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Studies on circular DNA
J. Mol. B&o.!. (1968) 33, 503-505
Studies on Circular II.?
Number
of Tertiary
DNA
Turns in Papilloma-DNA
Certain covalently closed circular DNA molecules have been found to possess a twisted structure$ (cf. recent review: Vinograd & Lebowitz, 1967). This structure arises from either a deficiency or an excess of helical turns in the molecule as compared to the normal Watson-Crick structure, since the sum of helical and tertiary turns is a topological constant in such a system. Partial unwinding of the helix, e.g. by denaturation, results in the loss of the twisted structure (Vinograd, Lebowitz, Radloff, Watson & Laipis, 1965; Crawford & Black, 1964) suggesting that such twists originated from a deficiency in helical turns and that the direction of the tertiary turns is righthanded (Vinograd & Lebowitz, 1967). Such a deficiency could arise in essentially two ways: (a) the number of helical turns per nucleotide at the time of ring closure in the cell is less than that in solution after extraction of the DNA; in this case the number of tertiary turns would be proportional to the molecular weight of the DNA; or (b) there is a region of a predetermined length in the molecule in which the bases do not base-pair in the form of the normal Watson--Crick double-helix before ring closure. The number of tertiary turns could then be independent of the molecular weight of the molecule. Based on alkaline denaturation analysis, Lebowitz, Watson & Vinograd (1966, 2nd Intern&. Biqhys. Congr., Abst. 1.1.2) estimated that in polyoma DNA (mol. wt 3.3 x 10s) there are 15 to 20 tertiary turns. Crawford t Waring (1967a) calculated the number of these turns by using the intercalating agent ethidium bromide. Intercalating molecules are thought to untwist the Watson-Crick helix of DNA (Lerman, 1961,1964). One would therefore expect that limited amounts of ethidium bromide would remove the twisted structure in DNA if it consists of right-handed turns. Further addition of ethidium bromide would then cause the DNA to resume again tertiary turns, but in the opposite (left-handed) orientation. The sedimentation velocity of a circular DNA molecule without twisted structure has to be smaller than that of a more compact twisted molecule. Therefore a plot of the sedimentation velocity of such a DNA against ethidium bromide concentration would exhibit a minimum where the concentration of the intercalating agent is just sufficient to remove the tertiary turns. The present communication describes the determination of the number of tertiary turns in bovine papilloma DNA, molecular weight 4.9 x lo6 (Lang, Bujard, Wolff & Russell, 1967) by titration with ethidium bromide. Bovine papilloma DNA was prepared and characterized as described earlier (Bujard, 1967). About 70% of the DNA molecules in the preparation existed in form I (right-handed twisted structures, fcv,2c = 28.1 s); the residual 30% were in form II (circular molecules without tertiary t~d%,m = 20.8 s). The interaction of such DNA with ethidium bromide was analyzed by boundary sedimentation in 0.05 M-Tris buffer, pH 8.0. A 30-mm cell was used in a t Paper I in this series is Bujard, $ In the present paper the term structure of papilloma DNA I.
1967. “tertiary
turns 503
” is used
to describe
the turns
in the twisted
504
R. BtJJARD
Spinco model E at 20°C and 40,000 rev./mm. The ethidium bromide (Boot’s Pure Drug Co. Ltd., Nottingham, England) was chromatographed on silica gel and the crystallized material was homogeneouson thin-layer chromatography (low activity silica gel/ethanol). The concentrations of DNA and ethidium bromide solutions were determined spectrophotometrically using molar extinction coefficients of 6600 (260 rnp, with respect to phosphorus) and 5600 (480 mp), respectively. The fraction of ethidium bromide bound to DNA was calculated by the relation (Klotz, 1953): r = c(n/k - r/k) where r is the number of ethidium bromide moleculesbound per nucleotide; c is the molar concentration of free ethidium bromide; n is the number of binding sites per nucleotide = 0.20 (LePecq & Paoletti, 1967); k is the dissociation constant of the ethidium bromide-DNA complex = 7 x lo-? moles/l. (Waring, 1965; LePecq t Paoletti, 1967). Figure 1 showsthe change of the sedimentation velocity of papilloma DNA at low concentrations of ethidium bromide. The sedimentation velocity of DNA I decreases initially and increases then at higher ethidium bromide concentrations, with the minimum at r = 0.037. Between r = O-028and r = O-041only one boundary could be observed for the two forms of DNA present in the mixture. Within this range, the slopeof the boundary tracings goesthrough a maximum at r = O-037,which coincides with the value at which the minimum in sedimentation velocity occurs. This is the
FICA 1. Change in the sedimentation velocity of papillomct DNA with increasing remounts of ethidium bromide (r). -O--O-, DNA I; -A-A-, DNA II.
LETTERS
TO THE
EDITOR
606
point at which the ethidium bromide concentration is just sufficient to untwist the tertiary turns of DNA I, which now cosedimentswith DNA II. Assuming that each intercalating ethidium bromide molecule untwists the DNA helix 12” (Fuller 6 Waring, 1964), one can calculate from these data that there are 18f3 superhelical turns in one papilloma DNA molecule. The expressed deviation represents the maximum error associatedwith the accuracy one can detect differences in the slope of the microdensitometer tracings of the boundary, and from the errors involved in concentration determination. Similar results from studies on human and rabbit papilloma DNA have recently been reported by Crawford & Waring (19673). For polyoma DNA, Crawford & Waring (1967a) found with the sameassumption and under comparable conditions 12 superhelical turns per molecule. Both DNA moleculestherefore have 3+3-&0*6tertiary turns per 1 x lo6 molecular weight (assuming a similar maximum error in the measurementsof Crawford & Waring on polyoma DNA). This would suggest that the tertiary turns originate in a manner similar to that discussedin alternative (a): before ring closure in Go, the DNA exists in a form which contains fewer helical turns per base-pair than under conditions employed in these experiments, where it is assumed to exist in the B-form (Luzzati, Mathis, Masson & Witz, 1964). I am very grateful to Drs D. Lang and M. Patrick of this Laboratory for their helpful discussion and to Mrs M. J. Hsrrod for dedicated technical assistance. This work was supported in part by research grants GB4388 from the National Science Foundation and GM13234 and FR5646 from the National Institutes of Health, U.S. Public Health Service. Southwest Center for Advanced Studies Dallas, Texas, U.S.A. Received
29 August
H. BUJARD
1967.
REFERENCES Bujard, H. (1967). J. Viral. 1, 1136. Crawford, L. V. & Black, P. H. (1964). l’iyobgy, 24, 388. Crawford, L. V. t Waring, M. J. (1967a). J. Mol. Bid. 25, 23. Crawford, L. V. & Waring, M. J. (1967b). J. Ben. VGoZ. 1, 387. Fuller, W. & Waring, M. J. (1964). Ber. Bunaenpaelkchajt, 68, 806. Klotz, I. (1953). In The Proteins, ed. by H. Neurath, vol. 2B, p. 748. New York: Academic Press. Lang, D., Bujard, H., Wolff, B. & Russell, D. (1967). J. Mol. Bid. 23, 163. LePecq, J.-B. t Paoletti, C. (1967). J. Mol. Biol. 27, 87. Len-man, L. S. (1961). J. Mol. Bid. 3, 18. Lerman, L. S. (1964). J. Mol. Bid. 10, 367. Luzzati, V., Mathis, A., Masson, F. & Witz, J. (1964). J. Mol. Bid. 10, 28. Vinograd, J. & Lebowitz, R. (1967). In Macromolecular Metubolima, The New York Heart Association, p. 103. Boston: Little, Brown 8z Co. Vinograd, J., Lebowitz, R., Redloff, R., Watson, R. & Laipis, P. (1966). Proc. Nat. Acad. Sci., Wash. 53, 1104. Waring, M. J. (1965). J. Mol. Bid. 13, 269.
Studies on Circular II.?
Number
of Tertiary
DNA
Turns in Papilloma-DNA
Certain covalently closed circular DNA molecules have been found to possess a twisted structure$ (cf. recent review: Vinograd & Lebowitz, 1967). This structure arises from either a deficiency or an excess of helical turns in the molecule as compared to the normal Watson-Crick structure, since the sum of helical and tertiary turns is a topological constant in such a system. Partial unwinding of the helix, e.g. by denaturation, results in the loss of the twisted structure (Vinograd, Lebowitz, Radloff, Watson & Laipis, 1965; Crawford & Black, 1964) suggesting that such twists originated from a deficiency in helical turns and that the direction of the tertiary turns is righthanded (Vinograd & Lebowitz, 1967). Such a deficiency could arise in essentially two ways: (a) the number of helical turns per nucleotide at the time of ring closure in the cell is less than that in solution after extraction of the DNA; in this case the number of tertiary turns would be proportional to the molecular weight of the DNA; or (b) there is a region of a predetermined length in the molecule in which the bases do not base-pair in the form of the normal Watson--Crick double-helix before ring closure. The number of tertiary turns could then be independent of the molecular weight of the molecule. Based on alkaline denaturation analysis, Lebowitz, Watson & Vinograd (1966, 2nd Intern&. Biqhys. Congr., Abst. 1.1.2) estimated that in polyoma DNA (mol. wt 3.3 x 10s) there are 15 to 20 tertiary turns. Crawford t Waring (1967a) calculated the number of these turns by using the intercalating agent ethidium bromide. Intercalating molecules are thought to untwist the Watson-Crick helix of DNA (Lerman, 1961,1964). One would therefore expect that limited amounts of ethidium bromide would remove the twisted structure in DNA if it consists of right-handed turns. Further addition of ethidium bromide would then cause the DNA to resume again tertiary turns, but in the opposite (left-handed) orientation. The sedimentation velocity of a circular DNA molecule without twisted structure has to be smaller than that of a more compact twisted molecule. Therefore a plot of the sedimentation velocity of such a DNA against ethidium bromide concentration would exhibit a minimum where the concentration of the intercalating agent is just sufficient to remove the tertiary turns. The present communication describes the determination of the number of tertiary turns in bovine papilloma DNA, molecular weight 4.9 x lo6 (Lang, Bujard, Wolff & Russell, 1967) by titration with ethidium bromide. Bovine papilloma DNA was prepared and characterized as described earlier (Bujard, 1967). About 70% of the DNA molecules in the preparation existed in form I (right-handed twisted structures, fcv,2c = 28.1 s); the residual 30% were in form II (circular molecules without tertiary t~d%,m = 20.8 s). The interaction of such DNA with ethidium bromide was analyzed by boundary sedimentation in 0.05 M-Tris buffer, pH 8.0. A 30-mm cell was used in a t Paper I in this series is Bujard, $ In the present paper the term structure of papilloma DNA I.
1967. “tertiary
turns 503
” is used
to describe
the turns
in the twisted
504
R. BtJJARD
Spinco model E at 20°C and 40,000 rev./mm. The ethidium bromide (Boot’s Pure Drug Co. Ltd., Nottingham, England) was chromatographed on silica gel and the crystallized material was homogeneouson thin-layer chromatography (low activity silica gel/ethanol). The concentrations of DNA and ethidium bromide solutions were determined spectrophotometrically using molar extinction coefficients of 6600 (260 rnp, with respect to phosphorus) and 5600 (480 mp), respectively. The fraction of ethidium bromide bound to DNA was calculated by the relation (Klotz, 1953): r = c(n/k - r/k) where r is the number of ethidium bromide moleculesbound per nucleotide; c is the molar concentration of free ethidium bromide; n is the number of binding sites per nucleotide = 0.20 (LePecq & Paoletti, 1967); k is the dissociation constant of the ethidium bromide-DNA complex = 7 x lo-? moles/l. (Waring, 1965; LePecq t Paoletti, 1967). Figure 1 showsthe change of the sedimentation velocity of papilloma DNA at low concentrations of ethidium bromide. The sedimentation velocity of DNA I decreases initially and increases then at higher ethidium bromide concentrations, with the minimum at r = 0.037. Between r = O-028and r = O-041only one boundary could be observed for the two forms of DNA present in the mixture. Within this range, the slopeof the boundary tracings goesthrough a maximum at r = O-037,which coincides with the value at which the minimum in sedimentation velocity occurs. This is the
FICA 1. Change in the sedimentation velocity of papillomct DNA with increasing remounts of ethidium bromide (r). -O--O-, DNA I; -A-A-, DNA II.
LETTERS
TO THE
EDITOR
606
point at which the ethidium bromide concentration is just sufficient to untwist the tertiary turns of DNA I, which now cosedimentswith DNA II. Assuming that each intercalating ethidium bromide molecule untwists the DNA helix 12” (Fuller 6 Waring, 1964), one can calculate from these data that there are 18f3 superhelical turns in one papilloma DNA molecule. The expressed deviation represents the maximum error associatedwith the accuracy one can detect differences in the slope of the microdensitometer tracings of the boundary, and from the errors involved in concentration determination. Similar results from studies on human and rabbit papilloma DNA have recently been reported by Crawford & Waring (19673). For polyoma DNA, Crawford & Waring (1967a) found with the sameassumption and under comparable conditions 12 superhelical turns per molecule. Both DNA moleculestherefore have 3+3-&0*6tertiary turns per 1 x lo6 molecular weight (assuming a similar maximum error in the measurementsof Crawford & Waring on polyoma DNA). This would suggest that the tertiary turns originate in a manner similar to that discussedin alternative (a): before ring closure in Go, the DNA exists in a form which contains fewer helical turns per base-pair than under conditions employed in these experiments, where it is assumed to exist in the B-form (Luzzati, Mathis, Masson & Witz, 1964). I am very grateful to Drs D. Lang and M. Patrick of this Laboratory for their helpful discussion and to Mrs M. J. Hsrrod for dedicated technical assistance. This work was supported in part by research grants GB4388 from the National Science Foundation and GM13234 and FR5646 from the National Institutes of Health, U.S. Public Health Service. Southwest Center for Advanced Studies Dallas, Texas, U.S.A. Received
29 August
H. BUJARD
1967.
REFERENCES Bujard, H. (1967). J. Viral. 1, 1136. Crawford, L. V. & Black, P. H. (1964). l’iyobgy, 24, 388. Crawford, L. V. t Waring, M. J. (1967a). J. Mol. Bid. 25, 23. Crawford, L. V. & Waring, M. J. (1967b). J. Ben. VGoZ. 1, 387. Fuller, W. & Waring, M. J. (1964). Ber. Bunaenpaelkchajt, 68, 806. Klotz, I. (1953). In The Proteins, ed. by H. Neurath, vol. 2B, p. 748. New York: Academic Press. Lang, D., Bujard, H., Wolff, B. & Russell, D. (1967). J. Mol. Bid. 23, 163. LePecq, J.-B. t Paoletti, C. (1967). J. Mol. Biol. 27, 87. Len-man, L. S. (1961). J. Mol. Bid. 3, 18. Lerman, L. S. (1964). J. Mol. Bid. 10, 367. Luzzati, V., Mathis, A., Masson, F. & Witz, J. (1964). J. Mol. Bid. 10, 28. Vinograd, J. & Lebowitz, R. (1967). In Macromolecular Metubolima, The New York Heart Association, p. 103. Boston: Little, Brown 8z Co. Vinograd, J., Lebowitz, R., Redloff, R., Watson, R. & Laipis, P. (1966). Proc. Nat. Acad. Sci., Wash. 53, 1104. Waring, M. J. (1965). J. Mol. Bid. 13, 269.