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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
The Flexibility of A-Form DNA a
c
Elliot Charney , Holly-Ho Chen & Donald C. Rau a
b
Laboratory of Chemical Physics
b
Laboratory of Biochemical Metabolism , NIDDK National Institutes of Health , Bethesda , Maryland , 20892 c
Department of Chemistry , George Mason University , 4400 University Drive, Fairfax , Virginia , 2203 Published online: 21 May 2012.
To cite this article: Elliot Charney , Holly-Ho Chen & Donald C. Rau (1991) The Flexibility of A-Form DNA, Journal of Biomolecular Structure and Dynamics, 9:2, 353-362, DOI: 10.1080/07391102.1991.10507917 To link to this article: http://dx.doi.org/10.1080/07391102.1991.10507917
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 9, Issue Number 2 (1991), @Adenine Press (1991).
The Flexibility of A-Form DNA Elliot Charney 1, Holly-Ho Chen 3 and Donald C. Rau 2 Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
1
Laboratory of Chemical Physics Laboratory of Biochemical Metabolism NIDDK National Institutes of Health Bethesda, Maryland 20892
2
3
Department of Chemistry George Mason University 4400 University Drive Fairfax, Virginia 2203
Abstract We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350-900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than forB-form DNA There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2-2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.
Introduction Over the past decade large variations have been found in the physical and structural properties of DNA, once thought to be interesting only for the fact that it codes genetic information by the sequential arrangement of the four nucleosides from which it is constructed. Greater attention is now given to the role of physical properties of different sequences and structures in modulating biological activity of genes (e.g., 1-4). In this paper we report that a variation in one physical property, flexibility or bending stiffness, differs by at least a factor of three for random sequence DNA in the A and B forms and, if one includes the synthetic polynucleotide, alternating poly(dA-dT), the range extends over a six-fold factor in stiffness. We determine the bending stiffness of the A-form of" random" sequence DNA using the technique of transient electric birefringence (TEB). We find that A-form DNA is characterized by an intrinsic "persistence length" of about 1500 ± 100 A The persistence length, L, characterizes the bending stiffness oflinear polymers and is related to the bending free energy per base pair, U, for a radius of curvature bend R (in A) by
353
Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
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Charney et a/.
U=3.4 LkT/ R2. The persistence length ofB-form DNA, determined by a variety of methods including TEB, is about 500 A Since it already takes considerable energy to bend B-ONA around the nucleosome core with a diameter of about 90 A (about 8 kT/ lObp), it is clear that the presence of an A-form family structure would be a strong deterrent to the formation of this and other folded nucleic acid or nucleoprotein structures. Contrariwise, it has been pointed out that the binding site within the 5s RNA gene for the regulatory protein TFIIIA has a G periodicity similar to the number of base pairs per helix turn found for A-form DNA (5) and that the circular dichroism (6) and the crystal x-ray structure (7) of an oligomer containing part of the recognition sequence has A-form characteristics. The suggestion is that TFIIIA is recognizing a DNA structure in the A-form family and the question of flexibility arises naturally. The persistence length can be determined from measurements of the birefringence decay of the molecules after they have become oriented in a pulsed electric field and the field is then sharply turned off(for a review of transient electric birefringence see ref. 8). The decay of the birefringence signal is exquisitely sensitive to the long dimension of rod-like molecules, approximately proportional to the 2.5 power of the molecular length. Flexibility effectively decreases the end-to-end distance of the molecule from its rigid rod limit. For a known DNA contour length (a known fragment length in base pairs and distance between base pairs), the relaxation time expected for the rigid rod ('trod) is calculated from Broersma's equation (9). The hydrodynamic calculations of Hagerman and Zimm (10) can be used to determine the ratio of persistence length and contour length necessary to account for the ratio ofthe observed relaxation time, (t'exp) and that expected for the rigid rod. Although measurement of crystals of nucleic acid oligomers have steadily yielded average values for the rise per base pairin the range of2.6-2.7 A, a significant, sequence specific variation (2.23.2 A) has been observed in crystal structures (11 ). We have, therefore, decided not to assume a base pair separation, but rather determine both a persistence length and rise per base pair (RPB) that best fits the data for four different DNA fragments ranging from 373 to 917 bp. This procedure assumes there is little variation in average RPB and persistence length among the four fragments. The validity of this physically reasonable assumption will be apparent in the self-consistency of the solution among the four fragments.
Materials and Methods DNA Isolation Four monodisperse sized DNA fragments were used in this work. Of these, two, 460 and 676 bp long, were purchased from Lofstrand Labs Lt. (Gaithersburg, MD). They were prepared by Rsa I restriction enzyme digestion of the DNA plasmid pG EM-3, separated by gel electrophoresis in 0.8% agarose, and isolated by electroelution. A 917 bp fragment was isolated from an Eco RI digestionofpUC18-INS-Ab2-2, a gift of W. Swiggard and M.H. Park. The Eco RI insert is derived from the eDNA for human eukaryotic initiation factor4D. The length was verified by direct sequencing. A 373 bp fragment was derived from a BalI- Bgl I digestion of the 917 bp fragment.
The Flexibility of A-Form DNA
355
Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
These latter fragments were separated on a 4% polyacrylamide gel and isolated by electroelution. All fragments were further purified by binding and elution from NACS PREPAC columns (Bethesda Research Laboratories). All fragments migrated as single bands on polyacrylamide gels (4%, TBE buffer), with molecular sizes, compared with known molecular weight markers, consistent with expectations. Nucleosomal length DNA fragments (about 145 bp long) were isolated from adult chicken erythrocytes as described in Shindo et al. (12). Circular Dichroism
Optical absorption circular dichroism (CD) measurements (Jasco J-500) were used to monitor the B- to A- form transition of the "random" sequence, 145 base pair DNA, with increasing trifluoroethanol (TFE) concentrations, at both 0.5 and 1 mM NaCacodylate (pH -7.0), with 0.05 mM EDTA, at 20 C. The available small quantities of the 373,460,676, and 917 bp fragments precluded extensive CD measurements on these samples. The results did not differ from our previous CD measurements on this system (13), but did establish the existence of a limiting CD spectrum for A-form DNA at 80-85% TFE, under salt conditions suitable for the transient electric birefringence experiment. Limiting A-form spectra at 85% TFE in 1 mM NaCacodylate were confirmed for the restriction fragments used for relaxation time measurements. Solution viscosity measurements
The calculation of stiff rod relaxation times requires values for solution viscosities of the TFE-water mixtures used in these experiments. Viscosities were determined to an accuracy sufficient for the precision of the birefringence measurements using a Cannon-Fenske viscometer (International Research Glassware, Kenilworth, New Jersey). Deionized H 20 was used to check calibration. Viscosities measured this way at 20.0 C were 1.0050 centipoise (cp ), 1.95 cp, and 1.92 cp, for solutions containing 0, 80 and 85. % volume of TFE. TEB measurements
The transient electric birefringence apparatus we employ has been described in detail elsewhere (14). Measurements are made at 632.8 nm using a 10 mW He-Ne laser (Spectra-Physics) as the light source. The optical system of two polarizers and a A/4 wave plate has an extinction coefficient of about 10. The particular optical cell used in the current measurements has stainless steel electrodes, with a separation of 2 mm and an optical path length of 1 em. The data was accumulated and averaged (usually over 128 pulses) using a LeCroy digitizing system, with 40-320 nsec/channel, depending on fragment length and relaxation time. There was no observable change in signal magnitude or kinetics during course of an experiment. Results
Figure I shows the change in the circular dichroism of 145 bp, random sequence DNA, derived from bulk chicken erythrocyte nucleosomes, at two different wavelengths
Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
356
Charney et a/.
. . . . . . . . . IIIII
CJ6
(v/v) TFE
Figure 1: The B-A transition of 145 bp DNA in TFE monitored by CD. Circular dichroic intensities (chart recorder units) at two wavelengths, 212 and 268 nm, are shown as dependent on volume per cent of trifluoroethanol (TFE). Measurements were done in 0.5 mM NaCacodylate buffer (pH~7.0) and 50 !1M EDTA at 20 C, with a DNA concentration ~ 15 J.!M bp for these nucleosomes size fragments derived from chicken erythrocytes. These are essentially the conditions used in the birefringence experiments. The transition is complete by 80% TFE. Limiting CD spectra forB-form (in water) and A-form (in 85% TFE) DNA are shown in the figure inset.
as the concentration oftrifluoroethanol (TFE) is increased. Full circular dichroic spectra of B- (upper) and A- form (lower) DNA are shown in the figure inset. The transition occurs over the same narrow range of TFE concentrations for the two wavelengths, clearly indicating a transition between two discrete structures. The curves show the transition is complete in the 80 - 85% TFE range. Figure 2 shows a comparison of the semilog plots of the birefringence decay data for each of the four DNA fragments, in 85% TFE and 0.5 mM NaCacodylate. The relaxation data are well described by single exponential fits for the 373, 460 and 676 bp fragments that correspond to rotation about the long molecular axis. The 917 bp
357
The Flexibility of A-Form DNA
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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
The Flexibility of A-Form DNA a
c
Elliot Charney , Holly-Ho Chen & Donald C. Rau a
b
Laboratory of Chemical Physics
b
Laboratory of Biochemical Metabolism , NIDDK National Institutes of Health , Bethesda , Maryland , 20892 c
Department of Chemistry , George Mason University , 4400 University Drive, Fairfax , Virginia , 2203 Published online: 21 May 2012.
To cite this article: Elliot Charney , Holly-Ho Chen & Donald C. Rau (1991) The Flexibility of A-Form DNA, Journal of Biomolecular Structure and Dynamics, 9:2, 353-362, DOI: 10.1080/07391102.1991.10507917 To link to this article: http://dx.doi.org/10.1080/07391102.1991.10507917
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 9, Issue Number 2 (1991), @Adenine Press (1991).
The Flexibility of A-Form DNA Elliot Charney 1, Holly-Ho Chen 3 and Donald C. Rau 2 Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
1
Laboratory of Chemical Physics Laboratory of Biochemical Metabolism NIDDK National Institutes of Health Bethesda, Maryland 20892
2
3
Department of Chemistry George Mason University 4400 University Drive Fairfax, Virginia 2203
Abstract We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350-900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than forB-form DNA There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2-2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.
Introduction Over the past decade large variations have been found in the physical and structural properties of DNA, once thought to be interesting only for the fact that it codes genetic information by the sequential arrangement of the four nucleosides from which it is constructed. Greater attention is now given to the role of physical properties of different sequences and structures in modulating biological activity of genes (e.g., 1-4). In this paper we report that a variation in one physical property, flexibility or bending stiffness, differs by at least a factor of three for random sequence DNA in the A and B forms and, if one includes the synthetic polynucleotide, alternating poly(dA-dT), the range extends over a six-fold factor in stiffness. We determine the bending stiffness of the A-form of" random" sequence DNA using the technique of transient electric birefringence (TEB). We find that A-form DNA is characterized by an intrinsic "persistence length" of about 1500 ± 100 A The persistence length, L, characterizes the bending stiffness oflinear polymers and is related to the bending free energy per base pair, U, for a radius of curvature bend R (in A) by
353
Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
354
Charney et a/.
U=3.4 LkT/ R2. The persistence length ofB-form DNA, determined by a variety of methods including TEB, is about 500 A Since it already takes considerable energy to bend B-ONA around the nucleosome core with a diameter of about 90 A (about 8 kT/ lObp), it is clear that the presence of an A-form family structure would be a strong deterrent to the formation of this and other folded nucleic acid or nucleoprotein structures. Contrariwise, it has been pointed out that the binding site within the 5s RNA gene for the regulatory protein TFIIIA has a G periodicity similar to the number of base pairs per helix turn found for A-form DNA (5) and that the circular dichroism (6) and the crystal x-ray structure (7) of an oligomer containing part of the recognition sequence has A-form characteristics. The suggestion is that TFIIIA is recognizing a DNA structure in the A-form family and the question of flexibility arises naturally. The persistence length can be determined from measurements of the birefringence decay of the molecules after they have become oriented in a pulsed electric field and the field is then sharply turned off(for a review of transient electric birefringence see ref. 8). The decay of the birefringence signal is exquisitely sensitive to the long dimension of rod-like molecules, approximately proportional to the 2.5 power of the molecular length. Flexibility effectively decreases the end-to-end distance of the molecule from its rigid rod limit. For a known DNA contour length (a known fragment length in base pairs and distance between base pairs), the relaxation time expected for the rigid rod ('trod) is calculated from Broersma's equation (9). The hydrodynamic calculations of Hagerman and Zimm (10) can be used to determine the ratio of persistence length and contour length necessary to account for the ratio ofthe observed relaxation time, (t'exp) and that expected for the rigid rod. Although measurement of crystals of nucleic acid oligomers have steadily yielded average values for the rise per base pairin the range of2.6-2.7 A, a significant, sequence specific variation (2.23.2 A) has been observed in crystal structures (11 ). We have, therefore, decided not to assume a base pair separation, but rather determine both a persistence length and rise per base pair (RPB) that best fits the data for four different DNA fragments ranging from 373 to 917 bp. This procedure assumes there is little variation in average RPB and persistence length among the four fragments. The validity of this physically reasonable assumption will be apparent in the self-consistency of the solution among the four fragments.
Materials and Methods DNA Isolation Four monodisperse sized DNA fragments were used in this work. Of these, two, 460 and 676 bp long, were purchased from Lofstrand Labs Lt. (Gaithersburg, MD). They were prepared by Rsa I restriction enzyme digestion of the DNA plasmid pG EM-3, separated by gel electrophoresis in 0.8% agarose, and isolated by electroelution. A 917 bp fragment was isolated from an Eco RI digestionofpUC18-INS-Ab2-2, a gift of W. Swiggard and M.H. Park. The Eco RI insert is derived from the eDNA for human eukaryotic initiation factor4D. The length was verified by direct sequencing. A 373 bp fragment was derived from a BalI- Bgl I digestion of the 917 bp fragment.
The Flexibility of A-Form DNA
355
Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
These latter fragments were separated on a 4% polyacrylamide gel and isolated by electroelution. All fragments were further purified by binding and elution from NACS PREPAC columns (Bethesda Research Laboratories). All fragments migrated as single bands on polyacrylamide gels (4%, TBE buffer), with molecular sizes, compared with known molecular weight markers, consistent with expectations. Nucleosomal length DNA fragments (about 145 bp long) were isolated from adult chicken erythrocytes as described in Shindo et al. (12). Circular Dichroism
Optical absorption circular dichroism (CD) measurements (Jasco J-500) were used to monitor the B- to A- form transition of the "random" sequence, 145 base pair DNA, with increasing trifluoroethanol (TFE) concentrations, at both 0.5 and 1 mM NaCacodylate (pH -7.0), with 0.05 mM EDTA, at 20 C. The available small quantities of the 373,460,676, and 917 bp fragments precluded extensive CD measurements on these samples. The results did not differ from our previous CD measurements on this system (13), but did establish the existence of a limiting CD spectrum for A-form DNA at 80-85% TFE, under salt conditions suitable for the transient electric birefringence experiment. Limiting A-form spectra at 85% TFE in 1 mM NaCacodylate were confirmed for the restriction fragments used for relaxation time measurements. Solution viscosity measurements
The calculation of stiff rod relaxation times requires values for solution viscosities of the TFE-water mixtures used in these experiments. Viscosities were determined to an accuracy sufficient for the precision of the birefringence measurements using a Cannon-Fenske viscometer (International Research Glassware, Kenilworth, New Jersey). Deionized H 20 was used to check calibration. Viscosities measured this way at 20.0 C were 1.0050 centipoise (cp ), 1.95 cp, and 1.92 cp, for solutions containing 0, 80 and 85. % volume of TFE. TEB measurements
The transient electric birefringence apparatus we employ has been described in detail elsewhere (14). Measurements are made at 632.8 nm using a 10 mW He-Ne laser (Spectra-Physics) as the light source. The optical system of two polarizers and a A/4 wave plate has an extinction coefficient of about 10. The particular optical cell used in the current measurements has stainless steel electrodes, with a separation of 2 mm and an optical path length of 1 em. The data was accumulated and averaged (usually over 128 pulses) using a LeCroy digitizing system, with 40-320 nsec/channel, depending on fragment length and relaxation time. There was no observable change in signal magnitude or kinetics during course of an experiment. Results
Figure I shows the change in the circular dichroism of 145 bp, random sequence DNA, derived from bulk chicken erythrocyte nucleosomes, at two different wavelengths
Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
356
Charney et a/.
. . . . . . . . . IIIII
CJ6
(v/v) TFE
Figure 1: The B-A transition of 145 bp DNA in TFE monitored by CD. Circular dichroic intensities (chart recorder units) at two wavelengths, 212 and 268 nm, are shown as dependent on volume per cent of trifluoroethanol (TFE). Measurements were done in 0.5 mM NaCacodylate buffer (pH~7.0) and 50 !1M EDTA at 20 C, with a DNA concentration ~ 15 J.!M bp for these nucleosomes size fragments derived from chicken erythrocytes. These are essentially the conditions used in the birefringence experiments. The transition is complete by 80% TFE. Limiting CD spectra forB-form (in water) and A-form (in 85% TFE) DNA are shown in the figure inset.
as the concentration oftrifluoroethanol (TFE) is increased. Full circular dichroic spectra of B- (upper) and A- form (lower) DNA are shown in the figure inset. The transition occurs over the same narrow range of TFE concentrations for the two wavelengths, clearly indicating a transition between two discrete structures. The curves show the transition is complete in the 80 - 85% TFE range. Figure 2 shows a comparison of the semilog plots of the birefringence decay data for each of the four DNA fragments, in 85% TFE and 0.5 mM NaCacodylate. The relaxation data are well described by single exponential fits for the 373, 460 and 676 bp fragments that correspond to rotation about the long molecular axis. The 917 bp
357
The Flexibility of A-Form DNA
Downloaded by [The University Of Melbourne Libraries] at 05:49 10 October 2014
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