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Base Extrusion Is Found at Helical Junctions Between Right- and Left-Handed Forms of DNA and RNA

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Base Extrusion Is Found at Helical Junctions Between Right- and Left-Handed Forms of DNA and RNA Base extrusion is found at helical junctions between right- and left-handed forms of DNA and RNA The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Kim, D. et al. “Base Extrusion Is Found at Helical Junctions Between Right- and Left-handed Forms of DNA and RNA.” Nucleic Acids Research 37.13 (2009): 4353–4359. Web. As Published http://dx.doi.org/10.1093/nar/gkp364 Publisher Oxford University Press Version Final published version Citable link http://hdl.handle.net/1721.1/72438 Terms of Use Creative Commons Attribution Non-Commercial Detailed Terms http://creativecommons.org/licenses/by-nc/2.5 Published online 21 May 2009 Nucleic Acids Research, 2009, Vol. 37, No. 13 4353–4359 doi:10.1093/nar/gkp364 Base extrusion is found at helical junctions between right- and left-handed forms of DNA and RNA Doyoun Kim1, Sanjith Reddy1, Dong Young Kim1, Alexander Rich2, Sangho Lee3, Kyeong Kyu Kim1,* and Yang-Gyun Kim4,* 1Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea, 2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, Departments of 3Biological Science and 4Chemistry, Sungkyunkwan University, Suwon 440-746, Korea Received February 1, 2009; Revised and Accepted April 24, 2009 ABSTRACT conformations. When this duplex repeat is disrupted, it creates a Z–Z junction (5–7). The Z–Z junction marks Base extrusion is a major structural feature at the the boundary between two out-of-phase Z-DNA regions junction between B- and Z-DNA (the B–Z junction) (6). In addition, the RNA duplex can have an A–Z junc- where a base pair is broken, and the two bases tion when Z-form RNA is formed. The detailed crystal are extruded from the double helix. Despite the structure of the B–Z junction was revealed in our previous demonstration of base extrusion at the B–Z junction, study (3); however, the structural features of B–Z, Z–Z it is not clear whether a similar base extrusion and A–Z junctions in solution are still to be investigated. occurs at other types of junctions involving the Formation of Z-DNA under physiological conditions left-handed Z conformation. Here, we investigate has remained a major experimental challenge for investi- structural changes of bases at three Z-form junc- gating Z-form-containing helical junctions of DNA and tions: DNA B–Z and Z–Z and RNA A–Z junctions. RNA. The use of a protein Z-DNA-binding domain By monitoring fluorescently labeled duplex nucleic (Za of human ADAR1) made it possible to determine acids using 2-aminopurines at various positions the crystal structure of B–Z junction (3). In that study, the double-stranded RNA adenosine deaminase relative to the junction point, we show that base (ADAR1) Za domain readily stabilized Z-DNA under extrusion occurs not only at the DNA B–Z junction, physiological condition, while the DNA segment with an but also at the RNA A–Z and DNA Z–Z junctions. Our unfavorable sequence for Z-DNA formation remained in data suggest that base extrusion is a general feature the B-conformation. Likewise, Za can convert an RNA of Z-form nucleic-acid junctions. duplex composed of r(CG) repeats into Z-form RNA (8). When salt is used to stabilize Z-RNA, it requires a higher salt concentration and temperature than is needed for INTRODUCTION DNA (9,10). Thus, Za is shown to be a powerful tool to The study of junction structures formed between helices in stabilize Z-RNA under physiological conditions. nucleic acid has progressed by understanding the struc- Nucleotide bases can also be removed from the stacked tures of Holliday junctions (1,2) and the B-DNA– bases at the center of nucleic-acid duplexes by a variety of Z-DNA junctions (3,4). Recent work by Ha et al. (3) base modifying enzymes. That process is generally referred revealed that base extrusion is a major structural feature to as ‘base flipping’ in that the enzyme actively promotes at the junction between B-DNA and Z-DNA. At the B–Z removal of the base from the stacked bases. However, junction a base pair is broken, and the two bases are in vivo, the bases at the B–Z junction are not actively extruded on opposite sides of the double helix. The junc- removed from the stacked bases by an external agent, tion then shows continuous stacking of base pairs between but are rather forced out by torsional strain of the the B-DNA and Z-DNA segments (Figure 1). duplex. Thus, we use the term ‘base extrusion’ rather There are helical junctions other than the B–Z junction than ‘base flipping’. in nucleic-acid duplexes. Very similar left-handed duplexes 2-Aminopurine (2AP) is a fluorescent base analog are formed in both DNA and RNA. Furthermore, the Z of adenine, and it can be incorporated in DNA with conformation has residues that alternate in syn and anti little effect on its structure (11) and function (12,13). *To whom correspondence should be addressed. Email: [email protected] Correspondence may also be addressed to Yang-Gyun Kim. Tel: +82 31 299 4563; Fax: +82 31 299 4575; Email: [email protected]; [email protected] ß 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 4354 Nucleic Acids Research, 2009, Vol. 37, No. 13 transformed into BL21(DE3). The cells were grown in LB containing 30 mg/ml kanamycin to an OD600 of 0.5–0.6 at 378C, and then induced with 0.5 mM isopropyl b-D- thiogalactoside (IPTG) at 378C for 4 h. Za was purified using methods described previously (21). After a His- affinity column (Amersham Bioscience) and removal of the N-terminal six histidines with thrombin (Boehringer Mannheim), the protein in buffer A (20 mM HEPES, pH 7.5 and 10 mM NaCl) was further purified using Resource S (Amersham Biosciences). The purified protein was dialyzed against buffer containing 5 mM HEPES, pH 7.5 and 10 mM NaCl. After dialysis, the protein was con- centrated to over 1 mM by ultrafiltration on a Centricon- YM3 device (Millipore). All of the buffer change steps were carried out using molecular porous membrane Figure 1. Crystal structure of the B–Z junction. Extruded bases at the tubing (Spectrum laboratories, Inc). The protein concen- junction are colored in red in the sequences. The conformations of the tration was measured spectroscopically using an extinc- DNA regions are indicated by a red line and bold letters. [The figure is À1 À1 reconstructed from PDB ID:2ACJ]. tion coefficient of 6970 M cm at 280 nm, calculated at www.expasy.org. In addition, 2AP has strong fluorescence compared to the Circular dichroism natural bases (14). 2AP fluorescence is significantly quenched in duplex nucleic acids due to stacking interac- The conversion of duplex oligonucleotides (Table 1) from tions with neighboring bases. When a 2AP is extruded B-DNA to Z-DNA was monitored by measuring the cir- from the duplex, its fluorescence considerably increases. cular dichroism (CD) spectrum using a Jasco J-810 CD Thus, it is an ideal probe to investigate junctions with spectrometer at 258C with 15 mM of dsDNA (BZ0, BZ1, Z-form nucleic-acid duplexes. 2AP has been used fre- BZ4 and ZZ substrates). Z-RNA measurements used quently for detecting dynamic structural changes of 7.5 mM of dsRNA (AZ0, AZ1 or AZ4). All solutions bases in DNA (11,15–17), as well as in base flipping of were in buffer containing 10 mM HEPES–NaOH, the targeted base by N6 adenine DNA methyltransferase pH 7.5, 10 mM NaCl. Za was added to dsDNA solution and EcoRI DNA methyltransferase (18–20). to a final concentration of 15 mM(1Â), 30 mM(2Â), 45 mM Here, we ask whether base extrusion is a general struc- (3Â), 60 mM(4Â) and 75 mM(5Â)Za was added to the tural feature for various Z-form-containing junctions RNA solution to a final concentration of 7.5 mM(1Â), by exploring base extrusion in three types of Z-form- 15 mM(2Â), 22.5 mM(3Â), 30 mM(4Â) and 37.5 mM containing junctions: B–Z, A–Z and Z–Z junctions. The (5Â). The mixtures were equilibrated for 1 h for dsDNA fluorescence of 2APs distinguishes between stacked bases and 2 h for dsRNA prior to the measurement. Spectra and unstacked bases. We show that base extrusion is a were recorded between 230 nm to 320 nm at 1 nm intervals common feature of junctions between opposite helical averaged over 2 s. The maximum volume of the protein handednesses of double-stranded nucleic acids. added to the sample did not exceed 5% of the total volume. MATERIALS AND METHODS Steady-state fluorescence Preparation of duplex oligonucleotides Fluorescence measurements were carried out at 258C using a Quartz Fluorometer Cell Microsquare 3 mm 2-aminopurine (2AP) modified and unmodified single- (Starna Cells, Inc., Atascadero, CA, USA). Titrations of stranded oligonucleotides (Table 1) were purchased from duplex oligonucleotides in buffer, which contains 20 mM Integrated DNA Technologies (IDT, San Diego, CA, HEPES–NaOH (pH 7.5) at each concentration of Za, USA). All oligonucleotides were equilibrated against were performed to reach equilibrium as monitored by buffer containing 50 mM Tris–HCl, pH 8.0, 50 mM the CD spectrum in which the wavelength was fixed at NaCl and 1 mM EDTA.
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