- Email: [email protected]
The structure of intramolecular triplex DNA: atomic force microscopy study1
doi:10.1006/jmbi.2001.5174 available online at http://www.idealibrary.com on
J. Mol. Biol. (2001) 314, 353±357
COMMUNICATION
The Structure of Intramolecular Triplex DNA: Atomic Force Microscopy Study William J. Tiner Sr1, Vladimir N. Potaman2, Richard R. Sinden2 and Yuri L. Lyubchenko1* 1
Department of Microbiology Arizona State University Tempe, AZ 85281-2701, USA 2
Institute of Biosciences and Technology, The Texas A&M University System Health Science Center, 2121 W. Holcombe Blvd, Houston TX 77030, USA
We applied atomic force microscopy (AFM) for direct imaging of intramolecular triplexes (H-DNA) formed by mirror-repeated purine-pyrimidine repeats and stabilized by negative DNA supercoiling. H-DNA appears in atomic force microscopy images as a clear protrusion with a different thickness than DNA duplex. Consistent with the existing models, H-DNA formation results in a kink in the double helix path. The kink forms an acute angle so that the ¯anking DNA regions are brought in close proximity. The mobility of ¯anking DNA arms is limited compared with that for cruciforms and three-way junctions. Structural properties of H-DNA may be important for promoter-enhancer interactions and other DNA transactions. # 2001 Academic Press
*Corresponding author
Keywords: atomic force microscopy; intramolecular triplexes; alternative DNA structures; H-DNA; DNA supercoiling
Although the predominant DNA structure is a B-DNA double helix, several alternative structures have been discovered that may play important biological roles.1 An intramolecular DNA triplex or H-DNA formed by homopurine-homopyrimidine (Pu Py) tracts is one biologically important alternative DNA structure.2,3 Potential biological roles of H-DNA are supported by a statistical overrepresentation of Pu Py tracts in the genomes of eukaryotes and prokaryotes.4 ± 6 Mirror-repeated Pu Py tracts that form H-DNA occur as frequently as 1 in 50,000 bp of human genomic DNA.7 In addition, several classes of Pu Py-binding proteins have been described.8 A number of models for the involvement of intramolecular triplexes in biological processes have been proposed.1 They include H-DNA roles in gene expression,9 DNA replication,10 recombination11 and stabilization of the structure of telomeres.12 Structural studies of intramolecular triplexes may provide an understanding of the potential for H-DNA involvement in these processes. DNA supercoiling induces a transition of B-DNA Pu Py mirror repeat tracts into H-DNA.3 It Abbreviations used: AFM, atomic force microscopy; Hy or Hr isomers, PyPuPy or PyPuPu H-DNA, respectively; EM, electron microscopy. E-mail address of the corresponding author: [email protected] 0022-2836/01/030353±5 $35.00/0
is suggested that H-DNA is formed by local unwinding in half of the Pu Py tract, and then the folding of a Py or a Pu single strand into the major groove of the duplex half of the Pu Py DNA, thus forming a PyPuPy or a PyPuPu H-DNA (Hy or Hr isomers, respectively). Hoogsteen hydrogen bond complementarity provides the sequence speci®city of triple helix formation.1,3 The major elements of H-DNA are a triplex and an unpaired half of the Pu or Py strand. Experimental and theoretical studies have provided a multi-faceted physical chemical characterization of the structural transition in Pu Py tracts (reviewed by Sinden1 and Soyfer & Potaman3). Numerous chemical and enzymatic probes have revealed the effects of DNA sequence and the state of DNA supercoiling, as well as pH and cations on the formation, stability, and isomerization of triplex DNA.3,13 There has been little effort to directly analyze the spatial geometry of H-DNA. Intramolecular triplexes are not thermodynamically stable in linear DNA. In addition, H-DNA in relatively large supercoiled DNA molecules are not amenable to studies by traditional structural biology methods, such as high-resolution NMR and crystallography. This structure is different from an intermolecular triplex that can be studied by different techniques including atomic force microscopy (AFM).14 Electron microscopy (EM) revealed the formation of a rather thick and a relatively long stem under the # 2001 Academic Press
354
AFM of H-DNA
conditions where H-DNA should be present in plasmid DNA.15 According to the existing models,1,3,16 the DNA molecule makes a U-turn at the site of H-DNA formation, and thus a sharp kink is introduced into DNA. The presence of single strands likely makes the kink ¯exible which provides a hinge between the ¯anking duplexes,1,16 although the extent of ¯exibility at the kink has not been determined. The position of an unpaired strand relative to the triple-stranded part of the H-DNA is also not known. It may be either loosely or closely associated with the triplex.17 Consistent with the U-turn model, it is possible that the H-DNA structure has a predominant, almost parallel orientation of the outgoing DNA duplexes. Indirect evidence of such an orientation was reported by Kohwi & Panchenko11 who described facilitated recombination between two homologous regions separated by the sequence capable of forming H-DNA. We have recently applied AFM for direct imaging of supercoil-stabilized cruciforms.18 These studies revealed the existence of two different cruciform conformations. The transition of the cruciform between these conformations can act as a molecular switch for global DNA geometry and dynamics.19 Here, using AFM, we analyze structural features of H-DNA in supercoiled plasmid. Plasmid pCW2966 containing a 46 bp mirror repeat (Figure 1(a)) from the PKD1 gene was used in this work.20 The transition of this insert into H-DNA is shown schematically in Figure 1(b). Two-dimensional gel electrophoresis data and chemical probing experiments showed that at pH 5 the insert forms H-DNA at a threshold superhelical density of s ÿ 0.04.20 For AFM, the DNA sample was incubated in 50 mM sodium acetate buffer at pH 5.0 and then deposited onto APS-mica21 in the same buffer. The images for plasmid containing H-DNA were acquired in air (Figure 2(a)). A clear feature in these images is the appearance of a sharp kink at the base of a thick protrusion indicated with arrows. Control images of plasmid pUC19 without the insert prepared in the same acidic buffer are shown in Figure 2(b). Although sharp kinks can be found in some of the molecules (e.g. molecule 1), these are small loops as shown in zoomed images in the Figure 2(b) inset. Higher resolution images of the H-DNA containing molecules with protrusions are shown in Figure 2(c) and (d). These structures can be unambiguously identi®ed in 30 % molecules. It should be noted that the superhelicity of the plasmid as isolated from Escherichia coli may not be suf®cient to induce H-DNA formation in 100 % molecules. In addition,
Figure 1. H-DNA structure used for AFM visualization. (a) Sequence of the 46 bp mirror-repeated PuPy insert. (b) At pH 5, the Hy3 structure forms in which the 30 Py end folds back as the third strand in the major groove of the duplex. The symbols * and indicate hydrogen bonds between duplex purines and third strand pyrimidines in A T and GC pairs, respectively.
the interaction of H-DNA with the slightly basic surface of APS-modi®ed mica may somewhat destabilize triplex formation which is favorable at acidic pH. Similar to the X-type cruciform, the thick protrusions (H-DNA) occur at the apex of the U-turn positions in plasmid molecules. This is not surprising because both alternative structures form sharp kinks in an otherwise smooth path of the DNA double helix. H-DNA was characterized with the following parameters: the protrusion length, width and height, and the angle between the outgoing arms (Table 1). The height and width were measured using a cross-section option of the AFM software, the width being measured at the halfheight (Figure 2(e)). Schematically, the measurements of the length of the protrusion and the kink angle are shown in the insert in Figure 2(f). Appropriate parameters for H-DNA (the width and the height) are compared with those for B-DNA measured for the same DNA molecules. The average protrusion length determined from AFM images is about 12 nm. This value can be compared with an expected length of H-DNA assuming that the helical parameters of the nonpolymeric triplex are close to those of the B-DNA.22 An estimated length of the H-DNA protrusion that consists of 21 triads and a 4 nt loop is about 7 nm, which is shorter than the measured length. Given uncertainties in de®ning the boundaries of the H-DNA stem and the image broaden-
Table 1. The parameters of AFM images of H-DNA and B-DNA Sample
Length (nm)
Width (nm)
Height (nm)
The kink angle (deg.)
H-DNA B-DNA
12.05 1.7
11.7 2.5 8.3 2.1
1.12 0.14 0.69 0.14
52.4 16.5
AFM of H-DNA
355
Figure 2. AFM images of DNA deposited at pH 5.0. (a) A largescale image of plasmid pCW2966 containing a 46 bp mirror repeat. (b) Images of plasmid pUC19 without the repeat; the inset is the highresolution image of molecules 1 and 2. (c) and (d) High-resolution images of H-DNA. Schematics of the height and width (e) and length and kink angle (f) measurements in the H-DNA. Plasmid pCW2966 contains a 46 bp mirror repeat from the PKD1 gene (bp 3461334658) cloned into the EcoRI site in pUC19.20 DNA at native supercoiling was isolated from the E. coli strain HB101 by alkaline lysis followed by equilibrium centrifugation in CsCl. To induce the transition into H-DNA for AFM, the DNA sample was incubated in 50 mM sodium acetate buffer at pH 5.0 for at least ten minutes, which was suf®cient for the complete H-DNA formation (data not shown), and then deposited onto APS-mica21 in the same buffer. The AFM imaging procedure has been described elsewhere.18 Brie¯y, DNA samples (3-5 ml) were placed onto APS-mica for two minutes, and the mica was rinsed with deionized water (Continental Water System Co., San Antonio, TX), after which the specimens were dried in an argon ¯ow. Images were acquired by MM SPM NanoScope 3a system (Veeco/Digital Instruments, Santa Barbara, CA) operating in Tapping Mode in air at ambient conditions using OTESPA probes (Digital Instruments, Inc.). The length, height and angle measurements were performed with the NanoScope software (version 4.43r8).
ing due to the tip convolution effects,18,19 the difference between the experimentally determined and expected values is acceptable. Judged from the measurements of the protrusions and DNA strands elsewhere in the plasmid, the folded H-DNA structure is about 1.5 times wider than B-DNA. Interestingly, this difference is not masked by the tip convolution effect. Another parameter, the height of DNA molecules, can be determined without taking into account the convolution effect. The H-DNA height is about 1.1 nm, whereas the B-DNA height is about 0.69 nm (Table 1), which clearly indicates a substantial difference in the thickness of duplex B-DNA and H-DNA composed of the triple helix
and the fourth unpaired strand. The heights of DNA duplex and another four-stranded DNA structure (quadruplex) in the AFM images were compared by Marsh et al.23 The symmetric fourstranded DNA quadruplex had more than twice the height of the DNA duplex. A smaller height difference between the duplex DNA and triplestrand/single strand H-DNA is consistent with the differences in the models of these two DNA structures, especially the positioning of the third strand in the major groove of intramolecular triplex DNA. The formation of a sharp kink is a characteristic feature of H-DNA. Due to the fold-back nature of intramolecular triplex, DNA strands make almost a complete U-turn. The angle between the two out-
356 going duplex strands (Table 1) is 52.4(16.5) . In comparison, the variability of the inter-arm angle in the X-type supercoil-stabilized cruciform18 is 71(36) , and it is even higher in the three-way DNA junctions.21,24 These data suggest that H-DNA is a relatively rigid structure. This study presents the ®rst AFM visualization of H-DNA. The only published EM images,15 although consistent with the H-DNA kinks in plasmid molecules, showed presumed H-DNA stems that were excessively long. Our AFM images allow clear visualization and thus facilitate characterization of the molecular structure around the site of H-DNA. Under conditions of stable triplex existence, the formation of a sharp kink and an H-DNA stem of expected size have been detected. As a result, the geometric parameters for the triple-helical structure and outgoing duplex strands have been determined to be about 52.4(16.5) . The model of H-DNA formation16 predicts that half of the Watson-Crick duplex in the purine-pyrimidine region is unpaired and the unwound polypyrimidine strand folds back into the major groove associating with the purine strand through Hoogsteen base-pairing. Similarly, another H-DNA isomer may form in which a half of polypurine strand may form Hoogsteen-type hydrogen bonds. The induction of a kink by H-DNA formation would be expected to affect the global DNA structure and dynamics in a manner similar to the cruciform.19 Indeed, the region of H-DNA formation is always located at the U-turn position of the DNA molecule, thereby bringing distant sequence elements in closer proximity than expected in a randomly coiled DNA. A sharp kink in the DNA strands is likely ¯exible due to the presence of single strands between the triple-stranded region and adjacent duplexes. DNA ¯exibility at the kink may provide relatively unrestricted mutual orientations of outgoing duplexes. Thus, the angle of the kink in H-DNA is expected to be variable, unlike that in the permanently bent regions such as the A-tracts. Consistent with the assumption of the so-called hinge model, the H-DNA containing fragments did not migrate in a polyacrylamide gel at a constant rate, likely due to variations in the angle of the kink.16 The AFM data presented here indicate a rather low variability of the angle between the two duplex strands so that the ¯exibility at the hinge, although present, is signi®cantly limited. The limited ¯exibility may re¯ect either some structural rigidity in the forth, unpaired strand, for example, signi®cant stacking of purine bases, or loose wrapping of the fourth strand around the triplex. At present, the conformation of unpaired H-DNA strand remains unknown. Several lines of evidence have been presented in support of H-DNA roles in genetic recombination, gene regulation, mutagenesis, and regulation of DNA replication.1 ± 3 Kink formation causes a change in DNA geometry, bringing ¯anking DNA sequences into close proximity and thereby stimu-
AFM of H-DNA
lating strand exchange.11,25 Bends and loops are also important gene regulatory control mechanisms, and the transition between B-DNA and H-DNA could promote loop formation. Our AFM data support these models. First, we have con®rmed the strong bend of the DNA duplex at the H-DNA region. Second, the limited mobility at the hinge region may provide extended periods of time for the ¯anking regions to probe for sequence homology or for interaction of proteins bound to DNA at distant locations.
Acknowledgments The work was supported by the grants GM 62235 (NIH) and DBI-0070356 (NSF). We are grateful to L. Shlyakhtenko for very useful comments and criticism, and V. Zhurkin and V. Vologodskii for valuable comments. The authors also thank J. J. Bissler for kindly providing the plasmid and the Keck Bioimaging Laboratory at ASU for the use of its facilities.
References 1. Sinden, R. R. (1994). DNA Structure and Function, Academic Press, San Diego. 2. Mirkin, S. M. & Frank-Kamenetskii, M. D. (1994). H-DNA and related structures. Annu. Rev. Biophys. Biomol. Struct. 23, 541-576. 3. Soyfer, V. N. & Potaman, V. N. (1996). Triple-helical Nucleic Acids, Springer, New York. 4. Birnboim, H. C. (1978). Spacing of polypyrimidine regions in mouse DNA as determined by poly(adenylate, guanylate) binding. J. Mol. Biol. 121, 541-559. 5. Bucher, P. & Yagil, G. (1991). Occurrence of oligopurine. oligopyrimidine tracts in eukaryotic and prokaryotic genes. DNA Seq. 1, 157-172. 6. Behe, M. J. (1995). An overabundance of long oligopurine tracts occurs in the genome of simple and complex eukaryotes. Nucl. Acids Res. 23, 689-695. 7. Schroth, G. P. & Ho, P. S. (1995). Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA. Nucl. Acids Res. 23, 1977-1983. 8. Guillonneau, F., Guieysse, A. L., Le Caer, J. P., Rossier, J. & Praseuth, D. (2001). Selection and identi®cation of proteins bound to DNA triple-helical structures by combination of 2D-electrophoresis and MALDI-TOF mass spectrometry. Nucl. Acids Res. 29, 2427-2436. 9. Lee, J. S., Woodsworth, M. L., Latimer, L. J. & Morgan, A. R. (1984). Poly(pyrimidine) poly(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH. Nucl. Acids Res. 12, 6603-6614. 10. Dayn, A., Samadashwily, G. M. & Mirkin, S. M. (1992). Intramolecular DNA triplexes: unusual sequence requirements and in¯uence on DNA polymerization. Proc. Natl Acad. Sci. USA, 89, 1140611410. 11. Kohwi, Y. & Panchenko, Y. (1993). Transcriptiondependent recombination induced by triple-helix formation. Genes Dev. 7, 1766-1778. 12. Veselkov, A. G., Malkov, V. A., Frank-Kamenetskll, M. D. & Dobrynin, V. N. (1993). Triplex model of chromosome ends. Nature, 364, 496.
357
AFM of H-DNA 13. Potaman, V. N. & Sinden, R. R. (1998). Stabilization of intramolecular triple/single-strand structure by cationic peptides. Biochemistry, 37, 12952-12961. 14. Hansma, H. G., Revenko, I., Kim, K. & Laney, D. E. (1996). Atomic force microscopy of long and short double-stranded, single-stranded and triple-stranded nucleic acids. Nucl. Acids Res. 24, 713-720. 15. Stokrova, J., Vojtiskova, M. & Palecek, E. (1989). Electron microscopy of supercoiled pEJ4 DNA containing homopurine. homopyrimidine sequences. J. Biomol. Struct. Dynam. 6, 891-898. 16. Htun, H. & Dahlberg, J. E. (1989). Topology and formation of triple-stranded H-DNA. Science, 243, 1571-1576. 17. Kohwi, Y. & Kohwi-Shigematsu, T. (1988). Magnesium ion-dependent triple-helix structure formed by homopurine- homopyrimidine sequences in supercoiled plasmid DNA. Proc. Natl Acad. Sci. USA, 85, 3781-3785. 18. Shlyakhtenko, L. S., Potaman, V. N., Sinden, R. R. & Lyubchenko, Y. L. (1998). Structure and dynamics of supercoil-stabilized DNA cruciforms. J. Mol. Biol. 280, 61-72. 19. Shlyakhtenko, L. S., Hsieh, P., Grigoriev, M., Potaman, V., Sunder, R. R. & Lyubchenko, Y. L. (2000). A cruciform structural transition provides a molecular switch for chromosome structure and dynamics. J. Mol. Biol. 296, 1169-1173.
20. Blaszak, R. T., Potaman, V., Sinden, R. R. & Bissler, J. J. (1999). DNA structural transitions within the PKD1 gene. Nucl. Acids Res. 27, 2610-2617. 21. Shlyakhtenko, L. S., Potaman, V. N., Sinden, R. R., Gall, A. A. & Lyubchenko, Y. L. (2000). Structure and dynamics of three-way DNA junctions: atomic force microscopy studies. Nucl. Acids Res. 28, 34723477. 22. Bhaumik, S. R., Chary, K. V., Govil, G., Liu, K. & Miles, H. T. (1998). A novel palindromic triplestranded structure formed by homopyrimidine dodecamer d-CTTCTCCTCTTC and homopurine hexamer d-GAAGAG. Nucl. Acids Res. 26, 29812988. 23. Marsh, T. C., Vesenka, J. & Henderson, E. (1995). A new DNA nanostructure, the G-wire, imaged by scanning probe microscopy. Nucl. Acids Res. 23, 696700. 24. Oussatcheva, E. A., Shlyakhtenko, L. S., Glass, R., Sunden, R. R., Lyubchenko, Y. L. & Potaman, V. N. (1999). Structure of branched DNA molecules: gel retardation and atomic force microscopy studies. J. Mol. Biol. 292, 75-86. 25. Rooney, S. M. & Moore, P. D. (1995). Antiparallel, intramolecular triplex DNA stimulates homologous recombination in human cells. Proc. Natl Acad. Sci. USA, 92, 2141-2144.
Edited by I. Tinoco (Received 3 August 2001; received in revised form 9 October 2001; accepted 10 October 2001)
J. Mol. Biol. (2001) 314, 353±357
COMMUNICATION
The Structure of Intramolecular Triplex DNA: Atomic Force Microscopy Study William J. Tiner Sr1, Vladimir N. Potaman2, Richard R. Sinden2 and Yuri L. Lyubchenko1* 1
Department of Microbiology Arizona State University Tempe, AZ 85281-2701, USA 2
Institute of Biosciences and Technology, The Texas A&M University System Health Science Center, 2121 W. Holcombe Blvd, Houston TX 77030, USA
We applied atomic force microscopy (AFM) for direct imaging of intramolecular triplexes (H-DNA) formed by mirror-repeated purine-pyrimidine repeats and stabilized by negative DNA supercoiling. H-DNA appears in atomic force microscopy images as a clear protrusion with a different thickness than DNA duplex. Consistent with the existing models, H-DNA formation results in a kink in the double helix path. The kink forms an acute angle so that the ¯anking DNA regions are brought in close proximity. The mobility of ¯anking DNA arms is limited compared with that for cruciforms and three-way junctions. Structural properties of H-DNA may be important for promoter-enhancer interactions and other DNA transactions. # 2001 Academic Press
*Corresponding author
Keywords: atomic force microscopy; intramolecular triplexes; alternative DNA structures; H-DNA; DNA supercoiling
Although the predominant DNA structure is a B-DNA double helix, several alternative structures have been discovered that may play important biological roles.1 An intramolecular DNA triplex or H-DNA formed by homopurine-homopyrimidine (Pu Py) tracts is one biologically important alternative DNA structure.2,3 Potential biological roles of H-DNA are supported by a statistical overrepresentation of Pu Py tracts in the genomes of eukaryotes and prokaryotes.4 ± 6 Mirror-repeated Pu Py tracts that form H-DNA occur as frequently as 1 in 50,000 bp of human genomic DNA.7 In addition, several classes of Pu Py-binding proteins have been described.8 A number of models for the involvement of intramolecular triplexes in biological processes have been proposed.1 They include H-DNA roles in gene expression,9 DNA replication,10 recombination11 and stabilization of the structure of telomeres.12 Structural studies of intramolecular triplexes may provide an understanding of the potential for H-DNA involvement in these processes. DNA supercoiling induces a transition of B-DNA Pu Py mirror repeat tracts into H-DNA.3 It Abbreviations used: AFM, atomic force microscopy; Hy or Hr isomers, PyPuPy or PyPuPu H-DNA, respectively; EM, electron microscopy. E-mail address of the corresponding author: [email protected] 0022-2836/01/030353±5 $35.00/0
is suggested that H-DNA is formed by local unwinding in half of the Pu Py tract, and then the folding of a Py or a Pu single strand into the major groove of the duplex half of the Pu Py DNA, thus forming a PyPuPy or a PyPuPu H-DNA (Hy or Hr isomers, respectively). Hoogsteen hydrogen bond complementarity provides the sequence speci®city of triple helix formation.1,3 The major elements of H-DNA are a triplex and an unpaired half of the Pu or Py strand. Experimental and theoretical studies have provided a multi-faceted physical chemical characterization of the structural transition in Pu Py tracts (reviewed by Sinden1 and Soyfer & Potaman3). Numerous chemical and enzymatic probes have revealed the effects of DNA sequence and the state of DNA supercoiling, as well as pH and cations on the formation, stability, and isomerization of triplex DNA.3,13 There has been little effort to directly analyze the spatial geometry of H-DNA. Intramolecular triplexes are not thermodynamically stable in linear DNA. In addition, H-DNA in relatively large supercoiled DNA molecules are not amenable to studies by traditional structural biology methods, such as high-resolution NMR and crystallography. This structure is different from an intermolecular triplex that can be studied by different techniques including atomic force microscopy (AFM).14 Electron microscopy (EM) revealed the formation of a rather thick and a relatively long stem under the # 2001 Academic Press
354
AFM of H-DNA
conditions where H-DNA should be present in plasmid DNA.15 According to the existing models,1,3,16 the DNA molecule makes a U-turn at the site of H-DNA formation, and thus a sharp kink is introduced into DNA. The presence of single strands likely makes the kink ¯exible which provides a hinge between the ¯anking duplexes,1,16 although the extent of ¯exibility at the kink has not been determined. The position of an unpaired strand relative to the triple-stranded part of the H-DNA is also not known. It may be either loosely or closely associated with the triplex.17 Consistent with the U-turn model, it is possible that the H-DNA structure has a predominant, almost parallel orientation of the outgoing DNA duplexes. Indirect evidence of such an orientation was reported by Kohwi & Panchenko11 who described facilitated recombination between two homologous regions separated by the sequence capable of forming H-DNA. We have recently applied AFM for direct imaging of supercoil-stabilized cruciforms.18 These studies revealed the existence of two different cruciform conformations. The transition of the cruciform between these conformations can act as a molecular switch for global DNA geometry and dynamics.19 Here, using AFM, we analyze structural features of H-DNA in supercoiled plasmid. Plasmid pCW2966 containing a 46 bp mirror repeat (Figure 1(a)) from the PKD1 gene was used in this work.20 The transition of this insert into H-DNA is shown schematically in Figure 1(b). Two-dimensional gel electrophoresis data and chemical probing experiments showed that at pH 5 the insert forms H-DNA at a threshold superhelical density of s ÿ 0.04.20 For AFM, the DNA sample was incubated in 50 mM sodium acetate buffer at pH 5.0 and then deposited onto APS-mica21 in the same buffer. The images for plasmid containing H-DNA were acquired in air (Figure 2(a)). A clear feature in these images is the appearance of a sharp kink at the base of a thick protrusion indicated with arrows. Control images of plasmid pUC19 without the insert prepared in the same acidic buffer are shown in Figure 2(b). Although sharp kinks can be found in some of the molecules (e.g. molecule 1), these are small loops as shown in zoomed images in the Figure 2(b) inset. Higher resolution images of the H-DNA containing molecules with protrusions are shown in Figure 2(c) and (d). These structures can be unambiguously identi®ed in 30 % molecules. It should be noted that the superhelicity of the plasmid as isolated from Escherichia coli may not be suf®cient to induce H-DNA formation in 100 % molecules. In addition,
Figure 1. H-DNA structure used for AFM visualization. (a) Sequence of the 46 bp mirror-repeated PuPy insert. (b) At pH 5, the Hy3 structure forms in which the 30 Py end folds back as the third strand in the major groove of the duplex. The symbols * and indicate hydrogen bonds between duplex purines and third strand pyrimidines in A T and GC pairs, respectively.
the interaction of H-DNA with the slightly basic surface of APS-modi®ed mica may somewhat destabilize triplex formation which is favorable at acidic pH. Similar to the X-type cruciform, the thick protrusions (H-DNA) occur at the apex of the U-turn positions in plasmid molecules. This is not surprising because both alternative structures form sharp kinks in an otherwise smooth path of the DNA double helix. H-DNA was characterized with the following parameters: the protrusion length, width and height, and the angle between the outgoing arms (Table 1). The height and width were measured using a cross-section option of the AFM software, the width being measured at the halfheight (Figure 2(e)). Schematically, the measurements of the length of the protrusion and the kink angle are shown in the insert in Figure 2(f). Appropriate parameters for H-DNA (the width and the height) are compared with those for B-DNA measured for the same DNA molecules. The average protrusion length determined from AFM images is about 12 nm. This value can be compared with an expected length of H-DNA assuming that the helical parameters of the nonpolymeric triplex are close to those of the B-DNA.22 An estimated length of the H-DNA protrusion that consists of 21 triads and a 4 nt loop is about 7 nm, which is shorter than the measured length. Given uncertainties in de®ning the boundaries of the H-DNA stem and the image broaden-
Table 1. The parameters of AFM images of H-DNA and B-DNA Sample
Length (nm)
Width (nm)
Height (nm)
The kink angle (deg.)
H-DNA B-DNA
12.05 1.7
11.7 2.5 8.3 2.1
1.12 0.14 0.69 0.14
52.4 16.5
AFM of H-DNA
355
Figure 2. AFM images of DNA deposited at pH 5.0. (a) A largescale image of plasmid pCW2966 containing a 46 bp mirror repeat. (b) Images of plasmid pUC19 without the repeat; the inset is the highresolution image of molecules 1 and 2. (c) and (d) High-resolution images of H-DNA. Schematics of the height and width (e) and length and kink angle (f) measurements in the H-DNA. Plasmid pCW2966 contains a 46 bp mirror repeat from the PKD1 gene (bp 3461334658) cloned into the EcoRI site in pUC19.20 DNA at native supercoiling was isolated from the E. coli strain HB101 by alkaline lysis followed by equilibrium centrifugation in CsCl. To induce the transition into H-DNA for AFM, the DNA sample was incubated in 50 mM sodium acetate buffer at pH 5.0 for at least ten minutes, which was suf®cient for the complete H-DNA formation (data not shown), and then deposited onto APS-mica21 in the same buffer. The AFM imaging procedure has been described elsewhere.18 Brie¯y, DNA samples (3-5 ml) were placed onto APS-mica for two minutes, and the mica was rinsed with deionized water (Continental Water System Co., San Antonio, TX), after which the specimens were dried in an argon ¯ow. Images were acquired by MM SPM NanoScope 3a system (Veeco/Digital Instruments, Santa Barbara, CA) operating in Tapping Mode in air at ambient conditions using OTESPA probes (Digital Instruments, Inc.). The length, height and angle measurements were performed with the NanoScope software (version 4.43r8).
ing due to the tip convolution effects,18,19 the difference between the experimentally determined and expected values is acceptable. Judged from the measurements of the protrusions and DNA strands elsewhere in the plasmid, the folded H-DNA structure is about 1.5 times wider than B-DNA. Interestingly, this difference is not masked by the tip convolution effect. Another parameter, the height of DNA molecules, can be determined without taking into account the convolution effect. The H-DNA height is about 1.1 nm, whereas the B-DNA height is about 0.69 nm (Table 1), which clearly indicates a substantial difference in the thickness of duplex B-DNA and H-DNA composed of the triple helix
and the fourth unpaired strand. The heights of DNA duplex and another four-stranded DNA structure (quadruplex) in the AFM images were compared by Marsh et al.23 The symmetric fourstranded DNA quadruplex had more than twice the height of the DNA duplex. A smaller height difference between the duplex DNA and triplestrand/single strand H-DNA is consistent with the differences in the models of these two DNA structures, especially the positioning of the third strand in the major groove of intramolecular triplex DNA. The formation of a sharp kink is a characteristic feature of H-DNA. Due to the fold-back nature of intramolecular triplex, DNA strands make almost a complete U-turn. The angle between the two out-
356 going duplex strands (Table 1) is 52.4(16.5) . In comparison, the variability of the inter-arm angle in the X-type supercoil-stabilized cruciform18 is 71(36) , and it is even higher in the three-way DNA junctions.21,24 These data suggest that H-DNA is a relatively rigid structure. This study presents the ®rst AFM visualization of H-DNA. The only published EM images,15 although consistent with the H-DNA kinks in plasmid molecules, showed presumed H-DNA stems that were excessively long. Our AFM images allow clear visualization and thus facilitate characterization of the molecular structure around the site of H-DNA. Under conditions of stable triplex existence, the formation of a sharp kink and an H-DNA stem of expected size have been detected. As a result, the geometric parameters for the triple-helical structure and outgoing duplex strands have been determined to be about 52.4(16.5) . The model of H-DNA formation16 predicts that half of the Watson-Crick duplex in the purine-pyrimidine region is unpaired and the unwound polypyrimidine strand folds back into the major groove associating with the purine strand through Hoogsteen base-pairing. Similarly, another H-DNA isomer may form in which a half of polypurine strand may form Hoogsteen-type hydrogen bonds. The induction of a kink by H-DNA formation would be expected to affect the global DNA structure and dynamics in a manner similar to the cruciform.19 Indeed, the region of H-DNA formation is always located at the U-turn position of the DNA molecule, thereby bringing distant sequence elements in closer proximity than expected in a randomly coiled DNA. A sharp kink in the DNA strands is likely ¯exible due to the presence of single strands between the triple-stranded region and adjacent duplexes. DNA ¯exibility at the kink may provide relatively unrestricted mutual orientations of outgoing duplexes. Thus, the angle of the kink in H-DNA is expected to be variable, unlike that in the permanently bent regions such as the A-tracts. Consistent with the assumption of the so-called hinge model, the H-DNA containing fragments did not migrate in a polyacrylamide gel at a constant rate, likely due to variations in the angle of the kink.16 The AFM data presented here indicate a rather low variability of the angle between the two duplex strands so that the ¯exibility at the hinge, although present, is signi®cantly limited. The limited ¯exibility may re¯ect either some structural rigidity in the forth, unpaired strand, for example, signi®cant stacking of purine bases, or loose wrapping of the fourth strand around the triplex. At present, the conformation of unpaired H-DNA strand remains unknown. Several lines of evidence have been presented in support of H-DNA roles in genetic recombination, gene regulation, mutagenesis, and regulation of DNA replication.1 ± 3 Kink formation causes a change in DNA geometry, bringing ¯anking DNA sequences into close proximity and thereby stimu-
AFM of H-DNA
lating strand exchange.11,25 Bends and loops are also important gene regulatory control mechanisms, and the transition between B-DNA and H-DNA could promote loop formation. Our AFM data support these models. First, we have con®rmed the strong bend of the DNA duplex at the H-DNA region. Second, the limited mobility at the hinge region may provide extended periods of time for the ¯anking regions to probe for sequence homology or for interaction of proteins bound to DNA at distant locations.
Acknowledgments The work was supported by the grants GM 62235 (NIH) and DBI-0070356 (NSF). We are grateful to L. Shlyakhtenko for very useful comments and criticism, and V. Zhurkin and V. Vologodskii for valuable comments. The authors also thank J. J. Bissler for kindly providing the plasmid and the Keck Bioimaging Laboratory at ASU for the use of its facilities.
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Edited by I. Tinoco (Received 3 August 2001; received in revised form 9 October 2001; accepted 10 October 2001)