Báo cáo Y học: Structural heterogeneity of pyrimidine/purine-biased DNA sequence analyzed by atomic force microscopy potx

PRIORITY PAPER Structural heterogeneity of pyrimidine/purine-biased DNA sequence analyzed by atomic force microscopy Mikio Kato 1,2 , Chad J. McAllister 2 , Shingo Hokabe 1 , Nobuyoshi Shimizu 3 and Yuri L. Lyubchenko 2 1 Department of Life Science, Osaka Prefecture University College of Integrated Arts and Sciences, Sakai, Japan; 2 Department of Microbiology, Arizona State University, Tempe, USA; 3 Department of Molecular Biology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan We report here the direct evidence for the formation of alternative DNA structures in a plasmid DNA, termed pTIR10, containing a 0.23-kb pyrimidine/purine-biased (Pyr/Pur) stretch isolated from the rat genome. Long Pyr/Pur sequences are abundant in eukaryotic genomes, and they may modulate the biological activity of genes and genomes via formation of various types of triplex-related structures. The plasmid DNA in sodium acetate buffer (pH 4.35) was deposited on APS-modified mica, and after drying it was imaged with an atomic force microscope in air. Various types of thick protrusions have been observed on pTIR10 DNA. Structural parameters (width and height) of DNA molecules suggest that the alternative structures observed here are variations on the theme of an intra- molecular triplex. The biological relevance of the structural features within Pyr/Pur stretches is discussed. Keywords: alternative DNA structure; atomic force micros- copy (AFM); H-DNA; intramolecular triplex DNA; poly- pyrimidine/ polypurine sequence. There is a wealth of evidence indicating that short pyrimidine/purine-biased (Pyr/Pur) mirror symmetry sequences adopt an intramolecular triplex conformation (H-DNA) [1,2]. Intramolecular triplexes play important roles in genome functions; e.g., triplex formation causes pausing of polymerases during replication [3] and tran- scription [4], and enhances homologous recombination [5,6]. Structural transition between B-DNA and triplex DNA may provide some target site for protein recogni- tion, as specific proteins are involved in homologous recombination mediated by triplex DNA [7]. Pyr/Pur regions of several hundred base pairs long are more abundant in eukaryotic genomes than expected from their base composition [8]. Pyr/Pur sequences in the intergenic regions in the genome are suggested to modulate replica- tion timing through the pausing or stalling of DNA polymerase, as they are often observed in the regions where replication timing switches [9]. The barrier region for replication-fork movement in human ribosomal RNA genes is also known to contain several simple repetitive sequences including Pyr/Pur tracts [10]. Long Pyr/Pur sequences cloned in the plasmid were sensitive to single strand-specific S1 nuclease, and led to the appearance of retarded, but diffused, bands in agarose gel electrophoresis under acidic conditions, suggesting the occurrence of alternative DNA structure and the presence of heteroge- neity in DNA conformations in vitro [11]. Characterization of the potential for forming unusual DNA structure is critical for understanding how the region works in the genome. Traditional chemical and enzymatic probe tech- niques were not efficient, however, in unraveling the structural organization of such long DNA sequences as the result was the sum of various conformers in solution. Recently, we have successfully characterized the intramo- lecular triplex structure formed in supercoiled DNA within 46 bp of Pyr/Pur mirror symmetry by atomic force microscopy (AFM) accompanied by an appropriate sample preparation procedure [12]. Here, we apply the same technique to visualize directly structural features of supercoiled DNA containing long Pyr/Pur sequence isolated from the rat genome. The studies revealed the formation of alternative local DNA structures of different shapes. MATERIALS AND METHODS DNA A pUC19 derivative, pTIR10, containing  0.23 kb of Pyr/ Pur region within a 0.5-kb insert isolated from the rat genome (GenBank accession number U22965 [11]) was used in this work. Southern blot analysis revealed that the pTIR10 sequence hybridized efficiently with fragments of rat and human genomic DNA, meaning that similar sequences were abundant in the genomes (M. Kato & M. Yuasa, unpublished observation). Supercoiled DNA was isolated from Escherichia coli strain JM107 transfor- mants by FlexiPrep Kit (Pharmacia). The electrophoretic mobility of pTIR10 DNA is shown in Fig. 1. In the acidic buffer, migration of pTIR10 was retarded and the molecules were diffused while it migrated normally in the neutral Correspondence to M. Kato, Department of Life Science, Osaka Prefecture University College of Integrated Arts and Sciences, 1-1 Gakuencho, Sakai 599-8531, Japan. Tel./Fax: + 81 72 254 9746, E-mail: mkato@rishiri.cias.osakafu-u.ac.jp or mikio_kato@mac.com Abbreviations: AFM, atomic force microscopy; Pyr, pyrimidine; Pur, purine. (Received 25 April 2002, revised 15 June 2002, accepted 20 June 2002) Eur. J. Biochem. 269, 3632–3636 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03063.x buffer, suggesting that pTIR10 molecules at acidic condi- tions are conformationally heterogenous and/or dynamic. AFM observation The samples for AFM studies were prepared at acidic pH favoring intramolecular triplex (H-DNA) formation (50 m M sodium acetate, pH 4.35) or at neutral pH (10 m M Tris/HCl/1 m M EDTA, pH 7.5). An aliquot (5 lL) of DNA solution (0.3–0.6 lgÆmL )1 ) was deposited on mica function- alized with aminopropyl silatrane (APS-mica) as described previously [13]. The AFM imaging procedure has been described elsewhere [14]. Images were acquired by MM SPM NanoScope III system (Veeco/Digital Instruments, Santa Barbara, CA, USA) operating in Tapping Mode in air Fig. 1. Electrophoretic mobility of pTIR10 DNA. Left panel, electro- phoresis on 1% agarose in 40 m M Tris/acetate/5 m M sodium acetate/ 1m M EDTA (pH 7.5); right panel, electrophoresis on 1% agarose in 30 m M sodium acetate/1 m M EDTA (pH 4.6). Lane 1, pUC19 DNA; lane 2, pTIR10 DNA; lane 3, pTIR10 DNA linearized by HindIII digestion; lane M, HindIII-digested lambda phage DNA size marker. Fully supercoiled molecules, linear molecules, open circles and super- coileddimermoleculesofpUC19aremarkedwitha,b,c,andd, respectively, in the left panel. The faint DNA band at the bottom of lanes 2 and 3 (marked with asterisk) might be the supercondensed structure reported previously [25,26]. Fig. 2. AFM images of pTIR10 and pUC19 DNA. Structural irregu- larities are indicated on large-scale images by arrows and the enlarged rescanned images of the molecules are inserted. (A) pTIR10 DNA prepared at acidic pH; (B) pTIR10 DNA prepared at neutral pH; (C) pUC19 DNA prepared at acidic pH. Thick protrusions were observed in pTIR10 samples (molecules 1–4). In the samples of pUC19, a few molecules retained a sharp-turn part (molecule 5) but it was distinct from the thick protrusions observed in pTIR10 samples. Ó FEBS 2002 AFM studies on alternative DNA structures (Eur. J. Biochem. 269) 3633 at ambient conditions using silicon probes from MikroMash Inc. (Estonia). The length and height measurements were performed with the FEMTOSCAN software (Advanced Tech- nologies Center, Moscow, Russia). RESULTS AND DISCUSSION Examples of AFM images of Pyr/Pur-containing super- coiled DNA (pTIR10) are shown in Fig. 2A,B, and those of control plasmid (pUC19) are shown in Fig. 2C. The most abundant among these structural features observed in pTIR10 DNA samples were thick protrusions (indicated by arrows in Figs 2A,B and 3A). Generally, more than 60% of the population retained this stem structure in pTIR10 samples prepared at acidic pH, and about 20% of the DNA molecules prepared at neutral pH had the stem structure, whereas the stem structure was rarely formed in pUC19 DNA samples. We have examined 43 molecules in total of pUC19 DNA prepared at acidic pH as a control and only one molecule had a stem part. Structural parameters for the stems (the width and the height) in comparison with the same parameters for regular DNA regions are defined as shown in Fig. 4D and listed in Table 1. Fiber diffraction analysis has proposed that the helix diameter of double- stranded DNA is about 2 nm in B-form DNA [2]. Due to the convolution effect of the probe tip [15,16], apparent width of DNA obtained by the AFM will be larger than actual size (Fig. 4D, right panel). In the present results, differences in the parameters between the stem part and regular DNA are close to those obtained for short intramolecular triplexes (H-DNA) earlier [12] suggesting that the structures observed are intramolecular triplexes. Efficient formation of the stem structures at acidic pH also supported the involvement of protonated bases that are required for H-DNA. It is noticeable that heterogeneity in size and shape of the stems occurred in the samples prepared at acidic pH. All types of local DNA structures obtained at acidic pH are shown in Fig. 3. An example of short stems is shown in Fig. 3A. Relatively long stems were often curved as shown in Fig. 3B. In the formation of intermolecular triplex structure, nontriplex forming sequence conjugated to the triplex forming oligonucleotide caused bending of target DNA at the junction [17]. The curved triplex stems observed in the present study might be caused by the presence of any mismatch in the triplex stem. Two clearly separated stems, twin stem structures, were also observed, and one example is shown in Fig. 3C. The two stems can be very close and form P-shaped and Y-shaped structures shown in Fig. 3D and E, respectively. We have identified 45 molecules having the triplex stems. Seven of these retained twin stems or noncanonical triplex stems (P-andY-shaped structures) and the rest had a single stem. Structural parameters for the twin stems or noncanonical triplex stems (14 stems in total) were similar to those for single stems. The average stem width is 6.86 nm (SD ¼ 0.49, n ¼ 14) and the average stem height is 1.43 nm (SD ¼ 0.21, n ¼ 14), whereas the average width and height of regular DNA regions are 5.69 nm (SD ¼ 0.71, n ¼ 7) and 0.90 nm (SD ¼ 0.09, n ¼ 7), respectively. The average length of stem part is 10.61 nm (SD ¼ 2.41, n ¼ 14), and the average of the total stem length in one molecule is 21.21 nm (SD ¼ 3.78, n ¼ 7). The observations suggest that the two stem structures on pTIR10 DNA formed independently within one large region due to the large size of the Pyr/Pur region. Moreover, as the junction of the triplex stem and outgoing arms may be highly flexible [18], certain twin stems are arranged in a tail- to-tail manner forming P-andY-shaped structures. Shimizu et al. [19] demonstrated that considerable structur- al diversity can exist in long (GA) n repeats; proposed higher order unusual DNA structure models containing multiple formation of triplex stems. A model for the formation of Fig. 3. High-resolution AFM images of the most representative families of the irregularities found in pTIR10 DNA. (A) short triplex-like stem; (B) long curved stem; (C) twin stems; (D) P-shaped structure; (E) Y-shaped structure. Thick protrusions are indicated by arrows. Scale bar (100 nm) is given at the bottom of panel e and common to all panels. Table 1. Structural parameters of pTIR10 DNA molecules defined by AFM. Mean values are reported in nm; SD is given in parentheses. Stem length Width Height No. of molecules Acidic sample deposition Triplex stem 14.55 (4.28) 8.14 (0.94) 1.52 (0.25) 38 a B-DNA b NA 6.03 (0.93) 0.98 (0.15) 45 Neutral sample deposition Triplex stem 10.90 (1.43) 7.92 (1.12) 1.41 (0.33) 19 B-DNA b NA 6.21 (0.90) 1.03 (0.25) 19 a The molecules having the single stem structure were used for determining stem parameters. b Width and height for B-DNA were obtained by measuring outgoing arms proximal to the stem part. 3634 M. Kato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 alternative DNA structures is shown in Fig. 4. The stem structures observed in the samples of pTIR10 DNA prepared at neutral pH were shorter than those obtained in the samples prepared at acidic pH, and heterogeneity in size and shape was not seen under neutral conditions. Long (> 200 bp) Pyr/Pur stretches are widely repre- sented at intergenic regions in the genomes of eukaryotic organisms. In the present work, one of the long Pyr/Pur sequences isolated from the rat genome is shown to adopt various types of alternative DNA structures including multiprotrusions, supposedly involving intramolecular tri- plex structure. Intermolecular interaction between two intramolecular triplexes of Pur/Pur/Pyr has been reported for (GAA/TTC) n triplet repeat in the first intron of the human frataxin gene (sticky DNA) [20], and formation of the sticky DNA directly correlates to the inhibitory effect on transcription [21,22]. As the triplex-forming Pyr/Pur regions exist in the genome widely and abundantly, certain Pyr/Pur loci may offer the triplex stems for inter- and intra-chromosomal interactions to modulate transcription and replication. Two independent triplex stems in the twin stem structure may be able to interact with each other to stabilize the alternative DNA structure in certain Pyr/Pur sequences. Conformational variability of triplex structures can provide tuning of the interaction of the triplex- forming regions. In addition, triplex formation absorbs the negative supercoils of the flanking regions, and the local changes in the DNA superhelicity can affect the global shape of the topological domain [23,24]. These regions with the potential for forming various types of intra- molecular triplex may function in some processes of genome regulation such as replication rate and timing, recombination and chromosome folding by modulating the local structure of DNA regions and the global topology of chromatin domains. ACKNOWLEDGEMENTS This work was supported in part by the fund from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (to M. K.), the fund for Research for the Future Program from the Japan Society for the Promotion of Science (JSPS) (to N. S.), and the National Institute of Health grant GM 62235 (to Y. L. L.). REFERENCES 1. Soyfer, V.N. & Potaman, V.N. (1996) Triple-Helical Nucleic Acids. Springer-Verlag, New York. 2. Sinden, R.R. (1994) DNA Structure and Function. Academic Press, New York. 3. Baran, N., Lapidot, A. & Manor, H. (1991) Formation of DNA triplexes accounts for arrest of DNA synthesis at d(TC)n tracts. Proc. Natl Acad. Sci. 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J. Biochem. 269) Ó FEBS 2002 . PRIORITY PAPER Structural heterogeneity of pyrimidine/purine-biased DNA sequence analyzed by atomic force microscopy Mikio Kato 1,2 , Chad J. McAllister 2 , Shingo Hokabe 1 , Nobuyoshi Shimizu 3 and. solution. Recently, we have successfully characterized the intramo- lecular triplex structure formed in supercoiled DNA within 46 bp of Pyr/Pur mirror symmetry by atomic force microscopy (AFM) accompanied by. (AFM); H -DNA; intramolecular triplex DNA; poly- pyrimidine/ polypurine sequence. There is a wealth of evidence indicating that short pyrimidine/purine-biased (Pyr/Pur) mirror symmetry sequences