Increased Negative Superhelical Density in Vivo Enhancesthe Genetic Instability of Triplet Repeat Sequences*

Received for publication, July 22, 2005, and in revised form, September 13, 2005 Published, JBC Papers in Press, September 13, 2005, DOI 10.1074/jbc.M508065200

Marek Napierala, Albino Bacolla, and Robert D. Wells1

From the Institute of Biosciences and Technology, Center for Genome Research, Texas A&M University SystemHealth Science Center, Houston, Texas 77030-3303

The influence of negative superhelical density on the geneticinstabilities of long GAA�TTC, CGG�CCG, and CTG�CAG repeatsequences was studied in vivo in topologically constrained plasmidsin Escherichia coli. These repeat tracts are involved in the etiologiesof Friedreich ataxia, fragile X syndrome, and myotonic dystrophytype 1, respectively. The capacity of these DNA tracts to undergodeletions-expansions was explored with three genetic-biochemicalapproaches including first, the utilization of topoisomerase I and/orDNA gyrase mutants, second, the specific inhibition of DNA gyraseby novobiocin, and third, the genetic removal of the HU protein,thus lowering the negative supercoil density (��). All three strate-gies revealed that higher �� in vivo enhanced the formation ofdeleted repeat sequences. The effectsweremost pronounced for theFriedreich ataxia and the fragile X triplet repeat sequences. Higherlevels of �� stabilize non-B DNA conformations (i.e. triplexes,stickyDNA, flexible andwrithedDNA, slipped structures) at appro-priate repeat tracts; also, numerous prior genetic instability inves-tigations invoke a role for these structures in promoting the slip-page of theDNAcomplementary strands. Thus, we propose that thein vivo modulation of the DNA structure, localized to the repeattracts, is responsible for these behaviors. Presuming that theseinterrelationships are also found in humans, dynamic alterations inthe chromosomal nuclear matrix may modulate the �� of certainDNA regions and, thus, stabilize/destabilize certain non-B confor-mations which regulate the genetic expansions-deletions responsi-ble for the diseases.

Genetic instability of microsatellite sequences have been widelyobserved throughout genomes of all organisms studied (1–3). In themajority of cases this phenomenon occurs without phenotypical conse-quences, but in some circumstances instability (mostly expansions ofthe repeat tracts) results in the development of disease (1, 4, 5). Expan-sions of tri-, tetra- and pentanucleotidemicrosatellites are related to theetiology of more than 20 neurological diseases including myotonic dys-trophy types I and II, fragile X syndrome, Friedreich ataxia, and spino-cerebellar ataxias (1, 4, 5).Studies on the mechanisms of trinucleotide repeat sequence (TRS)2

expansions have revealed that replication, recombination, and repair,probably acting in concert, are responsible for the instabilities of therepetitive tracts (1, 4–7). Several cis elements as well as trans-actingfactors influencing genetic instabilities were discovered (8–11). Thesequences of the repeats, their length, and the presence of polymor-phisms (interruptions) in the repeating tract are among themost impor-tant determinants of the extent of their instability (10, 12–15). In addi-tion, the orientation of the repeats relative to the origin of replication,their distance from the origin, transcription through the repeats, andDNA methylation status are factors (9, 16–21).Expansions of the repeats in affected individuals occur only at a sin-

gle, specific disease locus, suggesting the importance of the particularlocus-specific elements in contrast to the factors causing generalgenome-wide instability (22–24). Studies conducted in different pro-karyotic and eukaryotic model systems support these observations,implicating the importance of DNA itself in the repeat mutagenesisprocesses. Despite the progress in understanding mechanisms ofgenetic instability, several aspects of microsatellite expansions inhuman diseases, such as the exact timing of the expansion event or thetissue specificity of the somatic instabilities, remain to be elucidated.Virtually all models of TRS instability emphasize the capacity of the

sequences to adopt non-B DNA structures (1, 4, 5, 7, 25–27). Hairpins,slipped structures, triplexes, sticky DNA, tetraplexes, and unwoundDNA conformations formed by repetitive sequences can influence var-ious processes of DNAmetabolism (5, 7, 26, 27). Negative superheli-cal density (��) is a dominant factor in the stabilization of mostnon-B DNA structures, including those adopted by tandem repeatedtracts (28–31). Both global (overall) intracellular negative superheli-cal density as well as the transient waves of supercoiling associatedwith transcription influence the formation and stability of non-BDNA structures in vivo (28, 29). Moreover, �� facilitates structuraltransitions observed specifically for TRS associated with expansiondiseases (30, 32). Thus, these associations suggest a close relation-ship between the intracellular level of �� and the instability ofnon-B DNA structure-forming repeats.Herein, we used Escherichia coli strains harboring mutations in

topoisomerase I and/or DNA gyrase genes to analyze the influence of�� in vivo on the instability of GAA�TTC, CGG�CCG and CTG�CAGrepeats. The modulation of the transcription rate through the repeattracts enabled the analysis of the cumulative effect of the global ��

combined with local supercoiling. Both the global intracellular ��

alone as well as the effect of transcription on localized �� had a pro-found destabilizing influence on the TRS tracts. Hence, these studiesprovide further, strong evidence for the role of non-BDNAstructures inthe genetic instability of these repetitive sequences.

MATERIALS AND METHODS

Plasmids and E. coli Strains—The TRS containing plasmids used inthis study (seeTABLEONE) are derivatives of pUC19NotI or pGEM3Zf

* This research was supported by National Institutes of Health Grant ES11347 and grantsfrom the Robert A. Welch Foundation and the Friedreich Ataxia Research Alliance–Seek a Miracle (Muscular Dystrophy Association). The costs of publication of this arti-cle were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

1 To whom correspondence should be addressed: Center for Genome Research, Instituteof Biosciences and Technology, Texas A&M University Health Science Center, 2121 W.Holcombe Blvd. Houston, TX 77030-3303. Tel.: 713-677-7651; Fax: 713-677-7689;E-mail: rwells@ibt.tamhsc.edu.

2 The abbreviations used are: TRS, trinucleotide repeat sequence(s); GFP, green fluores-cent protein; IPTG, isopropyl-�-D-thiogalactopyranoside, IC05, inhibitory concentra-tion 5%.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 45, pp. 37366 –37376, November 11, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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(Promega) and were described in detail (12, 33, 34). The repeat tracts inpRW3546 (12), pRW3691 (34), and pRW3246 (33) are located down-stream of the functional lacZ promoter, thus allowing for the directcontrol of the transcription level by isopropyl-�-D-thiogalactopyrano-side (IPTG) induction and lacIQ repressor inhibition. The lacIQ repres-sor was encoded by the pIQ-kan plasmid (35). All plasmids used hereinwere characterized by restriction mapping (to determine the orienta-tion and lengths of the cloned TRS) and by dideoxy sequencing of bothstrands as described earlier (36) using pUC19- and pGEM-specificprimers (New England Biolabs, Inc.).All plasmidswere purified fromHB101 using standardmaxiprep pro-

cedures (Sigma), and plasmids were subsequently electrophoreticallyseparated overnight on long (30 cm) 1.2% agarose gels. The bands cor-responding to the full-length monomer plasmids (no TRS deletions)were excised without ethidium bromide staining, and DNA was recov-ered by electroelution (37) and purified by phenol/chloroform extrac-tion and ethanol precipitation. The size of the TRS insert and the extentof instability was determined using restriction digestion and polyacryl-amide gel electrophoresis followed by quantitative phosphorimaginganalyses as described below. Preparations containing more than 90% ofthe full-length, undeleted TRS tracts were used in the further experi-ments. E. coli JRY880 was a generous gift from Dr. J. Rouviere-Yaniv.

Determination of the Plasmid Superhelical Density in Vivo—pUC19was isolated fromdifferentE. coli strains grown to anA600 of 0.9, and thetopoisomer distributions were analyzed by agarose gel electrophoresis(1% w/v) in 90 mM Tris borate, 2 mM EDTA, pH 8.0, in the presence of3.5 �M chloroquine. The plasmids were separated on 30-cm-long gels(2–3 V/cm) for 36 h. After electrophoresis, the gels were rinsed withdistilled water, and DNA was stained with ethidium bromide. Thequantitation of the results was performed using FluorChemversion 3.04(Alpha Innotech Corp.). The average negative superhelical density wasobtained as described earlier (38).3

Determination of the Plasmid Copy Number—To evaluate the possi-ble influence of the supercoil-dependent replicative potential of differ-ent E. coli strains, the copy number of pUC19 was determined for eachstrain as described earlier (36, 39, 40). The quantitative analyses of theplasmid and genomic DNAs separated by agarose gels were performedusing FluorChem version 3.04 (Alpha Innotech Corp.).

Preparation of Bacterial Lysates and Fluorometric Quantitation ofGFP Expression—pGFPuv (Clontech) was used as a reporter plasmid tocharacterize the lacZ promoter expression levels in different E. colistrains and under varying transcription and supercoiling conditions.The preparation of the bacterial lysates and quantitation of the GFPlevels were performed precisely as described (41). Fluorescence meas-urements were conducted on a Turner Quantech fluorometer using anNB405 excitation filter and a SC500 emission filter. A standard curvewas made by supplementing GFP-free lysates with known amounts ofrecombinant GFPuv (Clontech), which gave the expected linear rela-tionship. Analyses of the GFPuv expression were performed in E. coliJTT1, RS2, SD7, and KD112 for cells grown in LB medium and in LBsupplemented with 2 mM IPTG. The amount of GFPuv expressed inE. coli grown in the presence of pIQ-kan (encoding the lacIQ repressor)was undetectable in all strains. In the typical conditions for our reculti-vation studies (LBmedium), the GFP expression level was�10-, 6-, and3-fold higher in the KD112, RS2, and SD7 strains, respectively, than inthe parental E. coli JTT1. Hence, we found no direct correlationbetween the in vivo superhelical density in these four strains and theGFP expression level from the lacZ promoter.

Assay for Genetic Instability—The plasmids containing various TRSwere transformed into the appropriate E. coli strains and were grown in10 ml of LB cultures for a number of generations, as described (16, 33).The efficiency of transformation was determined in every experimentby plating an aliquot of the transformation mixture onto the agar platecontaining ampicillin (100 �g/ml); the cultures were always initiatedwith the equivalent of 1000–3000 colony forming units. Importantly, toavoid the growth advantage effect of the cells harboring plasmids withshorter TRSs (33), the cells were always subcultured into the freshmedia at the logarithmic phase (A600 of 0.75–0.9, 10–16 h of growth).Cells were recultivated 3 times in 20-generation intervals, starting eachsubculture with 103–104 cells as an inoculum. An initial population ofthis size allowed the detection of several individual instability eventswhile preventing a potential “bottleneck” effect if the inoculum was toosmall (1–100 cells). The cultures were grown at 37 °C at a shaking rate of250 rpm in LB media supplemented with an appropriate antibiotic(ampicillin at 100 �g/ml, kanamycin at 50 �g/ml). The cells from eachculture were harvested and stored at �20 °C to the end of the experi-ment, and then all plasmid isolations and analyses were performedsimultaneously.In the experiments with inhibited transcription, the plasmid pIQ-kan

was first transformed into the appropriate E. coli strain, and the pIQ-kan-containing competent cells were prepared. When required, 2 mM

IPTGwas included in the growthmedium to induce transcription fromthe lacZ promoter.In the case of studies performed in the presence of novobiocin

(Sigma) the drug was added, during all stages of the experiment begin-ning with the preparation of competent cells, at the concentrationsequal to the strain-specific inhibitory concentration of 5% (IC05 � 4 and16 �M for SD7 and HB101 E. coli, respectively). Novobiocin inhibits theassembly of the active DNA gyrase (42, 43) and at the concentrationsused in this work has no influence on cell growth. IC05 values weredetermined experimentally for eachE. coli strain used. In contrast to thequinolones, novobiocin (as well as other coumarins) does not lead to theformation of DNA double-strand breaks that may influence the resultsof the TRS instability studies (44–47).

Simultaneous Analyses of the Plasmid Superhelical Density and TRSInstability from Single Colonies—Studies were conducted with E. coliJRY880, which is a deletion mutant of the hupA gene encoding the �

subunit of the HU protein, to evaluate its potential role in modulatingthe in vivo supercoil density of pRW3546 and thereby influencing theTRS genetic instability. The supercoil density and the GAA�TTC insta-bility weremonitored in single colonies since E. coli JRY880 is known torapidly acquire suppressor mutations resulting in an increase of theintracellular �� (48). Competent bacterial cells were co-transformedwith pRW3546 which contained (GAA�TTC)150 and with pACYC184.pACYC184 was used as a reference plasmid to monitor the ��. Thus,harboring both plasmids in the same cells allowed the simultaneousanalysis of the repeat instability at a precisely determined ��. Aftertransformation cells were plated on an agar plate containing ampicillin(100 �g/ml) for selection for pRW3546, tetracycline (20 �g/ml) forselection for pACYC184, and chloramphenicol (12.5 �g/ml) for selec-tion for E. coli JRY880. Single colonies were subsequently grown in liq-uid LB medium supplemented with the same concentrations of theantibiotics until the cultures reached an A600 of 0.9. Plasmids were iso-lated using standard techniques (Wizard Plus Miniprep DNA Purifica-tion System, Promega) and were subjected to restriction digestion. Aportion of the isolated DNA (containing a mixture of both pRW3546and pACYC184) was cleaved by a combination of five restriction endo-nucleases (AatII, AflIII, DraIII, PvuI, and NdeI) to completely remove3 P. Staczek, M. Napierala, A. Jaworski, and A. Bacolla, unpublished data.

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any supercoiled pRW3546 while leaving pACYC184 intact. This stepwas necessary since the presence of undigested pRW3546 could inter-ferewith the analyses of the pACYC184 topoisomer distributions on thechloroquine-containing agarose gels. After phenol/chloroform extrac-tion and ethanol precipitation, the topoisomer distributions ofpACYC184 were analyzed by agarose gel electrophoresis (1% w/v) in 90mM Tris borate, 2 mM EDTA, pH 8.0, in the presence of 3.5 �M chloro-quine as described above. In parallel, the remaining fraction of the plas-mid preparation was cleaved using BssHII/HindIII restriction endo-nucleases (New England Biolabs) to analyze the stability of the(GAA�TTC)150 tract in pRW3546. The repeat-containing inserts wereseparated on 5.5% polyacrylamide gels and analyzed as described below.

Analysis of Lengths of Genetic Instability Products—Plasmids wereisolated as described above and were subjected to restriction digestion.BssHII/HindIII restriction endonucleases (New England Biolabs) wereutilized to analyze the stability of the (GAA�TTC)150 tract in pRW3546,and EcoRI/BamHI were used in the case of pRW3691 and pRW3246.The repeat-containing inserts were radioactively labeled using with theKlenow fragment of E. coli DNA polymerase I and [�-32P]dATP andseparated on 5.5–7% polyacrylamide gels in TAE buffer (40 mM Trisacetate, 1 mM EDTA, pH 8.0). Results were analyzed using Storm 820and ImageQuant software (Amersham Biosciences). The statisticalanalyses were performed using SigmaStat Version 2.03.

RESULTS

Increased Negative Superhelical Density Enhances the Instability ofGAA�TTC and CGG�CCG Repeats but Not CTG�CAG—Four isogenicE. coli strains (parental (JTT1) aswell as cells harboringmutations in thetopoisomerase I and/orDNAgyrase genes (RS2, SD7, andKD112))weretransformed with plasmids containing (GAA�TTC)150, (CGG�CCG)73,or (CTG�CAG)98 (TABLE ONE) and were subjected to recultivationexperiments. The recultivation assay is a powerfulmethod for analyzingthe genetic instability of repetitive sequences and has been widely usedpreviously (12, 16, 18, 19, 33). The data from the recultivation experi-ments with pRW3546 containing (GAA�TTC)150 in the four strains areshown in Fig. 1. The strains used in this study differ in their intracellularlevel of negative supercoil density, which in turn will modulate the pro-pensity of repetitive DNA sequences to adopt any non-B DNA confor-mations with under-wound primary helices (6, 7, 49). Analysis of thetopoisomer distributions of the pUC19 reference plasmid on chloro-quine-containing agarose gels demonstrated that plasmids isolated

from the parental JTT1 and RS2 (topA mutation) strains are highlysupercoiled, whereas DNAs from SD7 and KD112 strains (both DNAgyrasemutants) aremuchmore relaxed (Fig. 2). Calculation of the aver-age �� revealed a significant difference between the plasmids grown inJTT1 (� � �0.057) or RS2 (� � �0.058) and SD7 (� � �0.049) orKD112 (� � �0.052).Analysis of the instability of the (GAA�TTC)150 tract in E. coli RS2

revealed that the full-length insert was entirely deleted from pRW3546,and only shorterDNA inserts (�150 repeats) could be detected after the3rd recultivation (Fig. 1). Similar results were obtained in the parentalJTT1 strain, confirming that the (GAA�TTC)150 tract is highly unstablegenetically when subjected to high superhelical tension. However, thestability of the TRS was much increased in the SD7 and KD112 strains,which maintained a lower level of negative superhelical density. Evenafter the 3rd recultivation, a substantial amount (�10–25%) of the plas-mids still harbored the full-length (GAA�TTC)150 repeats. Similarresults were obtained in the case of pRW3691 containing the(CGG�CCG)73 repeats (Fig. 3A), where the effect of �� was significantalthough not as pronounced as in the case of pRW3546 containing the(GAA�TTC)150 (Fig. 3A). No full-length fragment containing the TRSremained after the 3rd subculture step in E. coli JTT1 as well as in RS2;however, 5–8%of the undeleted (CGG�CCG)73 insertswere still presentafter identical cultivations in SD7 and KD112. As observed previously(12, 50), the (GAA�TTC)150 insert and especially the (CGG�CCG)73 tractare very unstable and difficult to maintain due to their propensity todelete in E. coli. No difference in the average negative superhelical den-sity between the wild type (JTT1) and the topoisomerase Imutant (RS2)of E. coli was detected. Indeed, previous reports demonstrated anincrease in superhelical density in chromosomal DNA and plasmidsexpressing the tetracycline resistance gene in the strains harboringtopoisomerase I mutation (51–54), whereas plasmids lacking the tetgene (such as pUC19 derivatives) showed normal supercoiled densities(52, 54).Interpretation of these results could be seriously complicated by the

differences in plasmid metabolism in the various E. coli strains evenafter the conditions of bacterial growth were standardized, and the cul-tures never reached stationary phase (“Materials and Methods”). Themain concern is the replicative propensity (copy number) of the plas-mids in the different E. coli strains. Results of the recultivation experi-ments can be properly interpreted if both the growth of the bacterialpopulation and the plasmid copy number are monitored. Therefore, we

TABLE ONE

Plasmids and E. coli strains used in this studyThe terms “orientation I” and “orientation II” refer to the orientation of the TRS relative to the origin of replication; for example, for the plasmids containing(GAA�TTC)n tracts in orientation I, theGAA repeat is in the leading strand template, whereas for the plasmids harboring (GAA�TTC)n tracts in orientation II, theGAA repeat is in the lagging strand template (16). NA, not applicable.

Plasmids Repeating sequence Orientation Derivative of Ref.

pRW3546 (GAA�TTC)150 II pGEM3Zf 12pRW3691 (CGG�CCG)73 I pUC19NotI 34pRW3246 (CTG�CAG)98 I pUC19 33pIQ-kan NA NA NA 35pGFPuv NA NA pUC19 NA

Strains Relevant genotype Source

JTT1 ((gal-25, ��, pyrF287, fnr-1, rpsL195(strR), iclR7(Const). trpR72(Am)) E. coli Genetic Stock CenterRS2 JTT1 (topA10) E. coli Genetic Stock CenterSD7 JTT1 (topA10, gyrB226) E. coli Genetic Stock CenterKD112 JTT1 (gyrB226) 68JRY880 JTT1 hupA�Cm 68HB101 (mcrB, mmr, hsdS20 (rB�, mB

�), recA1, supE44, ara14, galK2, lacY1, proA2, rplS20 (SmR), xyl5, ��, leuB6, mtl-1) E. coli Genetic Stock Center

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analyzed the copy numbers of pGFPuv in all four strains (Fig. 4). SD7and KD112 (the “relaxed DNA strains”) maintained 2.2 and 1.4 times,respectively, more copies of the plasmid than JTT1 and RS2, which hadessentially the same amount of plasmidmolecules. However, the formerstrains demonstrated a much higher stability of the TRS inserts. Thus,regardless of the higher copy numbers, which represent a larger numberof plasmid replication events, the (CGG�CCG)73 and (GAA�TTC)150tracts were substantially more stable in the SD7 and KD112 strains. Ifthe replication properties of the TRS-containing plasmids were similarin all four strains, the supercoil-dependent difference in the instability of

the repeat tracts might be even more pronounced than shown in Figs. 1and 3 since the gyrase-impaired strains (SD7 and KD112) demonstratedthe greatest TRS stability but had the highest number of plasmid repli-cation events per cell (Fig. 4).Surprisingly, the plasmid containing the (CTG�CAG)98 tract, the

most stable of the three inserts tested, was not influenced by the level ofintracellular �� (Fig. 3B). The overall stability of the (CTG�CAG)98tract is much higher in all strains than found for pRW3546 andpRW3691. In fact, after the 3rd subculture step, at least 50–70% of theplasmid preparations contained the full-length (CTG�CAG)98 insert(Fig. 3B). The reason for the weaker effect of negative superhelical den-sity on the genetic instability of the CTG�CAG repeats compared withtheGAA�TTCandCGG�CCG tractsmay be related to the differences inthe stabilities of the slipped hairpin structures formed by CTG�CAGrepeats (6, 16, 18, 49, 55–58) versus the triplex structures (adopted byGAA�TTC tracts) (30, 59–62) and the tetraplex and/or flexible andwrithed conformations (demonstrated for CGG�CCG repeats) (34, 63)under the pressure of negative superhelical density (see “Discussion”).

Transcription in Concert with High Negative Supercoil Density Facil-itates TRS Instability—The influence of transcription through therepeat tracts on their genetic instability under the various �� con-ditions was determined. Plasmids harboring (GAA�TTC)150,(CGG�CCG)73, or (CTG�CAG)98 were each transformed into E. coliJTT1, RS2, SD7, and KD112, and the cells were recultivated under threedifferent conditions; they are (i) transcription inhibition by expressionof the lacIQ repressor from the pIQ-kan plasmid, (ii) background level oftranscription (growth in LB medium with no inhibition or stimulationof transcription), and (iii) induced transcription from the lacZ promoterby 2 mM IPTG. The level of expression from the lacZ promoter wasanalyzed under all experimental conditions and in the fourE. coli strainsusing the pGFPuv plasmid, which expressed the GFPuv protein (“Mate-rials and Methods”).The results obtained with plasmids containing the (GAA�TTC)150

and (CGG�CCG)73 repeats showed that the background transcription(LB medium) from the lacZ promoter located upstream of the TRSsignificantly destabilized the repeat tracts in all E. coli strains analyzed(compare Figs. 1 and 5 along with Fig. 3A). This effect is in agreement

FIGURE 1. Increased negative superhelical density in vivo enhances the geneticinstability of the transcribed (GAA�TTC)150 triplet repeat tract. A, E. coli strains JTT1(parental strain), RS2 (JTT1: topA10), SD7 (JTT1: topA10, gyrB226), and KD112 (JTT1:gyrB226) were transformed with the purified non-deleted monomer of pRW3546 andcultured as described under “Materials and Methods.” The numbers of the subculturesteps are indicated above the gel lanes. M designates the 1-kilobase DNA Ladder (Invitro-gen) used as size marker. The arrowhead indicates the full-length DNA fragment contain-ing (GAA�TTC)150. Analysis of the TRS insert size was performed using BssHII/HindIIIdigestion followed by radioactive labeling of recessed ends and electrophoresis in 5.5%polyacrylamide gels as described under “Materials and Methods.” B, results of quantita-tion of the amount of undeleted TRS insert (% full-length fragment). Data from threeindependent experiments were quantitated using phosphorimaging, and the relativestability of the TRS tract is presented as the percentage of the full-length, undeletedstarting-length fragment remaining after subsequent recultivations. For control pur-poses, radioactively labeled DNA fragments were excised from a representative numberof polyacrylamide gel lanes, and the level of radioactivity was measured using a liquidscintillation counter (Beckman Instruments). The level of transcription from the lacZpromoter was neither inhibited (by lacIQ repressor) nor stimulated (by IPTG) through thecourse of the experiment. topol, topoisomerase; gyr., gyrase.

FIGURE 2. Determination of the average superhelical density of pUC19 grown to thelate logarithmic phase in the E. coli parental and topoisomerase/gyrase (gyr. topol)mutants. Bacterial cultures were grown to an A600 � 0.9, chilled quickly in ice/waterbath, and centrifuged at 4500 � g at 4 °C for 20 min. DNA was isolated using standardmaxiprep procedures (Sigma). Topoisomers were separated in a 1% agarose gel in thepresence of 3.5 �M chloroquine. 0 indicates nicked DNA. The average superhelical den-sity (��av) depicted underneath each electrophoretic lane was calculated using themethod described (38).

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with previous studies conducted with the CTG�CAG and GTC�GACrepeats (18, 19, 58). In the case of pRW3691, transcription stimulatedextensive TRS deletions; therefore, no full-length (CGG�CCG)73 frag-ment could be detected after the third subculture step in the parentalJTT1 and RS2 strains (Fig. 3A and data not shown). On the other hand,more than 7% of the undeleted TRS insert was detected after propaga-tion of this plasmid in the same E. coli strains but in the absence oftranscription from the lacZ promoter (i.e. in the presence of the lacIQ

repressor, Fig. 3A). A similar destabilizing effect of active transcriptionthrough the repeats was also observed for the plasmid containing the(GAA�TTC)150 insert in all strains (Figs. 1 and 5). Further stimulation oftranscription by 2 mM IPTG had no significant effect on the extent ofgenetic instabilities of the repeating tracts (data not shown).

Interestingly, the influence of transcription on the genetic instabilityof the TRS was more apparent in E. coli RS2 and JTT1 than in the SD7and KD112 strains (Figs. 1, 3, and 5). This difference is particularlyevident when the amount of undeleted progenitor (GAA�TTC)150 and(CGG�CCG)73 inserts are compared after the second subculture step; acomparison after the third growth step is less appropriate due to thecomplete loss of the full-length inserts in the experiments with activetranscription (Figs. 1 and 3). In the case of the plasmid containing the(GAA�TTC)150 repeats, only �2% of the progenitor insert was detectedin E. coli RS2 (high ��) after the 2nd round of growth in LB medium(Fig. 1). Inhibition of transcription increased this amount�10-fold (Fig.5). In contrast, identical experiments performed in E. coli SD7 (low��)resulted in�2.5-fold increase in TRS instability from the transcribed tothe non-transcribed conditions. For the plasmid harboring the(CGG�CCG)73 insert, the corresponding values were 3 versus 12% (inE. coli RS2) and 12 versus 17% (in E. coli SD7). These differences werestatistically significant (p � 0.05). Perhaps, the initial high level of neg-ative superhelical density in E. coli RS2 and JTT1, influenced by localchanges of DNA topology associated with transcription, further accel-erated TRS instability, whereas the transcription through the relaxedDNA template in E. coli SD7 and KD112 had a significantly less delete-rious effect on the stability of the repeats. These results show a stronglink between transcription and global superhelical density and theirsynergistic effect on the TRS instability.Hence, we conclude that transcription through the repeats strongly

induces the instability of the CGG�CCG and GAA�TTC repeats. Thedominant effect of transcription might mask the actual impact of thenegative superhelical density changes. To evaluate the influence of glo-bal �� on the TRS instability unbiased by changes in DNA topologyassociated with RNA synthesis, we conducted recultivation experi-ments in the presence of the lacIQ repressor. A comparison of the resultsbetween the strains, which differed in their average global ��, is shownin Figs. 3 and 5. Importantly, in the absence of transcription, the insta-bility of the TRS was greatly enhanced by the increase of the global ��.After the 3rd subculture step, the amount of the full-length(CGG�CCG)73 as well as the (GAA�TTC)150 fragments was �2 timesgreater in the SD7 and KD112 strains when compared with the E. coliRS2 and JTT1 strains (Figs. 3 and 5). Thus, regardless of the transcrip-

FIGURE 3. Influence of transcription and superhelical density on the instability of(CGG�CCG)73 (pRW3691) (A) and (CTG�CAG)98 (pRW3246) triplet repeats (B). Resultsof the quantitation are presented. The experiments and data analyses were conductedas described under “Materials and Methods.” � indicates active transcription from thelacZ promoter (LB medium), whereas � designates the inhibition of transcriptionthrough the repeat tract by overexpression of the lacIQ repressor from the separatereplicon (pIQ-kan). Note the difference in the scale of the y axes between A and B reflect-ing the general level of instability of the CGG�CCG versus the CTG�CAG tracts of similarlength. topol, topoisomerase; gyr., gyrase.

FIGURE 4. Influence of different superhelical density on plasmid copy number.Determination of the average copy number of the pGFPuv (lacking the TRS insert) wasperformed in four isogenic E. coli strains: JTT1 (parental strain), RS2 (JTT1: topA10), SD7(JTT1: topA10, gyrB226), and KD112 (JTT1: gyrB226). Plasmid copy numbers were ana-lyzed as described under “Materials and Methods” and presented relative to the copynumber determined for the JTT1 parental strain. topol, topoisomerase; gyr., gyrase.

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tional state of the plasmid, the global intracellular �� is an importantfactor in modulating TRS instability.In summary, these data show that global superhelical tension per se or

in concertwith transcription has a profound effect onCGG�CCGaswellas GAA�TTC instability. Also, the stability of pRW3246 containing the(CTG�CAG)98 tract was not significantly affected by these factors (datanot shown).

Effect of DNA Gyrase Inhibitor Novobiocin on TRS Instability—DNAgyrase is responsible for introducing negative supercoils into DNA (64,65). Because we demonstrated above that lower levels of superhelicaldensity facilitate more stable propagation of plasmids harboring TRStracts, we analyzed the effect of the specific inhibition of this enzyme onTRS instability. Novobiocin, a coumarin antibiotic, inhibits the assem-bly of the active gyrase (42, 43) and, in contrast to the quinolones, doesnot lead to the formation of DNA double-strand breaks (44–47).Because an increase in the global �� stimulates TRS instability, novo-biocin should stabilize the repeats through the inhibition of its primarytarget DNA gyrase.We analyzed the effect of novobiocin on the instability of the repeats

in E. coli JTT1, RS2, and SD7 as well as in the HB101 and DH5� strainsroutinely used for cloning and the stable propagation of plasmids con-taining the TRS tracts (32, 36, 66). Fig. 6 shows the influence of novo-

biocin on the instability of the (CGG�CCG)73 repeat and on the globalDNA �� in E. coli HB101 and SD7. Propagation of the plasmids con-taining (GAA�TTC)150 or (CGG�CCG)73 tracts in medium supple-mented with 16 �M novobiocin (which is the IC05 for E. coli HB101)resulted in a statistically significant increase of the TRS stability (Fig. 6, AandB, anddatanot shown). Furthermore, this concentrationof novobiocinreduced the intracellular��bymore than5% (from�0.056 to�0.054; Fig.6C, left panel) but had negligible consequences for the viability and growthof the cells. Similar results were obtained using the IC05 concentration ofnovobiocin for E. coli JTT1, RS2, and DH5� (data not shown).

FIGURE 5. Synergistic effects of inhibition of transcription through the repeats andrelaxation of the plasmid in vivo on the stability of (GAA�TTC)150 triplet repeattracts. A, inhibition of transcription through the repeat tract was attained by overexpres-sion of the lacIQ repressor from the separate replicon (pIQ-kan). The arrowhead indicatesthe full-length fragment DNA containing (GAA�TTC)150. The restriction fragment derivedfrom the pIQ-kan plasmid expressing the lacIQ repressor is indicated. Experiments wereconducted as described under “Materials and Methods” and the legend to Fig. 1. B, theresults of quantitation of the amount of undeleted TRS insert (% full-length fragment).topol, topoisomerase; gyr., gyrase.

FIGURE 6. Inhibition of DNA gyrase by novobiocin stabilizes the (CGG�CCG)73 tract.A, comparison of the (CGG�CCG)73 repeat instability in pRW3691 grown in E. coli HB101and SD7 strains in the absence and presence of IC05 concentrations of novobiocin (nov).The numbers of subculture steps are indicated above the gel lanes; M indicates the1-kilobase DNA ladder; the arrowhead indicates the full-length DNA fragment contain-ing (CGG�CCG)73. B, quantitation of the amount of the undeleted (CGG�CCG)73 insertfragment. C, influence of novobiocin on the in vivo superhelical density of pUC19 cul-tured in E. coli HB101 and SD7. The calculated average superhelical densities (��av) areshown on the bottom of the agarose gels. 0 indicates nicked DNA.

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Alternatively, the instabilities of neither CGG�CCG nor GAA�TTCrepeats were affected by the IC05 concentration of novobiocin (4 �M) inE. coli SD7. Likewise, this concentration of the drug did not change thetopoisomer distributions in the plasmid isolated fromSD7 cells (Fig. 6C,right panel). This result was expected since the product of the gyrB gene,which is targeted by novobiocin, is mutated in E. coli SD7. Even increas-ing the novobiocin concentration to 40 �M (inhibitory concentration of80%, IC80) did not influence the TRS instability but caused a significantlengthening of the doubling time of the cells (by �35%). This effect ispresumably due to the inhibition of topoisomerase IV, a secondary targetfor novobiocin (47). Such high concentrations of novobiocin were indeedshown to inhibit the activity of this enzyme, which is involved primarily indecatenation of DNA and removal of positive supercoils in front of amov-ing replication fork. However, it seems unlikely that inhibition of topoi-somerase IV could have influenced our results on TRS instability. Hence,blocking the activity of DNA gyrase by novobiocin decreased the intracel-lular ��, which then increased the stability of the repeats.

DNA Relaxation Associated with HU Deficiency Increases TRSStability—Thebinding of architectural proteins toDNA is an importantfactor in modulating DNA topology in vivo (29, 67). The histone-likeHU protein contributes to the plasmid superhelical density in E. coli intwo distinct ways; first, it constrains DNA through the physical bindingprocess, and second, it interferes with topoisomerase I and DNA gyrasereactions (29, 48, 68, 69). The removal of HU protein results in partialrelaxation of chromosomal and plasmid DNA (48, 68). To evaluate theinfluence of changes in the DNA topology caused by HU deficiency onTRS instability, E. coli JRY880 lacking the hupA gene transformed withpRW3546 containing (GAA�TTC)150 were used. The hupA geneencodes the � subunit of the HU protein, and its deletion dramaticallydecreases the level of HU in the cells to 5–10% of the wild type amount(68). Because the HU mutants readily acquire suppressor mutations,most likely in theDNAgyrase gene, leading to an increase in the average�� (48), population studies would reflect the effect of both low �� (inthe hupA mutants) and high �� (in the hupA mutants containing anadditional suppressor mutation). Therefore, single colony analysesinstead of the population assays (recultivation assays) were performed(“Materials andMethods”). The JRY880 cells were co-transformed withpRW3546 and a pACYC184 reporter plasmid followed by the simulta-neous analysis of the �� (as monitored with pACYC184 topoisomerdistributions, Fig. 7A) andTRS instability (asmonitored by the deletionsof the GAA�TTC repeat tract in pRW3546, Fig. 7B) using DNA isolatedfrom single colonies as described under “Materials andMethods.” Theseanalyses allow for a direct correlation between �� and TRS instability.Plasmids were isolated from a total of �60 colonies of E. coli JRY880;30%of the coloniesmaintained a low�� (see Fig. 7A, lane 1, for a typicalexample), whereas the remaining 70% of the plasmids demonstratedhigh �� (JRY880 revertants, Fig. 7A, lane 2).Fig. 7B shows the results of the GAA�TTC instability analyses per-

formed on plasmids isolated from 20 colonies of E. coli JRY880; 10 col-onies demonstrated low supercoil densities (left side), whereas the other10 colonies showed high levels of �� (right side), as determined by theanalyses of the pACYC184 reference plasmid on the chloroquine-con-taining agarose gels. The average amount of the full-length progenitorinsert was 14 and 2% for cells maintaining low and high levels of ��,respectively. Hence, the ��, rather than the HU protein per se, was thedominant factor for determining the instability of the GAA�TTC tract,since the suppressor gyrasemutation increased the�� and destabilizedthe repeats despite the absence of the HU protein.The global�� in E. coli JRY880 also had an influence on the extent of

deletions. The average size of the GAA�TTC tract in the plasmids iso-

lated from low �� colonies was 28 repeats longer than for the plasmidspropagated in the conditions of high�� (Fig. 7B). Thus, a higher level of�� facilitates the TRS instability by promoting large deletions, span-ning several repeat units. This behavior may be related to the propensi-ties of the GAA�TTC sequences to adopt more stable triplex structuresunder higher superhelical tension conditions (Refs. 30, 59, and 62; see“Discussion”).In summary, lowering the superhelical density by eliminating one of

the most abundant proteins involved in constraining negative super-coils in E. coli reduces the instability of the GAA�TTC repetitive tracts.However, this effect is caused by the change in �� rather than by theabsence of the HU protein. These results provide further evidence forthe strong association between �� and TRS instability.

DISCUSSION

Theroleofnegative supercoildensity in thegenetic instabilitiesof certaindisease-associated repeating sequenceshasbeenpostulated (58, 70, 71), butthe studies described herein present the first proof of this relationship, astransduced by non-B DNA conformations. Statistical mechanical calcula-tions of DNA molecules showed that CGG�CCG and CTG�CAG repeatsbegin to writhe at lesser free energy of supercoiling than random, non-repeating DNA (34, 70). Hence, these TRSs were proposed to act as sinksfor the accumulation of negative superhelical density. In addition, tran-scription is also accompanied by the accumulation of positive supercoils infront of the RNA polymerase complex and negative supercoils behind it(72–74).Thesewavesof supercoiling associatedwith transcription throughthe TRS were postulated to influence TRS instability (58).The effects of the changes in DNA topology on the genetic instabili-

ties of GAA�TTC, CGG�CCG, andCTG�CAG repeats were determined.Three different strategies were utilized: first, we showed that high ��

facilitates repeat instabilities by using topoisomerase I and/or DNAgyrase mutants of E. coli where various levels of intracellular �� weremaintained. This effect was even more pronounced when transcriptionthroughout the TRS was blocked. Second, specific inhibition of DNAgyrase using novobiocin, which resulted in the relaxation of the DNA,increased the stability of the TRS tracts in the plasmids. Third, we low-ered the �� of the target plasmid by eliminating the HU protein, whichis involved in constraining the negative supercoiling in E. coli as well asin the regulation of the topoisomerase I and DNA gyrase activity. Thisresulted in a significant stabilization of the GAA�TTC repeats in E. colihupAmutants. Thus, all three strategies revealed that an increase in thenegative superhelical density promotes the instability of triplet repeats.What is the mechanism of the ��-induced instability of the TRS

tracts? Negative supercoiling is a very powerful factor that enables seg-ments of the DNA double helix to acquire non-B DNA conformations(6, 28, 29, 75). The amount and/or the stabilities of these structuresincrease with increasing levels of negative supercoiling (Fig. 8). Cruci-forms, Z-DNA, triplexes, and sticky DNA conformations are stabilizedin the conditions of high�� (28–30, 75–79).Only certain types ofDNAsequences (e.g. direct or inverted repeats, polypurine�polypyrimidinetracts, G-rich sequences) are capable of adopting non-B DNA struc-tures (6, 7, 29, 41, 71, 80, 81). Also, TRS tracts, including GAA�TTC,CGG�CCG, and CTG�CAG repeats, have been demonstrated to adoptstable non-B DNA structures (6, 7, 25, 26, 56, 82).Hence, we postulate that �� plays an important role in the stimula-

tion of the genetic instability of the TRS tracts by favoring formation ofthe non-B DNA structures. The extent of the repeat instability can beinfluenced by both the level of �� and the different propensities of theTRS tracts to adopt the unusual structures. In addition to the global��,local or transient supercoiling, which is induced (for example by tran-

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scription), accelerates the formation of non-B DNA structures at theTRS (Fig. 8). It is likely that these changes in the �� affect TRS tractsmore than other DNA regions since these sequences (at leastCGG�CCG and CTG�CAG repeats) can locally accumulate a high levelof negative supercoiling (34, 70).The formation of stable non-BDNA structures is a critical element in

practically all models of microsatellite instability (1, 5). Supercoil-de-pendent DNA conformations can arrest replication fork progression,leading to nicks and/or double-strand break formation (83–85). Thesestructures can also be recognized and cleaved by repair proteins (Fig. 8,left site) (86–88). Moreover, stable structures existing on the leading orlagging strands during DNA replication can be bypassed by DNA poly-merases (Fig. 8, right site) (16). Hence, all the mechanisms describedabove would lead to the instability of the TRS tracts.The effect of the changes in the intracellular ��, regardless of the

methods used to promote them, was different depending on the

sequence studied. The (GAA�TTC)150 and the (CGG�CCG)73 tractswere sensitive to the changes in the level of ��. On the other hand, thegenetic stability of the (CTG�CAG)98 tract was unaffected by both theincrease of �� and the stimulation of transcription. These variations inthe extent of the �� influence on TRS instability may arise from thedifferent structural properties of the repeat tracts. Relatively short, singletracts of the (GAA�TTC)9–23 repeats can adopt an intramolecular triplexconformation, and its stability greatly depends on the superhelical density(30). At the high ��, tracts containing only 42 GAA�TTC repeats adopt avery stable bi-triplex structure (30). The CTG�CAG as well as CGG�CCGrepeats can adopt hairpin/cruciform conformations (slipped structures)(25, 26, 56, 82). The intrinsic thermodynamic stability of the CGGhairpinsis much greater than CTG and especially CAG hairpins (for the structurescomposed of identical number of repeat units), whichmay elicit the differ-ences in the �� dependent instability between the CGG�CCG andCTG�CAG sequences (82, 89). Additionally, the CGG hairpins can be fur-

FIGURE 7. DNA relaxation caused by HU proteindeficiency increases the stability of (GAA�TTC)150

triplet repeats. A, determination of the averagesuperhelical density of pACYC184 isolated fromsingle colonies of the JRY880 cells (lane 1) as wellas the JRY880 mutants containing an additionalsuppressor mutation, resulting in an increase ofthe intracellular �� (lane 2). Topoisomers wereseparated in a 1% agarose gel in the presence of3.5 �M chloroquine as described in the legend toFig. 2. The DNA fragments resulting from therestriction digestion of the pRW3546 with AatII,AflIII, DraIII, PvuI, and NdeI are indicated at the bot-tom of the gel. The average �� and the size of theGAA�TTC tracts were simultaneously analyzed.B, analysis of the genetic instability of the(GAA�TTC)150 triplet repeat tract in 10 JRY880 col-onies maintaining a low �� (left side) and a high�� (right side). The single colony analyses wereperformed as described under “Materials andMethods.” M designates the 1-kilobase DNA Lad-der (Invitrogen) used as size marker. The arrow-heads indicate the full-length DNA containing(GAA�TTC)150 and the approximate gel migrationof the insert lacking the entire repeat tract(0 repeats). Analysis of the TRS insert size was per-formed using BssHII/HindIII digestion followed byradioactive labeling of the recessed ends and elec-trophoresis of the fragments in 5.5% polyacryl-amide gels as described under “Materials andMethods.”

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ther stabilized by Hoogsteen interactions between the G residues in the“folded back” tetraplex structures (63, 90).In summary, we postulate that the formation of stable non-B DNA

structures at the TRS tracts driven by the energy of supercoiling stim-ulates the genetic instability of these sequences. Thus, the results pre-sented in this study are yet another example of the DNA structure-induced DNA mutagenesis.

Biological Significance—Expansions of microsatellites can reachthousands of repeats in some extreme cases (as many as 11,000 tet-

ranucleotide repeats, more than 40 kbp of DNA in DM2) (91). Howthese sequences expand to such a magnitude within a relative smallnumber of cell divisions is still unknown. What is responsible for thedifferences in instability at the various stages of development? Whydo repeats at only one particular locus expand, whereas identicalsequences of similar length located on different chromosomes donot? What determines the tissue specificity of the somatic instabilityevents? These and a number of other questions related to microsat-ellite instabilities remain unsolved (1, 5). We propose that the topol-

FIGURE 8. Model for the role of DNA topology in the instability of repetitive sequences. The instability is influenced by global and/or local (transcription driven) changes in DNAsuperhelical tension. Unusual DNA structures formed under the influence of negative supercoiling are crucial elements in the cascade of events leading to the repeat instabilities.Different types of under-wound non-B DNA structures (triplexes, tetraplexes, slipped structures, or hairpins) are shown as examples, since various TRS and other repetitive tracts mayadopt different conformations. These non-B DNA conformations may either serve as the instigators of the double-strand break (due to polymerase pausing, for example; see“Discussion”) or may be bypassed by polymerases during DNA synthesis. Both types of events will lead to the TRS instability.

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ogy of the chromatin at a particular site might be a predisposingfactor.Chromosomal domains (loop domains) are the basic structural and

topological units in eukaryotic chromatin and have been detected inmany organisms, including humans (92–98). These loops range in sizefrom 5 to 200 kbp with an average size of 80–100 kbp for somatic cellsand �30 kbp for male gametes (92–94, 99). The DNA in these domainsis attached to the nuclear matrix, or scaffold, to form topologicallyclosed segments (similar to the closed circular plasmids (TABLEONE))that have a deficit in linking numbers (superhelical turns) (98, 100, 101).Domain boundaries are defined by specific DNA regions, scaffold/ma-trix attachment regions (S/MAR) (97, 98). The sizes and organization ofthese chromosomal loops can undergo dramatic changes during variousstages of development and cell differentiation upon induction of geneexpression and in different tissues. These changes in the size and orga-nization of the chromosomal loops may modulate the unconstrainedsuperhelical density of a TRS tract. Consequently, these differences in�� may result in very distinct patterns of locus-specific genetic insta-bility observed in the repeat expansion disorders and, hence, account forsomatic, tissue-specific TRS instabilities.The analysis of topoisomerase/gyrase mutants and the use of specific

inhibitors of the enzymes involved in regulating DNA topology in vivotogether with reliable methods of monitoring the changes in DNAsupercoilingmade E. coli an appropriatemodel to study the influence ofDNA topology on genome instability. Parallel assays in eukaryotic cellsremain to be developed to conduct similar studies in mammaliansystems.

Acknowledgment—We thank Bozenna Jaworska for technical assistance.

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Marek Napierala, Albino Bacolla and Robert D. Wellsof Triplet Repeat Sequences

Enhances the Genetic Instabilityin VivoIncreased Negative Superhelical Density

doi: 10.1074/jbc.M508065200 originally published online September 13, 20052005, 280:37366-37376.J. Biol. Chem. 

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