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

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 s

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 stackbulge-bind-ing 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.

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 32P-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¢-32P-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)4C⁄ (GT)7G and (GT)4G⁄ (CA)7C, slipped

in such a way that they were unable to form stable secondary structures, such as (CAG)3⁄ (CTG)5 and (CTG)3⁄ (CAG)5tracts

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].

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 aglyagly-con 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)4G⁄

(CA)7C and (CA)4C⁄ (GT)7G 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 vitrostudies 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

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.

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¢-32P-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 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

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 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 *The 5¢- 32 P-end-labeled strand.

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

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)3and unlabeled template (ATT)5was 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 m M , respectively The amount of polymerase used was almost equal, i.e 0.0177 unitÆlL)1of 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, reacreac-tion 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 1A or 1B The concentration of 1A or DDI-1B is 60 l M Data are from experiments similar to those described in Figs 1 and 2 using32P-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

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)5tract 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 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 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

A B

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.

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 formaaddi-tion of

the bulge or stabilize the bulge structure The UV

melting temperature (Tm) 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 Tm(DTm) for the

ATT bulge increased by 3.4 and 1.7C in the presence

of DDI-1A and DDI-1B, respectively The increase in

DTm 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 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

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.

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 disassociassoci-ation 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-DITE 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 polymerpolymer-ase 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¢-32P-end labeled using [32P]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 unitÆlL)1of the reaction A mixture of 5-32P-end-labeled primer and unlabeled template, generally

in equimolar concentrations, was annealed by heating in Tris⁄ HCl and MgCl2 to 95C 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 37C 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