Structural and dynamic basis of a supercoiling-responsive DNA element

Published online January 9, 2006 254–261 Nucleic Acids Research, 2006, Vol. 34, No. 1 doi:10.1093/nar/gkj428 Structural and dynamic basis of a supercoiling-responsive DNA element Sung-Hun Bae, Sang Hoon Yun1, Dawei Sun, Heon M. Lim1 and Byong-Seok Choi* Department of Chemistry, KAIST, 373-1 Guseong-dong Yuseong-gu Daejeon 305-701, Republic of Korea and 1 Department of Biology, School of Biological Science and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea Received July 2, 2005; Revised and Accepted December 15, 2005 Downloaded from https://academic.oup.com/nar/article/34/1/254/2401609 by guest on 04 July 2022 ABSTRACT histone proteins or by the topological constraints imposed upon closed circular DNA, respectively. Supercoiling plays In both eukaryotes and prokaryotes, negative super- an important role in a variety of cellular processes, including coiling of chromosomal DNA acts locally to regulate transcription, replication, recombination and response to envi- a variety of cellular processes, such as transcrip- ronmental stresses (1,2). Global supercoiling changes are tion, replication, recombination and response to known to act locally and regulate the transcription of genes environmental stresses. While studying the interac- with promoters that are sensitive to supercoiling (
7% of the tion between the Hin recombinase and mutated Escherichia coli genome) (3). Although the mechanism by versions of its cognate DNA-binding site, we identi- which global supercoiling alters local DNA structure is not fied a mutated DNA site that binds Hin only when the clearly understood, it is well known that many proteins bind DNA is supercoiled. To understand the mechanism preferentially to supercoiled rather than relaxed or linear of this supercoiling-responsive DNA site, we used DNA. In addition, it has been shown that changes in protein binding affinity and/or specificity induced by supercoiling are NMR spectroscopy and fluorescence resonance dependent on the local DNA sequence [e.g. (4)]. energy transfer to determine the solution structures In our study of the DNA recombinase Hin from Salmonella and dynamics of three related DNA oligonucleotides. typhimurium, we observed supercoiling-induced local struc- The supercoiling-responsive DNA site formed a tural changes in the Hin DNA-binding site. Hin catalyzes a partially unwound and stretched helix and showed site-specific DNA inversion between two 26 bp inverted significant flexibility and base pair opening kinetics. sequences (hixL and hixR) that flank a 933 bp DNA segment. The single CAG/CTG triplet contained in this DNA This invertible segment contains a promoter that directs the sequence displayed the same characteristics as coordinate expression of the fljB and fljA genes, which encode do multiple CAG/CTG repeats, which are associated H2 flagellin and a repressor of the H1 flagellin gene (fljC), with several hereditary neuromuscular diseases. It respectively. By inverting this 933 bp segment, Hin regulates is known that short DNA sequence motifs that have the expression of two major flagellar structural proteins, the H1 and H2 flagellins, which allow the bacteria to escape the either very high or low bending flexibility occur pref- host’s immune system (5). During the first stage of DNA erentially at supercoiling-sensitive bacterial and inversion, Hin binds to each of the 26 bp hix DNA sites as eukaryotic promoters. From our results and these a dimer with high affinity (Kd
109) (6). It has been sug- previous data, we propose a model in which super- gested that the specificity of Hin binding results from direct or coiling utilizes the intrinsic flexibility of a short DNA water-mediated sequence-specific contacts made by the Hin site to switch the local DNA structure from an ineffi- protein with the major groove at positions 9–13 and with the cient conformation for protein binding to an efficient minor groove at positions 5–6 (numbering from the center of one, or vice versa. the inverted sequence, Figure 1A) (7,8). However, in the pre- sent study, we show that (i) a DNA mutation at the central positions (+1,1) of the hix site also modulates Hin binding INTRODUCTION and (ii) the mutated hix site, which does not bind Hin when The chromosomal DNA of both eukaryotes and prokaryotes is the DNA is relaxed, can bind Hin when the DNA is in a negatively supercoiled, either by the wrapping of DNA around supercoiled state. The negative supercoiling is required for *To whom correspondence should be addressed. Tel: +82 42 869 2828; Fax: +82 42 869 8120; Email: [email protected] Correspondence may also be addressed to Heon M. Lim. Tel: +82 42 821 6276; Fax: +82 42 822 9690; Email: [email protected]  The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected]  Nucleic Acids Research, 2006, Vol. 34, No. 1 255 formation of the complete Hin inversion complex (9,10); how- ever, it is not necessary for the initial recognition of the hix site by Hin (6,11). Therefore, the supercoiling-induced Hin bind- ing observed in our study is solely caused by the properties of the mutated DNA site and hence provides a model of DNA site that responds to supercoiling. To characterize this supercoiling-responsive DNA site, we have determined the solution structures and dynamics of three hix-related DNA oligonucleotides by NMR spectroscopy and fluorescence resonance energy transfer (FRET). Our results show that the supercoiling-responsive mutant hix site has a partially unwound and stretched helix structure and shows significant flexibility and base pair opening kinetics. The Downloaded from https://academic.oup.com/nar/article/34/1/254/2401609 by guest on 04 July 2022 single CAG/CTG triplet contained in the supercoiling- responsive hix site displayed the same characteristics as CAG/CTG repeats, which are associated with several hered- itary neuromuscular diseases such as myotonic dystrophy and Huntington’s disease (12). Our results suggest that supercoil- ing affects predominantly the highly flexible DNA site and that such changes can switch the local DNA structure from an inefficient conformation for protein interaction to an efficient one, or vice versa. MATERIALS AND METHODS Hin protein preparation Wild-type and G102A mutant Hin proteins were expressed and purified as described previously (13). Electrophoretic mobility shift assay (EMSA) PCR-amplified, 200 bp, double-stranded DNA (dsDNA) frag- ments containing the hix-AT, hix-AG and hix-CG sites were end-labeled with T4 polynucleotide kinase and [g-32P]ATP. Binding mixtures (20 ml) containing 0.8 nM of labeled DNA and 0–200 ng of Hin protein in binding buffer [0.2 M Tris–HCl (pH 7.5), 1 M NaCl, 10 mM EDTA, 100 mM DTT and 50 mM MgCl2] were incubated for 10 min at 25 C. The mixtures were subjected to electrophoresis on 5% polyacrylamide gels. DNase I footprinting Supercoiled plasmid DNA (20 nM) containing the hix-AT, hix-AG and hix-CG sites were pre-incubated with 0–50 ng of Hin protein in 50 ml of the binding buffer for 10 min at 25 C. After the addition of 1 U of DNase I, the incubation was continued for 2 min. The digested DNA was isolated by phenol/chloroform extraction and extended with a 32P-end- labeled primer. The extension products were separated on an 8% polyacrylamide/7 M urea gel. NMR experiments DNA oligonucleotides for NMR experiments were purchased Figure 1. Supercoiling-dependent Hin binding to hix-AG. (A) DNA-binding from Bioneer Co., Ltd. (Daejeon, Korea). Buffer conditions sites (hixL and hixR) of the Hin recombinase from S.typhmurium and the for the NMR experiments were 10 mM sodium phosphate mutant hix sites used in this study (hix-AT, hix-CG and hix-AG). Residues (pH 6.8) and 100 mM NaCl. All NMR spectra were obtained involved in sequence-specific contacts with Hin are marked by horizontal lines. on a Varian Inova 600 MHz spectrometer except for the 1H-31P (B) EMSAs with the 200 bp PCR-amplified DNA containing mutant hix sites heteronuclear correlation spectra, which were acquired on (0.8 nM) and 0–200 or 500 ng/ml of the Hin protein. (C) DNase I footprinting of supercoiled plasmids containing mutated hix sites in the presence of the Hin a Bruker DRX 600 MHz spectrometer. The 2D NOE spec- protein (0–50 ng/ml). Protected region from DNase I digestion is indicated by a troscopy (NOESY) (tm ¼ 180 ms) was carried out in 95% vertical bar in the left. H2O/5% D2O at 4 C. The 2D NOESY (tm ¼ 80, 160 and 256 Nucleic Acids Research, 2006, Vol. 34, No. 1 240 ms), 2D correlation spectroscopy (COSY), 2D total fluorescein-C6-50 -TTA TCA AAA ACC ATG GTT TTC correlation spectroscopy (TOCSY) (tm ¼ 80 ms) and AAG AA-30 , TAMRA-50 -TTC TTG AAA ACC ATG GTT 1 H-31P heteronuclear COSY were conducted in 100% D2O TTT GAT AA-30 , fluorescein-C6-50 -TTA TCA AAA ACC at 22 C. All the acquired spectra were processed by NMRPipe CGG GTT TTC AAG AA-30 , TAMRA-50 -TTC TTG AAA (14) and analyzed by Sparky 3 (T. D. Goddard and ACC CGG GTT TTT GAT AA-30 , fluorescein-C6-50 -TTA D. G. Kneller, University of California, San Francisco). TCA AAA ACC AGG GTT TTC AAG AA-30 and TAMRA-50 -TTC TTG AAA ACC CTG GTT TTT GAT Resonance assignments and structure calculation AA-30 . Equimolar concentrations of each of the complemen- All of the slowly exchanging imino and amino resonances were tary DNA strands in 10 mM Tris–HCl, pH 7.5 (20 C) and 0.1 assigned with the H2O NOESY, and all non-exchangeable mM EDTA were incubated at 95 C for 10 min and then slowly base and most of the sugar proton resonances were assigned cooled to room temperature. Complete annealing was checked by using the D2O NOESY and TOCSY spectra. The distance by non-denaturing PAGE. All FRET experiments were carried constraints were derived from the integrated NOE peak out with 0.5 mM of singly- or doubly-labeled dsDNA. Downloaded from https://academic.oup.com/nar/article/34/1/254/2401609 by guest on 04 July 2022 volumes and three assumed isotropic correlation times Acquired data were processed as described previously (24). (tc ¼ 3, 4 and 5 ns) using a relaxation matrix analysis pro- gram, MARDIGRAS (15). The d dihedral angles were derived from the H10 -H20 scalar couplings from regular 2D COSY RESULTS (16). The c dihedral angles were constrained to 220 ± 45 , on the basis of the medium to weak intra-residue NOE between The mutated Hin binding site, hix-AG, is recognized H6 or H8 and H10 . The a and z angles were unconstrained, and by Hin only if it is supercoiled other backbone dihedral angles were loosely constrained to the EMSAs using Hin and 200 bp linear dsDNA fragments standard B-form [b (180 ± 45 ), g (60 ± 30 ), e (230 ± 70 )]. showed that Hin binds to the symmetric hix site, which has All the structure calculations were carried out using XPLOR- AT as its central +1/1 residues (hix-AT) (Figure 1A and B). NIH (17). Two extended single DNA strands were used as a Although the native hixL and hixR sites have AA sequences at starting structure and were subjected to 60 ps of torsion angle their centers, the symmetric hix-AT site has been tested in dynamics (TAD) at 20 000 K, followed by 150 ps of TAD in vitro DNA-binding assays, such as EMSAs and methylation cooling from 20 000 to 0 K. The final structures were obtained protection assays, and found to bind Hin as well as the wild- after 20 000 cycles of energy minimization. The distance force type hix sites (25). Furthermore, the hix-AT sequence exhibits constant was 50 kcal mol1·A2 throughout the calculation biological activity equivalent to that of the native hix site in and the dihedral angle force constant, which initially was 5, invertasome formation, and inversion reactions (13). was scaled to 250 kcal mol1·rad2 during cooling. The If the central AT sequence is changed to AG (hix-AG), Hin database potential of mean force base–base positional cannot recognize the hix site even though the other residues interactions was adopted with a force constant of 0.1 (18). important for sequence-specific contacts between Hin and hix For self-complementary DNAs (hix-AT and hix-CG), a non- are preserved. However, DNA binding by Hin can be recov- crystallographic symmetry force constant of 10 was used. ered by replacing the central AG with CG (hix-CG). Consistent From 100 starting structures, 25, 15 and 13 structures for with results obtained for linear DNA fragments, our DNase I hix-AT, hix-CG and hix-AG, respectively, were converged footprinting experiments using supercoiled plasmid DNA to root mean square deviations (r.m.s.d.) of 0.56 ± 0.29, showed that Hin binds to hix-AT and hix-CG DNAs. However, 0.61 ± 0.34 and 0.92 ± 0.34 s, respectively. The final struc- quite unexpectedly, Hin also bound to supercoiled hix-AG tures were analyzed by Curves 5.3 (19), 3DNA (20), Madbend with comparable affinity (Figure 1C). A mutated version of (21) and MOLMOL (22). The atomic coordinates have been the Hin protein (G102A), which does not bind to the hix site deposited in the Protein Data Bank [PDB ID codes 1ZYF (26), also did not bind to supercoiled hix-AG. This confirms (hix-AT), 1ZYG (hix-CG) and 1ZYH (hix-AG)]. that the protection from DNase I digestion that was observed with wild-type Hin at the hix-AG site is not an artifact caused Base pair kinetics by experimental conditions and/or non-specific binding (data Selective longitudinal relaxation times were measured at not shown). increasing concentrations of ammonia ranging from 0 to 0.19 M at 12, 17 and 22 C while maintaining a pH of 8.9– hix-AT, hix-AG and hix-CG have different 9.0. The water signal was suppressed by the jump-and-return overall structures pulse sequence. Interpretation and analysis of the data fol- In order to understand the structural basis of the sensitivity of lowed the previously reported method (23). hix-AG to supercoiling, we determined and compared the solution structures of three dodecamer DNAs (hix-AT, hix- Fluorescence resonance energy transfer AG and hix-CG) (Figure 2). The NOE connectivity and chemi- Half of the single-stranded oligonucleotides were labeled with cal shifts of imino protons in the D2O and H2O NOESY fluorescein on their 50 ends through a six-carbon linker (C6), spectra showed that hix-AT, hix-AG and hix-CG have the and their complementary sequences were labeled with expected right-handed helix structures. Also, nearly all of 6-carboxytetramethylrhodamine (TAMRA) on their 50 ends. the residues of hix-AT and hix-CG showed H10 -H20 scalar These and all non-labeled oligonucleotides of the same couplings larger than 8–9 Hz, indicating that they have C20 - sequences were purchased from Bioneer Co., Ltd. (Daejeon, endo sugar puckerings typical of B-form DNA. However, most Korea). The constructs used for FRET experiments were of the residues in hix-AG showed smaller H10 -H20 scalar  Nucleic Acids Research, 2006, Vol. 34, No. 1 257 Downloaded from https://academic.oup.com/nar/article/34/1/254/2401609 by guest on 04 July 2022 Figure 2. Superimposed overall structures of the hix sites. View into the minor groove of (A) hix-AT (25 structures), (B) hix-CG (15 structures) and (C) hix-AG (13 structures). Adenines are colored in red, guanines in blue, cytidines in cyan and thymidines in yellow. Rise and twist are shown for each of the central 5 bp steps. Table 1. Structure determination statistics had similar depths (
4 Å), but hix-AG had a very shallow (
1.5 Å) depth in the middle of the sequence. hix-AT had a hix-AT hix-CG hix-AG narrow (width,
7 Å) and deep (depth,
5.3 Å) minor groove, Total number of NOE 222 205 354 which is similar to that of average B-form DNA, while hix-CG distance restraints had a wide (width,
10 Å) and shallow (depth,
3.5 Å) minor Intra-residue 86 76 159 groove, which is similar to that of average A-form DNA (27). Sequential residue 94 93 161 Interstrand 42 36 34 The minor groove of hix-AG was of intermediate width and Dihedral restraints 116 113 116 depth, when compared with those of hix-AT and hix-CG (b, g, d, e and c) (Figure 2). Base pair planarity restraints 12 12 12 Total number of restraints 350 330 482 Pairwise r.m.s.d. for all 0.56 ± 0.29 0.61 ± 0.34 0.92 ± 0.34 hix-AG is partially unwound and stretched heavy atoms (Å) r.m.s.d. to the mean structure (s) 0.65 0.54 0.79 The converged structures of hix-AT, hix-CG and hix-AG Average NOE violations (s) 0 (>0.5s) 0 (>0.5s) 0 (>0.5s) showed sequence-dependent structural differences in quanti- Average dihedral angle 0 (>5 ) 0 (>5 ) 0 (>5 ) tative helical analyses. Because of the possible inaccuracy of violations (degrees) the structure defined for the terminal residues, we assessed Mean deviation from covalent geometry only the central 6 bp and their 5 bp steps for each DNA. Bond lengths (s) 0.007 0.007 0.007 The accumulation of rises of central base pair steps from Angles (degrees) 0.9 0.9 1.0 C4–C5 to G8–G9 was 18.1, 18.3 and 19.1 Å for hix-AT, Impropers (degrees) 2 2 2 hix-CG and hix-AG, respectively (Figure 2). This increased helical rise could result from a twist. The sum of the twist angles of the base pair steps from C4–C5 to G8–G9 were couplings in a range of 6–8 Hz (data not shown), which sug- 163.5 for the hix-AG, which is less than that calculated for gests that hix-AG undergoes a dynamic equilibrium between hix-AT by 12.7 (Figure 2). The lack of a twist in hix-AG the C20 - and C30 -endo sugar puckers. Because typical A-form compared with hix-AT is consistent with previous analyses of DNA, which has the C30 -endo sugar pucker, shows H10 -H20 the local helix parameters of high-resolution DNA crystal scalar couplings of <2.0 Hz, hix-AG appears to spend most of structures, in which the mean twist angles of AT, CG its time in a B-form-like structure rather than an A-form-like and AG base pair steps are 33.4 ± 3.5 , 31.1 ± 4.7 and structure. A total of 350, 330 and 482 restraints, respectively, 30.5 ± 4.9 , respectively (28). If we assume the standard A- for the hix-AT, hix-CG and hix-AG were derived from NMR or B-form DNA structures (27) keep their helical diameter data and were used for structure calculations to obtain well- constant upon unwinding, then a decrease in the twist angle converged ensemble structures (Table 1). of 12.7 could be transformed into an increase of 1.0–1.2 Å The major groove widths of hix-AT, hix-CG and hix-AG in the helical rise. Supporting this assumption, hix-AT and were within a ±2 Å boundary of the major groove width of hix-AG had nearly identical helical diameters, in that the average B-form DNA (27). hix-AG showed the largest major average interstrand P–P distances of base pairs from C4pC5 groove width (
14 Å), which is significantly larger than that to G8pG9 were 18.0 ± 0.2 and 18.1 ± 0.2 Å, respectively. of hix-AT (
10 Å). The major grooves of hix-AT and hix-CG Similar mechanism of DNA stretching has been observed in 258 Nucleic Acids Research, 2006, Vol. 34, No. 1 several DNA complexes, such as those that contain catabolic groove width variation and deformation (35). Furthermore, activator protein (29) or TATA-box binding protein (30). Such sequence-directed bending in DNA has been reported as an DNA-binding proteins can make use of the natural coupling of inducible, not a static, phenomenon (36,37). Thus we inves- twist and roll with slide and/or shift to stretch DNA at selected tigated the effect of salts on the deformation of 26 bp hix-AT base pair steps. and hix-AG DNAs, where the change of end-to-end distance The accumulated twist angle of hix-CG for residues from was monitored by a change in FRET efficiency (38). Increas- C4–C5 to G8–G9 was 154.4 , which is even smaller (by 9.1 ) ing the salt concentration of Na+, NH+4 and Mg2+ from 0.0 to than that of hix-AG. However, unlike hix-AG, hix-CG showed 0.5 M augmented the FRET efficiency for all DNAs, hix-AT, no significant increase in the helical rise. It is possible that hix-CG and hix-AG (Figure 3A–C). Salt-dependent changes in the increased helical diameter or interstrand P–P distance we FRET efficiency may result not only from changes in the end- observed for hix-CG (18.4 ± 0.2 Å) can accommodate the to-end distance, but also from changes in the fluorescence unwinding without further stretching of the DNA. characteristics of the fluorophores; however, the absorbance and fluorescence of rhodamine used here are known to be very stable at least up to 0.5 M NaCl (24). In the case of Mg2+, Downloaded from https://academic.oup.com/nar/article/34/1/254/2401609 by guest on 04 July 2022 hix-AG has high potential for deformation FRET efficiencies of hix-AT, hix-CG and hix-AG increased so Because partially unwound DNA sites are easily bent (31,32), rapidly that no significant differences were observed between we sought to determine whether DNA bending differed among them. However, for both Na+ and NH+4 , the FRET efficiency of hix-AG, hix-AT and hix-CG by assessing their roll, tilt and hix-AG increased more rapidly than that of the hix-AT or hix- twist angles (21). Because the distance constraints used CG, suggesting that hix-AG is more flexible and, therefore, in structural calculations cover a relatively short distance more easily deformed as the salt concentration increases. range (<6 Å) and thus cannot define a long-range curvature accurately, we included only the central 5 bp steps in our DNA Base pair opening of hix-AG is very fast curvature calculations. Both hix-AT and hix-AG, which exhib- ited negative global rolls (8 ± 5 and 10 ± 7 ), were bent DNA deformation occurs concurrently with base pair opening, toward the minor groove by 8 ± 5 and 12 ± 6 , respectively. and the propensity of base pair opening is also related to the hix-CG, which had a positive roll (8 ± 6 ), was bent toward the thermodynamics and kinetics of DNA deformation (21,30,39). major groove by 9 ± 5 . Considering that the bending flexi- In order to explore the differential dynamics of the three hix bility of generic B-DNA, which was estimated from Monte sites, we measured the base pair lifetimes of the common G8– Carlo simulations using a static bend model, is 5 (33), our C17 base pair using ammonia as a base catalyst (Figure 4A). In results indicate that all three oligonucleotides are only slightly a stacked helix, the imino protons are protected from exchange bent and the magnitude of bending of the hix-AG is not with a base catalyst, but in the presence of higher concentra- remarkably different from that of the hix-AT or hix-CG. tions of a base catalyst, exchange of imino protons may take However, statistics of crystal structures of DNA and DNA– place each time a base pair opens. When we consider that protein complexes have demonstrated that AG dinucleotide the typical base pair lifetimes of A–T and G–C base pairs steps tend to undergo significant translational and tilt changes, are 0.5–7 and 4–50 ms, respectively (23), the lifetimes of while AT and CG dinucleotide steps have essentially no base G8–C20 base pairs measured at 17 C for hix-AT (5.0 ms), pair displacement (34). Therefore, we suspected that hix-AG hix-CG (4.4 ms) and hix-AG (2.6 ms) and those values might have higher potential of deformation than hix-AT or hix- measured at 12 C for hix-AT (2.8 ± 3.0 ms), hix-CG CG even though hix-AG did not appear to have significant (12.8 ± 4.6 ms) and hix-AG(-7.0 ± 6.4 ms) imply that the intrinsic curvature. Because the DNA phosphate backbone is central base pairs of hix-CG and hix-AG are rapidly opened negatively charged, electrostatic interactions with monovalent and closed, with hix-AG undergoing the fastest local motion. or divalent cations are important in DNA bending, twisting, It appears consistent with the previous observation that a 0.16 0.16 0.16 0.14 A 0.14 B 0.14 C FRET efficiency FRET efficiency FRET efficiency 0.12 0.12 0.12 0.10 0.10 0.10 0.08 0.08 0.08 0.06 0.06 0.06 0.04 hix-AT 0.04 hix-AT 0.04 hix-AT hix-CG hix-CG hix-CG 0.02 hix-AG 0.02 hix-AG 0.02 hix-AG 0.00 0.00 0.00 0 100 200 300 400 500 0 100 200 300 400 500 0 20 40 60 80 100 Na+(mM) NH4+(mM) Mg 2+(mM) Figure 3. End-to-end distances of hix-AT and hix-AG. (A–C) Dependence of FRET efficiency on the concentration of NaCl, NH4Cl and MgCl2 in the buffer [10 mM Tris–HCl (pH 7.5, 20 C) and 0.1 mM EDTA].  Nucleic Acids Research, 2006, Vol. 34, No. 1 259 differential binding of Hin to these sites in the presence or absence of supercoiling. Supercoiling confers topological constraints on the local DNA structure. As protein binding induces structural changes in the DNA-binding site, supercoil- ing should also induce structural changes in the local DNA site where flexibility of the DNA including and surrounding this site would be critical for a response to supercoiling. Sup- porting this idea, the partially unwound and stretched structure of hix-AG shows high potential for deformation and very fast kinetics of base pair opening. Furthermore, in the present study, we have revealed that a single CAG/CTG triplet sequence motif contained in hix-AG shows essentially the same characteristics as do multiple CAG/CTG repeats Downloaded from https://academic.oup.com/nar/article/34/1/254/2401609 by guest on 04 July 2022 which are associated with several hereditary neuromuscular diseases, including myotonic dystrophy and Huntington’s disease; a block of multiple CAG/CTG repeats is present near or within genes associated with such diseases (12). The gel mobility and cyclization kinetics of DNA that contains short tracts of CAG/CTG repeats revealed that the CAG/CTG repeats are intrinsically straight but extremely flexible (41). Also, a stretch of multiple CAG/CTG repeats shows an unusu- ally high affinity for the histone octamer, forming a tight nucleosome (42) in which DNA wraps around a histone core in a left-handed configuration that produces a negative toroidal supercoiling. The free energy of supercoiling for the CAG/CTG repeats calculated by statistical mechanics is only 66% of that of random B-DNA at a length of 104 bp (43). How might the structural and dynamic properties of the CAG/CTG site explain the supercoiling-dependent interaction between Hin and hix-AG? There may be a few possible reasons for why hix-AG is not bound by Hin when the DNA is relaxed, but is bound by Hin when the DNA is super- coiled. First, the spacing between the half-sites in the hix sequence might be critical for DNA inversion, as demonstrated by a mutant hix site that contains AAA sequence rather than Figure 4. Base pair opening kinetics of hix-AT, hix-CG and hix-AG. (A) Exchange time of the G8 imino proton was measured at varying concentrations AA sequence at the center of an otherwise normal hix site (13). of base catalyst, ammonia, at pH 8.9, 12 C. Base pair lifetime was calculated However, the Hin dimer is flexible enough to bind to both by extrapolation to infinite ammonia concentration, [NH3]. (B) Imino spectra half-sites of hix sequences that contain a 2 (wild type), 3 or of hix-AT and hix-AG at increasing temperature. For the 17–32 C spectra, 5 bp spacer between the half-sites, albeit the following inver- the buffer contained 0.136 M NH3/NH+4 (at pH 9.0, [NH3] ¼ 45 mM) and sion process is aborted (6). The same flexibility has been 100 mM NaCl. reported for the gd resolvase, which shares
40% amino acid sequence identity with Hin and which binds to three G-C base pair within consecutive G-C base pairs has an unusu- res subsites, each with a different spacer length (7, 10 and ally short base pair lifetime (40). The opening of the central 16 bp) between two half-sites (44). Therefore, the differential G8–C17 base pair of hix-AG did not seem to follow the simple spacing or the helical rise may not be relevant at least with two-state model (23), and the measured exchange times were respect to Hin-hix-AG binding. not extrapolated to a positive value at an infinite concentration Second, the bending property of hix-AG may differ from of ammonia. However, we confirmed, by monitoring the cen- that of hix-AT and hix-CG. Several studies on the homologous tral imino proton while increasing the temperature of samples gd resolvase (44) and Gin invertase (45) and preliminary at a given concentration of base catalyst, that hix-AG base pair results of circular permutation assays on the Hin recombinase exhibited faster base pair opening kinetics than did the hix-AT suggest that the hix site should be bent toward the major base pair (Figure 4B). groove upon Hin binding (6). We showed that all three hix sites are similarly bent with a magnitude ranging from 8 to 12 . However, hix-AT and hix-AG are bent toward the minor groove, which is opposite to the expected orientation of the bending in the Hin-bound hix site. Also, kinetics of base pair DISCUSSION opening and fluctuations of roll and tilt indicated that hix-AG The three mutant hix sites (hix-AT, hix-CG and hix-AG) is significantly more disordered than hix-AT. Therefore, a studied here are the same except for one or two base pairs. great entropy loss due to the constraints imposed by complex However, we have shown that their structural and dynamical formation as well as additional energy required for inverting properties are remarkably different, which may explain the the orientation of bending may explain the unfavorable 260 Nucleic Acids Research, 2006, Vol. 34, No. 1 binding between Hin and relaxed hix-AG site. Effect of super- charges for this article was provided by the National Creative coiling on a local DNA structure has been shown to be mim- Research Initiative from the Ministry of Science and icked by a nick which is known to hardly alter the bending Technology, Korea. flexibility but to substantially increase twist flexibility (46,47). Conflict of interest statement. None declared. Our FRET data and the preferred occurrence of CAG/CTG triplet at the dyad of nucleosome suggest that CAG/CTG trip- let has high twist and bending flexibility (48). Combined REFERENCES together with this intrinsic flexibility of CAG/CTG site, extrin- sic twist flexibility provided by supercoiling would facilitate 1. Dorman,C.J. (1996) Flexible response: DNA supercoiling, transcription and bacterial adaptation to environmental stress. Trends Microbiol., 4, further unwinding of hix-AG, which shall increase roll and 214–216. thus change the orientation of bending through strong negative 2. Travers,A. and Muskhelishvili,G. (2005) DNA supercoiling—a global correlation between twist and roll (28,34). This structural transcriptional regulator for enterobacterial growth? Nature Rev. change will reduce the energetic cost of Hin binding. CAG/ Microbiol., 3, 157–169. 3. 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