcharge a f te r condensa t ion .th erea tm ent f Record St 27 ) They show th a t he on t r ib u t i on o f i on conden-s a t i o n o h e AG o f h- ' i s iRI n (Na* ) and tha t hec o n t r i b u t i o n o fs c r e e n i n gi s (- 71 12) RT ?kS(Na*). Thus th ec o n d e n s a t i o nc 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 h eDNA. T he n ete f f e c to f (N a+) b e c m s ( i - 7) 2) RT I n (Na'). Experim ental alues ors 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 .h e e l i xw i t h e s p e c t oh e o i lo ri n e a rd i f f e r e n t i a l o e f f i c i e n t s e r i v e d r a n h e t he nn od yn am lc t r e at m e n t (27) y i e l d i and9 we obtain. C m b i n i n g h ed e f i n i t i o n o f q frm eqs. (4 ) and ( 5 ) w i t h e q. (21 a n d n g l l e c t r n g

T h e q u a n t i t i e s i nd 7) a r e d e n t i f i e d n e l a t i o n o t he nn od yn am i c q u a n t i t i e s b y

1 ' E h + ' (6 1


8/8

Denaturation of Covalently Closed Circular D N AA va lue o f 1 fran eq. (6 ) ca e used w i th he e f in i t ion s rese n ted above too b t a i nbehav io r . q . * (6 )a p p l ie s t o o t h e u t r a l a nd a l k a l i n e e n a t u r a t i o n fi n e a r ONAt h e a r m e t e r s t c t c a nd tc, h a t h a r a c t e r i z e h e COY1 a nd I t s O ~ y e l e c t r o l Y t eb are employed.p r o v i d e da o e f f i c i e n t o f 2 i s a p p li e d o1 and the a p p r o p r i a t e d e f i n i t i o n so fd and

T he a p p l i c a t i o n o f h i s r e s u l t o h e - I d r a n s i t i o n c anbe made usin g

derived by Record eta l . (27). T h i sd i f f e r e n t i a l o e f f i c i e n t h as b ee n t a k en t o e q u alApHmlAlog L f o r uh??hThe val ue -0.91 was der ived frm ou rda ta Tab le 11). Combiningt h i s e s u l t w i t h e q s ( 5 ) (6) nd ( 7 ) we o b t a i n ? = 0.045 and 1 = 1.19 a t p b wherek = 0.25. Th is es u i t ha; been ex tended in Tab le 111 t o h e c a l c u l a t i o n Of 7) a nd a l lr e l a t e d p ar a me te rs f o r r b i t r a r y a l u e s f on the assumpt ion t ha t eqs. ( 4 ) . ( 5 )and (7 ) a r e v a li d o v e r h e e n t ir e i t r a t i o n .

Table I1 1I o n c o n de n s at i on a nd s c r e en i n q p a r a m e te r s f o r h e a l k a l i n e d e n a t u r a t i o n

of orm I DNAQ u a n t i t i e s h a veb e en c a l c u l a t e d o r a r b i t r a r y v a l u e s o f h e r a c t i o n a lt i t r a t i o n , k . frm theexper imenta l Ap&/A log Na+) lT as exp la ined in he ex t ,

k 7 1 d t c b t c ii i i - 7 1 20.1.018 1.08 3.7 1.7 0.23 -0.08 -0.090.2 0.036 1.15 3.9 1.6 0.23 -0.16 -0.180.25.045 1.19 4.0 1.6 0.23 -0.20 -0.230.3 0.054 1.23 4.2 1.6 0.22 -0.25 -0.270.4.072 1.30 4.4 1.6 0.22 -0.33 -0.360.5 0.090 1.38 4.7 1.5 0.22 -0.41 -0.45

Constan t incetc = ( l l l + k ) Zbh. then c r e a se i n 1 i s i n e a rw i t h ( 1 + kc).l i n e a r ONA (27). It can e seen in Tab le 111 t h a tb tc and & i1re pproximatelyThe same ' resu l t was noted y ecord & a. 27 ) f o r i t r a t e d i n e a r DNA. A d i r e c tc a n pa r is o n n i t h d c a n e m ad e b y a l c u l a t i o n Of if frm t h e i r d a t a . I n both casesthe cons tancy o f & iL i n d i c a t e s h a t h e same f r a c t i o n o f h e o t a l c h a r g e o n t h e c o i lrema ins nneut ra l i zed y ondensa t ion . u t ince he o t a l c ha r ge i s i n c r e a s i n g . h eac tua l e t charge a lso n c r e a s e s u r i n gh ei t r a t i o n . h i sesu l t may p rov ide ane l e c t r o s t a t i c a s i sor hem o l e c u l a r x t e n s i o n o f t h e i n g l e - s tr a n d e d DNA. I n th ec as e o f I d we a t t r i b u t e it i np a r t o h e m a gn it ud e s Of the average unber f basea b o u t a c h t h e r a t h e i g h t n e s s o f w i nd i ngha to rceshe ormat ion Of p a s i t i v ep a i r s p e r u r n t h e e q u i v a l e n t o f a h e I i c a l e p e a t ) Of t h e n o n i n t e r a c t i n g s i n g l e s t r a n d s

d i ff e re n c es i n t h e f f e c t o f s a l t o n t h e l k a l i n e e n a t u r a t i o n fhe n o e r i v esuperco i ls .incehen c re a s e i n l e n g t h i s s i m i l a r o r i n e a r DNA an d f o r d h em a i n l y frm th ed i f f e r e n c e s i n c o n f i g u r a t i o no f h e e s p e c t i v e co i l species. The fac tt b a t d r e t a i n s t s d u pl ex c h a ra c t er e s u lt s i n t s r e t e n t i o n o f ah ighchargedens i ty .= 4.3 - 4.6, s l i g h t l y g r e a t e r h a n h a t OFth e doub le s t randedhe l ix . AS a e s u l tbu tn c r e a s e s sh e e t e s i d u a l h a r g e i s i n c r e a se d b y t i t r a t i o n . T heHowever . the ncrease d charge dens i ty and the ncrease in t o ta l charge be fo re condensa-t i o n c c u bi n e t o e v e r s e h eo n o n d e n s a t i o n f f e c t so t h a t i s n e g a t i ve v erhee n t i r e i t r a t i o n and Na+ i o n s r eaken up dur ing ena tu r a t iona t h e rh a n e i n greleased. The canbinedparameter (i 7 1 2 ) is . o f c o u rs e . s t i l l m or e n e g a t iv e b ec au sebothondensa t ion and sc ree n ingm b i n eot a b i l i z eh eo i l . I n c o n t r a s t itr m a i n e d o s i t i v e o r h e l k a l i n e i n e a r DNA t r a n s i t i o n a nd o n l y e c m e e g i t i v etoward the end of t h e i t r a t i o n ( F ig . 1 of Ref. p 7 , 34 ) where 7) s l a rg e .th eoub le -s t randedo i l .hi s i s c o n s i s t e n t 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 .The value Of 1 f o r h e u l l y i t r a t e d d m p l i e sa 40% i nc rease i n l e n g t h o rAn a c t u a l a l u e o r h e u p e r h e l i c a ld e n s i t y a n n o tb e a l c u l a t e dw i thou t an es t imateo f h e e f f e c t i v e h e l i c a l e p e a t .

7 1 2 . t h u s S t a b i l i z e s h e c o i l o r m a s i n o t h e r DNA t r a n s i t i o n s .

denatu ra t ion Of form I ONA. The differences between th e a l k a l i n e d e n a t u r at i o n o fQ u a l i t a t i v e l y h e io n condensa t ion heory accounts fo r th ee f f e c t o f s a l t on th el i n e a r an d c i r c u l a r ON d t h a t de pe nd o n t h e i f f e r e n c e n t r u c t u r e o f t h e t w o c o i lf o m s r e a r t i c u l a r l yw e l l e f i n e d b y t h eh e o r y . T h er e i s a s y e t no independentc o n f i r m a t i o n Of the uan t i ta t i ve red ic t ion s f imens iona l change. It i s a s s m ed i nthe above Ca lC u la t ionS ha t hea x i a lc h a r g ed e n s i t y s t i l l d e t er m i n es i o n c o n de n s a ti o naccord ing to he Mann ing theor y i n sp i te o f he h i gh ly compact, superco i led a r rangmentof theo u b l e- s t ra n d e d c o i l f Id. Rough ca lc u la t ion s o f the averaae sma ra t i onbetween charges on d i f f e re n t segments o f a c o i le d - up o u b le t r an d i n n e q h i v a l en thydrodynan icphere fhe i z e o f I i n d i c a t e s h a t it i s m u ch g r e a t e rha nh eaxi al phosphate pacing and proba bly wi n ot mod i fy on condensat ion .denatu ra t ion o f about 8 k c a ler bp et&loyfng the r e l a t i o n o f Record & fi. (27) .The value o f the c o e f f i c i e n t (ApH /AT) giv en i n T a b le I I p r e d i c t s a A HO f o r

a r e d e n t i c a l w i t h h o s e c T cc uT a te d fr m t h e i r d a t aemploy ingour eq ( 6 ) and the e la t -2 I n Table 1 of Record e t a l . (27) th ev a l u e sg i v e n o r h eaxi al phosphate pacinged d e f i n i t i o n s . H ow ev er . i n h e a b l e h i s u a n t i t y i s designated.as p s h i c h i n

A nm ber o f conc lus ions can be d rawn frm Tab le 111 and frm a m p a r i s o n o f t h e e t a i n h eo r i g i n a ld e f i n i k i o n o f b 129) as an average pacing er harge and t o i n c l u d eou rerm ino logy qua ls t - (1 + k ) for ingle -stra nded DNA Ue have Fre fe r red or e s u l t sw i t h h o s e Of Record e t a1 (27) f o r ha l k a l i n ed e n a t u r a t i o n o f l i n e a r DNA e x p l i c i t l y h e u a n t i t y , (1 + k c ) , oa d j u s t it t o a per ucle otid e asis where necess-a s d e s c r ib e d i n T a b l e I a nd F G . 7 ' o f R ef. (27) t . The axial phosphate separation ofId ,d t c . n c r e a s e sw i t h n c r e a s i n g i t r a t i o n O nl y a l i t t l e e s s t h a n h a t o r i t r a t e d a ry . E ; : i se s s e n t i a l l y h e sam e a s l~',)" efined y Record St. 27).

j. biol. chem.-1986-camien-6026-33

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