30
GEL ELECTROPHORESIS AND TOPOLOGICAL METHODS
[2]
[2] A n a l y z i n g D N A C u r v a t u r e in P o l y a c r y l a m i d e G e l s
By STEPHAN DIEKMANN Introduction This chapter focuses on sequence-induced DNA curvature or intrinsically bent DNA, terms which refer to net (time-averaged) deflections of the helix axis from straight linearity. The DNA structure is assumed to be in the equilibrium state with no external force applied; this is different from bent DNA structures which are formed due to external forces (e.g., by bound proteinsl). Bending effects due to external forces result from different sequence-specific base pair properties than sequence-induced curved DNA. Because curved DNA migrates slower than straight DNA in polyacrylamide gels, this experimental effect has been used to analyze the properties of curved DNA. In this chapter the mobility analysis of DNA fragments in polyacrylamide gels is described in some detail, and the origin of DNA curvature is discussed. Experimental Approach to Curved DNA The different molecular properties of dA. dT and dG. dC base pairs result in a sequence-specific local modulation of the DNA structure. 2 For particular sequences or structural motifs this can lead to a deflection of the helix axis from straight linearity. When such sequences or motifs are repeated in phase with the helix repeat [-10.5 base pairs (bp)3], the local deflections point toward the same direction so that the DNA appears generally curved. Short tracts of the homopolymer dA. dT confer intrinsic curvature on the axis of the DNA double helix (for reviews, see Ref. 4-8). This ability is assumed to be a consequence of such tracts adopting a stable B'-DNA conformation which is distinct from that normally assumed by other I~NA sequences. A major distinguishing feature of the B' structure I A. A. Travers, Annu. Reo. Biochem. 58, 427 (1989). 2 R. E. Dickerson and H. R. Drew, J. Mol. Biol. 149, 761 (1981). 3 S. D i e k m a n n and J. C. Wang, J. Mol. Biol. 186, 1 (1985). 4 E. N. Trifonov, Crit. Reu. Biochem. 19, 89 (1986). 5 S. D i e k m a n n , in " N u c l e i c Acids and Molecular Biology" (F. Eckstein and D. M. J. Lilley, eds.), Vol. 1, p. 138. Springer-Verlag, Berlin, 1987. 6 A. A, T r a v e r s and A. Klug, Philos. Trans. R. Soc. London, Ser. B 317, 537 (1987). 7 p. j. Hagerrnan, Annu. Rev. Biochem. 59, 755 (1990). 8 D. M. Crothers, T. E. Haran, and J. G. N a d e a u , J. Biol. Chem. 2,65, 7093 (1990).
METHODS IN ENZYMOLOGY.VOL. 212
Copyright © 1992by AcademicPress, Inc. All rights of reproduction in any form reserved.
[2]
ANALYZING DNA CURVATURE IN POLYACRYLAMIDE GELS
31
is a high propeller t w i s t T M that optimizes stacking interactions and narrows the minor groove. In principle such a propeller twist can only be readily adopted by base pairs with two, but not three, hydrogen bonds. The naturally occurring base pair with this property is dA- dT, but other base pairs, notably dI. dC, also share this characteristic. The B' structure might also have a large wedge roll angle between the dAA dinucleotides.a, 5,8 D N A curvature can be detected using several experimental methods: circularization of D N A molecules, 15-18 electron microscopy, 19'2° and gel migration in gel electrophoresis. 3'21-27 Results obtained from these measurements can be compared with D N A structures obtained from nuclear magnetic resonance (NMR) H'28 and X-ray crystallographic analysis. ~2-14 Other techniques like Raman, 29circular dichroism (CD),29,30 and N M R 9'31'32 spectroscopy yield relevant information on the structural properties of curved D N A sequences. 9 R. Behling and D. R. Kearns, Biochemistry 25, 3335 (1986). 10 D. G. Alexeev, A. A. Lipanov, and I. Y. Skuratovski, Nature (London) 325, 821 (1987). 11 A. A. Lipanov and V. P. Chuprina, Nucleic Acids Res. 15, 5833 (1987), 12 H. C. M. Nelson, J. T. Finch, B. F. Luisi, and A. Klug, Nature (London) 330, 221 (1987). 13 M. Coil, C. A. Frederick, A. H.-J. Wang, and A. Rich, Proc. Natl. Acad. Sci. U.S.A. 84, 8385 (1987). 14 A. D. DiGabriele, M. R. Sanderson, and T. A. Steitz, Proc. Natl. Acad. Sci. U.S.A. 86, 1816 (1989). is K. Zahn and F. Blattner, Nature (London) 317, 451 (1985). I6 L. Ulanovsky, M. Bodner, E. N. Trifonov, and M. Choder, Proc. Natl. Acad. Sci. U.S.A. 8, 862 (1986). 17 S. D. Levene and D. M. Crothers, J. Mol. Biol. 189, 61 (1986). 18 K. Zahn and F. Blattner, Science 236, 416 (1987). 19 1. Griffith, M. Bleyman, C. A. Rauch, P. A. Kitchin, and P. T. Englund, Cell (Cambridge, Mass.) 46, 717 (1986). 2o B. Theveny, D. Coulaud, M. LeBret, and B. Revet, in "Structure and Expression Volume 3: DNA Bending and Curvature" (W. K. Olson, M. H. Sarma, R. H. Sarma, and M. Sundaralingam, eds.), p. 39. Adenine Press, Schenectady, New York, 1988. 21 j. (2. Marini, S. D. Levene, D. M. Crothers, and P. T. Englund, Proc. Natl. Acad. Sci. U.S.A. 79, 7664 (1982). 22 j. (2. Marini and P. T. Englund, Proc. Natl. Acad. Sci. U.S.A. 80, 7678 (1983). 23 H. M. Wu and D. M. Crothers, Nature (London) 308, 509 (1984). 24 p. j. Hagerman, Biochemistry 24, 7033 (1985). 25 S. Diekmann, FEBS Lett. 195, 53 (1986). 26 H. S. Koo, H. M. Wu, and D. M. Crothers, Nature (London) 320, 501 (1986). 27 p. 3. Hagerman, Nature (London) 321, 449 (1986). 28 M. H. Sarma, G. Gupta, and R. H. Sarma, Biochemistry 27, 3423 (1988). 29 S. Brahms and J. G. Brahms, Nucleic Acids Res. 18, 1559 (1990). 30 S. R. Gudibande, S. D. Jayasena, and M. J. Behe, Biopolymers 27, 1905 (1988). 31 J.-L. Leroy, E. Charretier, M. Kochoyan, and M. Gueron, Biochemistry 27, 8894 (1988). 32 j. G. Nadeau and D. M. Crothers, Proc. Natl. Acad. Sci. U.S.A. 86, 2622 (1989).
32
GEL ELECTROPHORESIS AND TOPOLOGICAL METHODS
[2]
From the migration analysis in polyacrylamide gels, absolute values are not obtained for structural DNA parameters. This is a principal drawback of the gel assay. Therefore, conclusions obtained from this technique should be verified by other methods based on better understood physical techniques. However, rigorous techniques are often elaborate and do not detect the local structural variations which can be resolved in the gel (e.g., see Diekmann and P6rschke33). The gel assay is very convenient, and small amounts (a few nanograms) of DNA can be analyzed. Furthermore, the mobility of DNA molecules in gels is not affected by the presence of other DNA fragments or a variety of chemical compounds present in small quantities. The gel migration anomaly of considerably curved DNA fragments is a rather large effect since the anomaly increases with the square of the degree of the curvature. 3
GEL ELECTROPHORESIS AND TOPOLOGICAL METHODS
[2]
[2] A n a l y z i n g D N A C u r v a t u r e in P o l y a c r y l a m i d e G e l s
By STEPHAN DIEKMANN Introduction This chapter focuses on sequence-induced DNA curvature or intrinsically bent DNA, terms which refer to net (time-averaged) deflections of the helix axis from straight linearity. The DNA structure is assumed to be in the equilibrium state with no external force applied; this is different from bent DNA structures which are formed due to external forces (e.g., by bound proteinsl). Bending effects due to external forces result from different sequence-specific base pair properties than sequence-induced curved DNA. Because curved DNA migrates slower than straight DNA in polyacrylamide gels, this experimental effect has been used to analyze the properties of curved DNA. In this chapter the mobility analysis of DNA fragments in polyacrylamide gels is described in some detail, and the origin of DNA curvature is discussed. Experimental Approach to Curved DNA The different molecular properties of dA. dT and dG. dC base pairs result in a sequence-specific local modulation of the DNA structure. 2 For particular sequences or structural motifs this can lead to a deflection of the helix axis from straight linearity. When such sequences or motifs are repeated in phase with the helix repeat [-10.5 base pairs (bp)3], the local deflections point toward the same direction so that the DNA appears generally curved. Short tracts of the homopolymer dA. dT confer intrinsic curvature on the axis of the DNA double helix (for reviews, see Ref. 4-8). This ability is assumed to be a consequence of such tracts adopting a stable B'-DNA conformation which is distinct from that normally assumed by other I~NA sequences. A major distinguishing feature of the B' structure I A. A. Travers, Annu. Reo. Biochem. 58, 427 (1989). 2 R. E. Dickerson and H. R. Drew, J. Mol. Biol. 149, 761 (1981). 3 S. D i e k m a n n and J. C. Wang, J. Mol. Biol. 186, 1 (1985). 4 E. N. Trifonov, Crit. Reu. Biochem. 19, 89 (1986). 5 S. D i e k m a n n , in " N u c l e i c Acids and Molecular Biology" (F. Eckstein and D. M. J. Lilley, eds.), Vol. 1, p. 138. Springer-Verlag, Berlin, 1987. 6 A. A, T r a v e r s and A. Klug, Philos. Trans. R. Soc. London, Ser. B 317, 537 (1987). 7 p. j. Hagerrnan, Annu. Rev. Biochem. 59, 755 (1990). 8 D. M. Crothers, T. E. Haran, and J. G. N a d e a u , J. Biol. Chem. 2,65, 7093 (1990).
METHODS IN ENZYMOLOGY.VOL. 212
Copyright © 1992by AcademicPress, Inc. All rights of reproduction in any form reserved.
[2]
ANALYZING DNA CURVATURE IN POLYACRYLAMIDE GELS
31
is a high propeller t w i s t T M that optimizes stacking interactions and narrows the minor groove. In principle such a propeller twist can only be readily adopted by base pairs with two, but not three, hydrogen bonds. The naturally occurring base pair with this property is dA- dT, but other base pairs, notably dI. dC, also share this characteristic. The B' structure might also have a large wedge roll angle between the dAA dinucleotides.a, 5,8 D N A curvature can be detected using several experimental methods: circularization of D N A molecules, 15-18 electron microscopy, 19'2° and gel migration in gel electrophoresis. 3'21-27 Results obtained from these measurements can be compared with D N A structures obtained from nuclear magnetic resonance (NMR) H'28 and X-ray crystallographic analysis. ~2-14 Other techniques like Raman, 29circular dichroism (CD),29,30 and N M R 9'31'32 spectroscopy yield relevant information on the structural properties of curved D N A sequences. 9 R. Behling and D. R. Kearns, Biochemistry 25, 3335 (1986). 10 D. G. Alexeev, A. A. Lipanov, and I. Y. Skuratovski, Nature (London) 325, 821 (1987). 11 A. A. Lipanov and V. P. Chuprina, Nucleic Acids Res. 15, 5833 (1987), 12 H. C. M. Nelson, J. T. Finch, B. F. Luisi, and A. Klug, Nature (London) 330, 221 (1987). 13 M. Coil, C. A. Frederick, A. H.-J. Wang, and A. Rich, Proc. Natl. Acad. Sci. U.S.A. 84, 8385 (1987). 14 A. D. DiGabriele, M. R. Sanderson, and T. A. Steitz, Proc. Natl. Acad. Sci. U.S.A. 86, 1816 (1989). is K. Zahn and F. Blattner, Nature (London) 317, 451 (1985). I6 L. Ulanovsky, M. Bodner, E. N. Trifonov, and M. Choder, Proc. Natl. Acad. Sci. U.S.A. 8, 862 (1986). 17 S. D. Levene and D. M. Crothers, J. Mol. Biol. 189, 61 (1986). 18 K. Zahn and F. Blattner, Science 236, 416 (1987). 19 1. Griffith, M. Bleyman, C. A. Rauch, P. A. Kitchin, and P. T. Englund, Cell (Cambridge, Mass.) 46, 717 (1986). 2o B. Theveny, D. Coulaud, M. LeBret, and B. Revet, in "Structure and Expression Volume 3: DNA Bending and Curvature" (W. K. Olson, M. H. Sarma, R. H. Sarma, and M. Sundaralingam, eds.), p. 39. Adenine Press, Schenectady, New York, 1988. 21 j. (2. Marini, S. D. Levene, D. M. Crothers, and P. T. Englund, Proc. Natl. Acad. Sci. U.S.A. 79, 7664 (1982). 22 j. (2. Marini and P. T. Englund, Proc. Natl. Acad. Sci. U.S.A. 80, 7678 (1983). 23 H. M. Wu and D. M. Crothers, Nature (London) 308, 509 (1984). 24 p. j. Hagerman, Biochemistry 24, 7033 (1985). 25 S. Diekmann, FEBS Lett. 195, 53 (1986). 26 H. S. Koo, H. M. Wu, and D. M. Crothers, Nature (London) 320, 501 (1986). 27 p. 3. Hagerman, Nature (London) 321, 449 (1986). 28 M. H. Sarma, G. Gupta, and R. H. Sarma, Biochemistry 27, 3423 (1988). 29 S. Brahms and J. G. Brahms, Nucleic Acids Res. 18, 1559 (1990). 30 S. R. Gudibande, S. D. Jayasena, and M. J. Behe, Biopolymers 27, 1905 (1988). 31 J.-L. Leroy, E. Charretier, M. Kochoyan, and M. Gueron, Biochemistry 27, 8894 (1988). 32 j. G. Nadeau and D. M. Crothers, Proc. Natl. Acad. Sci. U.S.A. 86, 2622 (1989).
32
GEL ELECTROPHORESIS AND TOPOLOGICAL METHODS
[2]
From the migration analysis in polyacrylamide gels, absolute values are not obtained for structural DNA parameters. This is a principal drawback of the gel assay. Therefore, conclusions obtained from this technique should be verified by other methods based on better understood physical techniques. However, rigorous techniques are often elaborate and do not detect the local structural variations which can be resolved in the gel (e.g., see Diekmann and P6rschke33). The gel assay is very convenient, and small amounts (a few nanograms) of DNA can be analyzed. Furthermore, the mobility of DNA molecules in gels is not affected by the presence of other DNA fragments or a variety of chemical compounds present in small quantities. The gel migration anomaly of considerably curved DNA fragments is a rather large effect since the anomaly increases with the square of the degree of the curvature. 3
