Báo cáo khoa học: In vitro expansion of DNA triplet repeats with bulge binders and different DNA polymerases pdf

In vitro expansion of DNA triplet repeats with bulge binders and different DNA polymerases Di Ouyang, Long Yi, Liangliang Liu, Hong-Tao Mu and Zhen Xi State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, Nankai University, Tianjin, China Triplet repeats are the most abundant simple sequence repeats in the coding and non-coding sequences of all known eukaryotic genomes [1]. The frequency of spe- cific types of triplet repeats and their localization in genes vary significantly between genomes, reflecting their important role in genome evolution [1,2]. Expan- sions of DNA triplet repeat sequences are associated with  16 inherited neurological disorders known as triplet repeat expansion diseases [3–5], which can lead to total disability and death. The severity of a triplet repeat expansion disease is increased anticipatively and the age of onset is reduced with each successive generation [6,7]. The high mutation rate of triplet repeats makes them a rich source of quantitative genetic variation [8–11]. The tendency for repeating DNA strands to form hairpin loops or slipped confor- mations, and their inherent conformational properties, for example their high degree of flexibility, writhing and the stability of the hairpin formation, are impor- tant in the investigation of DNA slippage phenomena [3,11,12]. Among the non-B-form DNA conformations formed by triplet repeats, simple bulged structures (one or more unpaired bases) have been postulated as inter- mediates in the synthesis of slipped DNA and are associated with the unstable expansion of triplet repeats on the basis of their entropy [13]. Several groups have shown an interest in developing small molecules that possess specific effects for DNA triplet repeat strand slippage [14–23]. The most promising and successful bulge-specific agent discovered to date originated from studies on the enediyne natural prod- uct neocarzinostatin chromophore (NCS-chrom) [24]. Its isostructural mimic, NCSi-gb (Scheme 1A) binds bulge DNA at sub-micromolar concentrations [25], and is also able to induce formation of the bulge-bind- ing pocket by stacking between the base pairs that flank the bulge site in the oligonucleotide [26,27]. Keywords bulge binder; DNA polymerase; DNA slippage; drug–DNA interaction; repeat sequences Correspondence Z. Xi, State Key Laboratory of Elemento- Organic Chemistry and Department of Chemical Biology, Nankai University, Tianjin, 300071, China Fax: +86 022 2350 4782 Tel: +86 022 2350 4782 E-mail: zhenxi@nankai.edu.cn (Received 11 May 2008, revised 8 July 2008, accepted 10 July 2008) doi:10.1111/j.1742-4658.2008.06593.x The expansion of DNA repeat sequences is associated with many genetic diseases in humans. Simple bulge DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. To probe the possible role of bulged structures in DNA slippage, we designed and synthesized a pair of simple chiral spirocyclic compounds [Xi Z, Ouyang D & Mu HT (2006) Bioorg Med Chem Lett 16, 1180–1184], DDI-1A and DDI-1B, which mimic the molecular architecture of the enediyne antitumor antibiotic neocarzinostatin chromophore. Both compounds strongly stimu- lated slippage in various DNA repeats in vitro. Enhanced slippage synthesis was found to be synchronous for primer and template. CD spectra and UV thermal stability studies supported the idea that DDI-1A and DDI-1B exhibited selective binding to the DNA bulge and induced a significant conformational change in bulge DNA. The proposed mechanism for the observed in vitro expansion of long DNA is discussed. Abbreviations DDI, Double Deck Intercalater; NCS-chrom, neocarzinostatin chromophore. 4510 FEBS Journal 275 (2008) 4510–4521 ª 2008 The Authors Journal compilation ª 2008 FEBS Molecular studies have shown that the affinity of NCSi-gb for DNA bulges is mostly dependent on the spirocyclic ring junction being at an appropriate angle, the pendant aminosugar group that enhances binding at the bulged site, and the two discrete aromatic moie- ties for p-stacking that mimic a base pair. Molecules that mimic the wedge-shaped natural product have been designed and synthesized, with the expectation that they may be used to study the role of bulged structures in nucleic acid function [16]. For example, the compound double deck intercalater (DDI), which has an spirocyclic backbone almost identical to that of NCSi-gb (Scheme 1B), was able to enhance slippage synthesis of various repeat DNA strands [16,20,28]. Analogs of NCSi-gb, the aminoglucose in a-glycosidic linkage or the natural sugar N-methylfucosamine in b-glycosidic linkage to the backbone, were found to interfere with bulge-specific cleavage by NCS-chrom and to inhibit DNA synthesis involving DNA poly- merase-dependent primer extension on two-base bulge templates [14]. Of these, enantiomers possessing the natural sugar in a b-glycosidic linkage have been shown to be the most potent inhibitors. An NMR study [17] found that another designed stable analog of NCSi-gb, SCA-R2, binds specifically and tightly at a two-base bulge in DNA via stacking of its helically oriented aromatic ring systems on the bulge-flanking base pairs that define the long sides of the triangular prism binding pocket, with its amino sugar anchored in the major groove of the DNA pointing toward the 3¢-bulge-flanking base pair. We were interested in small molecules that can selec- tively bind bulge DNA and control DNA repeat expansion. In our previous studies, some molecules targeted at the bulge site were found to enhance repeat nucleotide slippage during in vitro DNA replication [20,29]. DDI-1A and DDI-1B (Scheme 1C,D) with one benzene ring fewer than the spirocyclic backbone of DDI, showed comparative activities in simulating ATTÆAAT triplet expansion [20]. To gain insight into the stimulation effect of DDI-1A and DDI-1B on DNA strand slippage synthesis, we studied the effect of drug-stimulated DNA slippage synthesis using vari- ous repeat sequences (DNA doublet or triplet with 3–7 repeats) and different prokaryotic DNA polymerases (sequenase, Taq, pfu, T4, T7, etc.) on the DNA exten- sion reaction by using 32 P-labeling primer or template in the presence or absence of DNA-binding agents (DDIs and doxorubicin). The DNA bulge binding of both compounds was detected by CD and UV melting experiments. Possible slippage mechanisms are discussed. Results and Discussion Effect of DDI-1A and DDI-1B on repeats expansion DDI-1A and DDI-1B were tested for their effect on the expansion of several doublet and triplet repeats in the presence of the Klenow fragment of DNA poly- merase I. The reaction contained 5¢- 32 P-end-labeled 9-mer primer, unlabeled template, dNTPs and the Klenow fragment. Figure 1 shows the extension prod- ucts on a denaturing polyacrylamide gel. Band intensi- ties in each lane were measured using a Phosphor Imager. In the control reaction (Fig. 1, lane 2), the 9-mer primer with different sequences was extended to different lengths. Sequences with relatively unstable secondary structures, such as the triplet repeats (AAT) 3 ⁄ (ATT) 5 and (ATT) 3 ⁄ (AAT) 5 and doublet repeats (CA) 4 C ⁄ (GT) 7 G and (GT) 4 G ⁄ (CA) 7 C, slipped in such a way that they were unable to form stable secondary structures, such as (CAG) 3 ⁄ (CTG) 5 and (CTG) 3 ⁄ (CAG) 5 tracts. In the presence of DDI-1A and DDI-1B, slippage synthesis was greatly enhanced, as indicated by the presence of much longer DNA products (Fig. 1A–F, lanes 3 and 4). Slippage enhancement for sequences with less stable secondary structures was much stronger (Fig. 1A–D) than for sequences with relatively stable secondary structures (Fig. 1E,F). The stimulation effect Scheme 1. (A) DNA bulge-specific compound derived from NCS- chrom upon base catalysis. (B–D) Synthetic compounds mimicking NCS-chrom, which showed selectivity for binding to DNA bulge site [16], and strongly enhanced the repeat nucleotide slippage during in vitro DNA synthesis [20]. D. Ouyang et al. Expansion of DNA repeat sequences FEBS Journal 275 (2008) 4510–4521 ª 2008 The Authors Journal compilation ª 2008 FEBS 4511 of DDI-1A was better than that of DDI-1B, presum- ably because of the different conformation of the agly- con moiety. DDI-1A has a right-handed aglycon helix with geometry mimicking the DNA helix, and is there- fore more effective at intercalating into DNA base pairs [20]. Although DDI-1B also mimics the structure of NCSi-gb, it has a left-handed aglycon helix and may be less effective at base pair intercalation. It should be noted that 2-deoxy-2-aminoglucose (with concentrations of 10–1000 lm) or the aglycon back- bone (concentrations of 10–100 lm) of DDI-1A and DDI-1B did not affect DNA slippage (data not shown). Interestingly, there was a similar hierarchy of intensities for the three bands in the (AAT) 3 ⁄ (ATT) 5 and (ATT) 3 ⁄ (AAT) 5 systems (Fig. 1A,B), each appar- ently separated by two nucleotides, and this was repeated every three nucleotides. This band spacing appeared to reflect the triplet repeat unit, implying that the in vitro DNA strand slippage syntheses of (AAT) 3 ⁄ (ATT) 5 and (ATT) 3 ⁄ (AAT) 5 tracts mainly occurred by triplet step expansion. Addition of both DDI-1A and DDI-1B did not influence this pattern (Fig. 1, inset). Similarly, the doublet repeat (GT) 4 G ⁄ (CA) 7 C and (CA) 4 C ⁄ (GT) 7 G also produced a regular two-band repeat (Fig. 1C,D), suggesting that slippage of these repeats occurred by two nucleotides each time. Extension products from other two-triplet repeat sequences, (CAG) 3 ⁄ (CTG) 5 and (CTG) 3 ⁄ (CAG) 5 , were too short to generate a similar pattern on the gel. Doxorubicin, an anthracycline glycoside that inter- calates between DNA base pairs [30], inhibited the expansion of all the repeat sequences used (Fig. 1, lane 5). When both DDI-1A or DDI-1B and doxo- rubicin were present, similar inhibition was found at experimental concentrations (data not shown). In vitro studies show that single-stranded tracts con- taining (CTG) n repeats have a higher propensity to form hairpin structures than similar tracts containing the complementary (CAG) n repeats [31]; possibly accounting for the orientation-dependent behavior of these repeats in replication. Hairpin stability is attrib- uted to the TÆT mismatch which stacked more effi- ciently on the CTG strand than the AÆA mispair on the complementary CAG strand, resulting in expanded CTG fragments that are shorter than those of the CAG strand (Fig. 1E,F). This rule is also the same for other repeat sequences. As a result, the slippage effects of AAT and CA repeats (Fig. 1A,D) are better than those of their complementary strands, ATT and GT (Fig. 1B,C). As such, the enhancement effects of AB CDE F Fig. 1. Expansion of the various repeats and the effect of diastereomers DDI-1A and DDI-1B with 32 P-primer strands. A standard reaction (23 °C, 24 h) containing 5¢- 32 P-end-labeled primer and unlabeled template was catalyzed by the Klenow fragment at 0.0177 unitÆlL )1 . (A–F) Lane 1, control to which no DNA polymerase was added; lane 2, control reaction system lacking compound, but receiving an equal volume of dimethyl sulfoxide; lanes 3 and 4, reaction system to which DDI-1A and DDI-1B at a concentration of 60 l M were added, respectively; lane 5, reaction system to which 40 l M doxorubicin was added. The products were resolved on a 15% sequencing gel. The numbers indi- cate size markers of 26 and 41 nucleotides (random sequence) in length. *The 5¢ - 32 P-end-labeled strand. (Inset) Special attention of triple band pattern in the gel. Expansion of DNA repeat sequences D. Ouyang et al. 4512 FEBS Journal 275 (2008) 4510–4521 ª 2008 The Authors Journal compilation ª 2008 FEBS DDI-1A and DDI-1B on AAT, CAG and CA repeats are relatively better than for ATT, CTG and GT. State of the template during slippage extension Several repeated templates with 5¢- 32 P-end labeling were used to investigate template extension. Figure 2 shows the extension result of six different sequences under similar reaction conditions to those in Fig. 1. In the control reaction, the 15-mer template of various sequences (Fig. 2, lane 2) was extended to different lengths depending on the stability of the secondary structure formed between the primer and template (Fig. 1). Enhancement of sequences with less stable secondary structures was stronger than that with rela- tively stable secondary structures. After addition of DDI-1A and DDI-1B, slippage synthesis was greatly enhanced for all sequences, as reflected by the presence of much longer products (Fig. 2, lanes 3 and 4) in comparison with the control. The stimulation effect of DDI-1A in the template extension was obviously better than that of DDI-1B, which was similar in the primer extension reaction. As expected, doxorubicin inhibited template expansion for all the repeated sequences chosen (Fig. 2, lane 5). Again, the gel band pattern of the synthesized DNA products reflected the particular nucleotide repeat unit. A similar band pattern in both the labeled primer and the template expansion system implied that template and primer extension took place synchronously. Time course of DNA expansion A time course for the extension of the repeat sequences was performed (Table 1) in the assays shown in Figs 1 and 2. In the control, longer DNA fragments were generated with the increase in reaction time, indicating that primer and template slippage occurred during DNA synthesis. In the presence of DDI-1A and DDI- 1B, radioactivity bands (both primer and template) with long fragments increased steadily over time for all the sequences tested. The slippage of less stable repeat sequences almost reached saturation after being incu- bated for > 48 h, and the differences in length between the drug-containing samples and the control was remarkable. Effect of different polymerases on drug- stimulated replication of the ATTÆAAT triplet As shown in Fig. 3, we also investigated the effect of a series of different prokaryotic polymerases proficient or deficient in 3¢ to 5¢ exonuclease activity on ATTÆAAT triplet slippage synthesis in vitro. The exten- ABC DEF Fig. 2. Expansion of various templates with 32 P-template strands. (A–F) Lane 1, control to which no DNA polymerase was added; lane 2, control reaction system lacking the compound, but receiving an equal volume of dimethyl sulfoxide; lanes 3 and 4, reaction system to which DDI-1A and DDI-1B (60 l M) was added, respectively; lane 5, reaction system to which 40 lM doxorubicin was added. Products were resolved on a 15% sequencing gel. The numbers indicate size markers of 26 and 41 nucleotides (random sequence) in length. *The 5¢- 32 P- end-labeled strand. D. Ouyang et al. Expansion of DNA repeat sequences FEBS Journal 275 (2008) 4510–4521 ª 2008 The Authors Journal compilation ª 2008 FEBS 4513 sion behavior of different polymerases was completely different. The primer itself slipped in the control reaction when using polymerases deficient in 3¢ to 5¢ exonuclease activity, such as the Klenow fragment (Fig. 3B, lane 2), Sequenase Version 2.0 DNA poly- merase (Fig. 3C, lane 2), Taq DNA polymerase (Fig. 3E, lane 2) and pfu DNA polymerase (Fig. 3F, lane 2). The addition of DDI-1A and DDI-1B strongly increased the slippage effect in these polymerase systems. Among these, Sequenase showed the weakest ABCDEF Fig. 3. Effect of different polymerases on the stimulation of a triplet repeat expansion. A standard reaction (23 °C, 24 h) containing 5¢- 32 P- end-labeled (AAT) 3 and unlabeled template (ATT) 5 was catalyzed by different prokaryotic polymerases (indicated). The concentration of pri- mer–template and deoxynucleoside triphosphates in the reaction system is 4 l M and 1 mM, respectively. The amount of polymerase used was almost equal, i.e. 0.0177 unitÆlL )1 of each enzyme. (A–F) Lane 1, control to which no DNA polymerase was added; lane 2: control reac- tion system lacking drug, but with an equal volume of dimethyl sulfoxide; lanes 3 and 4, reaction system to which DDI-1A or DDI-1B (60 l M) was added; lane 5, reaction system to which 40 l M doxorubicin was added. Products were resolved on a 15% sequencing gel. The numbers indicate size markers of 26 and 41 nucleotides (random sequence) in length. Table 1. Time course of primer ⁄ template expansion in the presence or absence of DDI-1A or DDI-1B. The concentration of DDI-1A or DDI- 1B is 60 l M. Data are from experiments similar to those described in Figs 1 and 2 using 32 P-labeled primer ⁄ templates. After gel analysis of the products, the band intensities were quantitated by Phosphor Imager (Molecular Dynamics). *5¢- 32 P-end-labeled primer or template. Primer ⁄ template Fragments > 15-mer (%) at 23 °C 12 h 24 h 48 h Control DDI-1A DDI-1B Control DDI-1A DDI-1B Control DDI-1A DDI-1B (AAT) 3 * ⁄ (ATT) 5 23.3 56.4 32.2 29.8 86.4 45.5 33.4 92.1 63.5 (AAT) 3 ⁄ (ATT) 5 * 16.8 76.7 44.6 22.3 95.9 62.8 26.7 97.6 85.4 (ATT) 3 * ⁄ (AAT) 5 17.6 52.2 30.5 18.9 72.2 35.5 22.6 88.4 56.3 (ATT) 3 ⁄ (AAT) 5 * 9.7 50.2 25.9 15.6 71.1 42.6 19.2 89.4 67.7 (CAG) 3 * ⁄ (CTG) 5 10.3 12.9 8.2 15.0 25.9 21.4 18.5 33.6 28.7 (CAG) 3 ⁄ (CTG) 5 * 2.2 25.1 12.3 3.5 38.7 19.4 5.2 49.6 26.8 (CTG) 3 * ⁄ (CAG) 5 0 7.8 1.0 0 15.8 2.0 0 23.2 4.5 (CTG) 3 ⁄ (CAG) 5 * 6.9 37.5 18.1 9.4 52.0 28.2 10.9 67.2 44.3 (GT) 4 G* ⁄ (CA) 7 C 0 32.7 9.4 1.5 67.7 12.0 12.0 89.7 36.8 (GT) 4 G ⁄ (CA) 7 C* 32.9 70.4 55.7 46.3 93.1 83.7 52.5 95.2 90.7 (CA) 4 C* ⁄ (GT) 7 G 15.7 50.2 19.5 18.6 85.2 28.5 23.7 92.2 48.7 (CA) 4 C ⁄ (GT) 7 G* 25.6 69.3 51.8 35.8 89.7 80.7 37.3 96.4 90.5 Expansion of DNA repeat sequences D. Ouyang et al. 4514 FEBS Journal 275 (2008) 4510–4521 ª 2008 The Authors Journal compilation ª 2008 FEBS slippage effect under uniform conditions, whereas, the difference between DDI-1A and DDI-1B is undistin- guishable for Taq DNA polymerase. An inhibition effect of doxorubicin was also observed (Fig. 3, lane 5). Under the extension conditions used, pfu DNA polymerase did not excise the extruded nucleo- tides in the oligomer or eliminate the secondary struc- ture formed by the repeated sequences, but it did amplify the ATT repeats faithfully. By contrast, for T7 DNA polymerase (Fig. 3D) and T4 DNA polymerase (data not shown), although their extension activities are very similar to that of the Kle- now fragment [32], the 3¢ to 5¢ exonuclease activity was so strong that the overhanging nucleotides were excised completely from the 3¢-terminus in the anneal- ing oligomer, and consequently, no expanded band was observed during incubation. Doxorubicin did not inhibit the exonuclease activity of T7 DNA polymer- ase, whereas for Escherichia coli DNA polymerase I, both exonuclease and polymerase activities were seen. In the control (Fig. 3A, lane 2) and drug-addition (Fig. 3A, lanes 3 and 4) reactions, the enzyme both extended the primer to some extent, and excised the 3¢ overhung nucleotides from the duplex to give smaller fragments. Because of the presence of excised short oligomers, the extension bands in these lanes were much lighter than the others, and various types of duplex were formed by the primer and template. We did not observe any strong stimulation to DNA slip- page synthesis in the gel pattern by the addition of DDI-1A and DDI-1B in these cases. These results may be due to DNA polymerases with strong 3¢ to 5¢ exonuclease activity (including T7 DNA polymerase and DNA polymerase I) degrading the product. To our surprise, the addition of doxorubincin did not obviously inhibit expansion, but did inhibit the exonu- clease activity of DNA polymerase I to some extent; the excised short oligomers were obviously less (Fig. 3A, lane 5) than in the control and drug-addition reactions. Again, a triple band pattern was apparent through- out the gel. Although the pattern in the Taq and pfu DNA polymerase system differed from that in the Escherichia coli DNA polymerase I-based system, the expanded primary bands were almost all seen in the three-nucleotide unit, which indicated that the in vitro DNA strand slippage synthesis of (ATT) 3 ⁄ (AAT) 5 tract was mainly a triplet expansion pattern. It is suggested that the complete complementary structure formed by the two triplet complementary strands might be more stable than the others during slippage synthesis, and be similar whatever DNA polymerases are used. Selective binding of DDI-1A and DDI-1B to bulge DNA Because formation of the bulge structure might be important in DNA slippage [16], we speculate that the enhancement of repeat slippage by DDI-1A and DDI- 1B might be caused by their specific recognition of bulge DNA. Accordingly, CD spectropolarimetry can be used to monitor conformational transitions as the ligand–nucleic acid complex is formed. To gain insight into the binding of drugs to bulge DNA, several bulge- containing oligonucleotides were selected as binding hosts for DDI-1A and DDI-1B. CD spectroscopy of DDI-1A (Fig. 4) showed a positive Cotton effect at 246 and 310 nm, and a neg- ative Cotton effect at 220 and 290 nm, whereas the CD of DDI-1B was almost complementary to that of DDI-1A. These peaks are Cotton effect-associated with corresponding p to p* transitions in the UV spectra. The positive CD spectra for DDI-1A suggests that the helix was right-handed, hence in the P con- formation [14]. In order to observe the conformational transitions of DNA directly and to eliminate drug interference, the CD spectra of native DNA and altered DNA, after subtracting the spectrum for the drug alone from that of the complex, are also presented, assuming that the conformation of the drug was not significantly altered because the molecular models of DDI-1A and DDI-1B are fairly rigid. The differential CD spectra of the complex formed between DNA and the drugs are shown in Fig. 4. The observed CD spectrum of the native DNA (solid line) consists of a distinct positive band at 280 nm caused by base stacking and a negative band at 250 nm caused by helicity [33], which is char- acteristic of DNA in the right-handed B-form. CD spectra of DNA with DDI-1A (dashed line) and DDI- 1B (dotted line) consistently revealed an isoelliptic point at  260 nm, except for the oligomer without a bulge structure (Fig. 4A), suggesting formation of a drug–DNA complex. For oligomer with a hairpin structure (HT3AT), the band at 252 nm shifted to 241 nm (Fig. 4A), whereas for DNA with simple bulge structures (one to three unpaired bases), the band at 252 nm shifted to 244 nm for DDI-1A and to 248 nm for DDI-1B. There was no overall change in ellipticity detected from the differential spectra of DNA (Fig. 4B) for the oligomer HT3AT. In this case, the binding of DDI-1A or DDI-1B to DNA might be via simple groove binding and ⁄ or electrostatic interaction that showed fewer or no perturbations on the base stacking and helicity bands [34], ruling out the possi- bility of conformational change. D. Ouyang et al. Expansion of DNA repeat sequences FEBS Journal 275 (2008) 4510–4521 ª 2008 The Authors Journal compilation ª 2008 FEBS 4515 AB CD EF GH IJ Fig. 4. CD spectra and differential spectra of DDI-1A and DDI-1B and their complexes with selected DNA sequences. Solid line, 20 l M free DNA. Dashed line: (A,C,E,G,I) complex of DNA with DDI-1A (50 l M), (B,D,F,H,J) drug-alone has been subtracted. Dotted line: (A,C,E,G,I) complex of DNA with DDI-1B (50 l M), (B,D,F,H,J) drug-alone has been subtracted. The numbers indicate size markers of 26 and 41 nucleotides (ran- dom sequence) in length. Expansion of DNA repeat sequences D. Ouyang et al. 4516 FEBS Journal 275 (2008) 4510–4521 ª 2008 The Authors Journal compilation ª 2008 FEBS For oligomers containing single (one to three base) bulges, the differential spectrum (dashed line for DDI- 1A and dotted line for DDI-1B) was changed signifi- cantly, compared with that for the native DNA (solid line). As shown in Fig. 4D, the new band at 305 nm proved the formation of a DNA–drug complex [33]. The significant change in the band at 250 and 280 nm implied an alteration in the DNA conformation because of an overall bending of the DNA backbone [33,35]. Both DDI-1A and DDI-1B exhibited binding behaviors obviously different to that of the bulge DNA host. The addition of DDI-1A and DDI-1B to the two-base bulge (HT3AGTT) and three-base bulge (HT3AATTT and HT3AAATT) caused the DNA spectrum to be altered significantly. The trend and characteristics of the conformational transformation were similar to that of the one-base bulge oligomer. However, the aglycon unit of DDI-1A and DDI-1B (10–500 lm), lacking any CD signal itself, did not affect the conformation of DNA (data not shown). From the CD results, both compounds can interact with oligomers containing a simple bulge and induce significant conformational change. Therefore, the addi- tion of DDI-1A and DDI-1B may induce formation of the bulge or stabilize the bulge structure. The UV melting temperature (T m ) of oligonucleotides with a three-base bulge increased upon intercalating with DDI-1A and DDI-1B (Table 2), implying that DNA secondary structures were stabilized by interaction with the drug. For example, the change in T m (DT m ) for the ATT bulge increased by 3.4 and 1.7 °C in the presence of DDI-1A and DDI-1B, respectively. The increase in DT m for DDI-1A was higher than that for DDI-1B, implying that intercalation to the bulge site was better for DDI-1A than for DDI-1B due to its right-handed aglycon helix, which might be suitable for stacking into natural helical bases. The CD and melting temper- ature data were consistent with the greater stimulation effect of DDI-1A than of DDI-1B in the repeat slip- page. Conclusion It has previously been shown that long DNA prod- ucts can be generated in polymerase extension reac- tions containing short complementary oligomers (e.g. 9-mer ⁄ 15-mer combinations) of di- or trinucleotide repeats [36]. The efficiency of reiterative synthesis depended on several factors including the length of the repetitive unit, its sequence and the characteristics of polymerase. In vitro studies on the expansion of triplet repeats such as CAG, CGG and GAA, which are associated with human hereditary disease genes, helped in understanding the possible mechanism of slippage and the molecular basis of the diseases [37,38]. Given the size of DNA products made by the DNA polymerase-based system using short repeat primers and templates, slippage must be involved during repli- cation. Furthermore, slippage occurs synchronously on both strands. Slippage synthesis was enhanced mark- edly by our synthetic diastereomers DDI-1A and DDI- 1B, which bind preferentially to simple bulges of one to three unpaired bases in DNA. These results suggest a process of stimulated slippage synthesis (Scheme 2). After denaturing and annealing, the primers and tem- plates form various types of duplex DNA. The small DNA primer–template may have gone through multi- ple rounds of slippage to reach the large expanded products observed. Each cycle is initiated by the disso- ciation of polymerase to re-associate at a new inter- mediate. The intermediate is a combination of various DNA strands with an unorthodox structure, such as hairpin, bulged and slipped DNA, and may be the main contributor to expansion. Under the experimen- tal conditions used, various combinations of these unstable intermediates are in homeostasis. When one round of extension finishes, the extended primer and template separate and realign to form new intermedi- ates for the next round of replication, and longer extended products are obtained through multiple rounds of replication. For example, following bulge ⁄ hairpin formation on the AAT strand of an AAT ⁄ ATT repeat tract, replication extends the fore- shortened AAT strand. The AAT bulge ⁄ hairpin may then come apart to allow the complementary ATT strand to be extended by DNA polymerase along the previously extended AAT strand, and vice versa. In fact, template extension is the same as primer exten- sion. We call it template extension to distinguish the Table 2. T m values of oligomers (P3 and P4) and DT m values by addition of DDI-1A and DDI-1B. DNA sequence P3 5¢-GTCCGATGCGTG-3¢ 3¢-CAGGCTACGCAC-5¢ ATT P4 5¢-GTCCGATGCGTG-3¢ 3¢-CAGGCTACGCAC-5¢ TAA Native 20 l M DDI-1A 20 lM DDI-1B Native 20 lM DDI-1A 20 lM DDI-1B T m (°C) 27.5 30.9 29.2 29.2 30.7 29.8 DT m (°C) 3.4 1.7 1.5 0.6 D. Ouyang et al. Expansion of DNA repeat sequences FEBS Journal 275 (2008) 4510–4521 ª 2008 The Authors Journal compilation ª 2008 FEBS 4517 expanded product of labeled primer from that of labeled template. If doxorubicin intercalates between DNA base pairs, a bulge structure cannot form. As a result, DNA expansion of the repeated sequences is inhibited (Scheme 2). The propensity for the unwinding of DNA unwind- ing elements, for example AATÆATT triplet repeats [39,40], enables accessibility to chemical probes within the region, as well as oligonucleotide hybridization, which lead to aberrant DNA replication. At the reac- tion temperature, the bulge ⁄ hairpin structures of these types of sequences form and come apart easily, as does realignment of the expanded primer and template, allowing the complementary strand to be extended fur- ther (Scheme 2). By contrast, the repeat sequence CTGÆCAG associated with myotonic dystrophy type 1 has been observed to form slipped structures and hair- pins in a length- and orientation-dependent manner under physiological conditions [41–43]. Once the non- B structure has formed, it is difficult for the CTG or CAG strand to re-anneal to its complementary strand, nor would realignment of primer and template and further extension be easy. Thus, the expanded frag- ments are relatively short. Scheme 2. Mode for primer and template extensions stimulated by drug. The crooked region of two swallow-tailed shapes represent the unstable intermediates that are composed of bulge, hairpin and slipped DNA etc. The compound formula represents DDI-1A or DDI-1B. One cycle of simple extension and drug stimulation is shown for each pathway. It is assumed that multiple cycles through these pathways are required to reach the dramatic expansion. Expansion of DNA repeat sequences D. Ouyang et al. 4518 FEBS Journal 275 (2008) 4510–4521 ª 2008 The Authors Journal compilation ª 2008 FEBS Once simple extension of the primer and template is accomplished, slippage synthesis in the presence of DDI-1A and DDI-1B becomes more pronounced as incubation proceeds. In our experiment, the more DDI-1A and DDI-1B were added and the longer incu- bation time, the longer the expanded products obtained; this may be due to two or more bulged inter- mediates formed or induced by the additional drug (Scheme 2). Compared with stimulation slippage and bulge binding specificity, it is proposed that the associ- ation and disassociation of the compound with the bulged structure is also in a dynamic equilibrium, whereas a molecule with moderate binding affinity and binding dynamics to the bulged structure would facili- tate further slippage, and yield a good stimulation result [29]. In summary, DDI-1A and DDI-1B were designed according to a DNA bulge binder, the enediyne natu- ral product NCS-chrom [28], which exhibited selective bulge-binding properties. To date, both compounds are the smallest bulge-binding molecules shown to suc- cessfully stimulate DNA strand slippage [20]. Detailed investigation into the effects on in vitro DNA replica- tion leads to several conclusions: (a) DNA sequences with relatively unstable secondary structures slipped more than DNA sequences with stable secondary structures; (b) slippage of these repeats occurred by two or three nucleotides each time, depending on the DNA sequences; (c) template and primer extension were synchronous; (d) the stimulation effect of DDI- 1A containing a right-handed aglycon helix was greater than that of DDI-1B; (e) the enhancement effects of DDI-1A and DDI-1B on AAT, CAG and CA repeats are stronger than that of the ATT, CTG and GT strand, which may be attributed to the TÆT mismatch as opposed to the AÆA mismatch; (f) doxo- rubincin inhibited the exonuclease activity of DNA polymerase I to some extent. Considering these results and previous publications [16,17,20,28,29], we propose that the bulge selectivity of drugs is due to the wedge- shaped spirocyclic part which fits into the DNA bulge pocket, and aromatic aminosugar compounds with bulge-binding selectivity may be anticipated to stimu- late DNA slippage synthesis. This study provides insight into the development of agents that interfere with nucleotide expansion, as found in various disease states. Given the relationship between repeat length and both disease severity and age of onset, treatment that interferes with triplet expansion or the generation of ineffectual DNA triplet templates, might make sense for RNA regulation and prevent the formation of toxic proteins, such as polyglutamine [44] and polyalanine tract [45]. Experimental procedures Materials Oligodeoxyribonucleotides were synthesized on a EXPE- DITEÔ 8909 nucleic acids synthesis system (Applied Biosystems, Foster City, CA, USA), and purified by elec- trophoresis on a denaturing polyacrylamide gel using a standard procedure [46]. The product was recovered from the gel by phenol ⁄ chloroform extraction and ethanol pre- cipitation. T4 polynucleotide kinase, E. coli DNA polymer- ase I, the Klenow fragment of E. coli DNA polymerase I lacking 3¢ to 5¢ exonuclease activity, Taq DNA polymerase and pfu DNA polymerase were from Takara Biotechnology (Dalian City, China). T7 DNA polymerase was from MBI Company (Tangshan City, China). Sequenase Version 2.0 DNA polymerase was from U.S. Biochemical Corporation (London, UK). Radioactive materials were from Beijing Furui Biological Engineering Company (Beijing, China). Other chemicals were from Sigma (St Louis, MO, USA). The oligonucleotides were 5¢- 32 P-end labeled using [ 32 P]ATP[cP] and polynucleotide kinase. DNA polymerase assays A standard reaction (15 lL) contained 4 lm each of the primer and template and 1 mm each of deoxynucleoside tri- phosphate, DNA polymerase and the corresponding reac- tion buffer. The DNA was in a several fold molar excess of the enzyme. Unless otherwise indicated, the enzyme was at a level of  0.0177 unitÆ lL )1 of the reaction. A mixture of 5- 32 P-end-labeled primer and unlabeled template, generally in equimolar concentrations, was annealed by heating in Tris ⁄ HCl and MgCl 2 to 95 °C for 5 min followed by slow cooling to room temperature. Following the addition of dithiothreitol and deoxynucleoside triphosphates to the annealed mixture, it was distributed for assays. The com- pounds to be tested were added as a solution in dimethyl sulfoxide. Controls lacking the compound received an equal volume of dimethyl sulfoxide, the final concentration of which was 2%. The reaction was started by addition of the enzyme. Incubation was at 23 or 37 °C for the times indi- cated. To terminate the reaction, 98% formamide contain- ing 100 mm EDTA and marker dyes was added to the reaction mixtures at a 1 : 1 vol. The reaction mixtures with formamide, EDTA and marker dyes were loaded onto a 15% polyacrylamide sequencing gel for analysis. Gels were exposed to a storage phorsphor screen, and the band inten- sities were quantitated on a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA, USA). UV melting experiments Ultraviolet absorptions of 2 lm oligonucleotides were mea- sured using a Cary-Bio100 UV-Visible spectrophotometer D. Ouyang et al. Expansion of DNA repeat sequences FEBS Journal 275 (2008) 4510–4521 ª 2008 The Authors Journal compilation ª 2008 FEBS 4519 [...].. .Expansion of DNA repeat sequences D Ouyang et al with heating at 0.5 °CÆmin)1 in phosphate buffer containing 10 mm phosphate, 10 mm NaCl, pH 7.0 The Tm for DNA in the presence of DDI-1A or DDI-1B was determined when the concentration of drug was 10-fold that of DNA, and calculated using the derivative method supplied in the cary winuv software package for Tm calculation... helical ligand binding at a two-base bulge site in DNA Biochemistry 46, 4793–4803 18 Lin Y, Jones GB, Hwang GS, Kappen L & Goldberg IH (2005) Convenient synthesis of ncs-chromophore metabolite isosteres: binding agents for bulged DNA microenvironments Org Lett 7, 71–74 19 Trotta E, Grosso ND, Erba M, Melino S, Cicero D & Paci M (2003) Interaction of DAPI with individual strands of trinucleotide repeats. .. RD (1995) Pausing of DNA synthesis in vitro at specific loci in CTG and CGG triplet repeats from human hereditary disease genes J Biol Chem 270, 27014–27021 32 Tuntiwechapikul W & Salazar M (2002) Mechanism of in vitro expansion of long DNA repeats: effect of temperature, repeat length, repeat sequence, and DNA polymerase Biochemistry 41, 854–860 33 Fasman GD (1996) Circular Dichroism and the Conformational... 1319–1321 25 Yang CF, Stassinopoulos A & Goldberg IH (1995) Specific binding of the biradical analog of neocarzinostatin chromophore to bulged DNA: implications for thiolindependent cleavage Biochemistry 34, 2267–2275 26 Gao X, Stassinopolous A, Ji J, Kwon Y, Bare S & Goldberg IH (2002) Induced formation of a DNA bulge structure by a molecular wedge ligand-postactivated neocarzinostatin chromophore Biochemistry... genetic instability of triplet repeat sequences J Biol Chem 280, 37366–37376 10 Parniewski P, Jaworski A, Wells RD & Bowater RP (2000) Length of CTGÆCAG repeats determines the in uence of mismatch repair on genetic instability J Mol Biol 299, 865–874 11 Wells RD (1996) Molecular basis of genetic instability of triplet repeats J Biol Chem 271, 2875–2878 12 Perutz MF (1996) Glutamine repeats and inherited... (1992) Slippage synthesis of simple sequence DNA Nucleic Acids Res 20, 211– 215 37 Lyons-Darden T & Topal MD (1999) Effects of temperature, Mg2+ concentration and mismatches on tripletrepeat expansion during DNA replication in vitro Nucleic Acids Res 27, 2235–2240 38 Pearson CE & Sinden RR (1998) Slipped strand DNA, dynamic mutations, and human disease In Genetic Instabilities and Hereditary Neurological... (2006) Development of new simple molecular probes of DNA bulged structures Bioorg Med Chem Lett 16, 2895– 2899 23 Hwang GS, Jones GB & Goldberg IH (2004) Stereochemical control of small molecule binding to bulged DNA: comparison of structures of spirocyclic enantiomer-bulged DNA complexes Biochemistry 43, 641–650 24 Kappen LS & Goldberg IH (1993) DNA conformationinduced activation of an enediyne for... TTAÆTAA triplet repeats in plasmids form a non-H bonded structure J Biol Chem 271, 16784– 16791 40 Trotta E, Grosso ND, Erba M & Paci M (2000) The ATT stand of AATÆATT trinucleotide repeats adopts stable hairpin structures induced by minor groove binding ligands Biochemistry 39, 6799–6808 41 Tam M, Montgomery ES, Kekis M, Stollar DB, Price GB & Pearson CE (2003) Slipped (CTG)Æ(CAG) repeats of the myotonic... Jasco-715 spectropolarimeter, using a cylindrical quartz cell of 1 mm path length The cell compartment was purged continuously with dry N2 Data were recorded at a bandwidth of 1.0 nm and measured every 0.2 nm over 210–325 nm at 20 ± 1 °C in TE buffer (10 mm Tris, 1 mm EDTA, pH 8.0) containing 10 mm NaCl All oligonucleotides were heated to 95 °C in the same buffer for 5 min and then cooled slowly to room... Conformations of bulge DNAbound drug were obtained by subtracting the drug-only CD signal from that of the complex made by 20 lm of DNA mixed with 50 lm drug Acknowledgements We thank the two referees for the useful discussion This study was supported by the National Key Project for Basic Research of China (2003CB114403), National Natural Science Foundation of China (20272029, 20572053, 20421202, 20432010), Ministry . In vitro expansion of DNA triplet repeats with bulge binders and different DNA polymerases Di Ouyang, Long Yi, Liangliang Liu, Hong-Tao Mu and Zhen. reaction by using 32 P-labeling primer or template in the presence or absence of DNA- binding agents (DDIs and doxorubicin). The DNA bulge binding of both compounds