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Originally published In Press as doi:10.1074/jbc.M309595200 on November 18, 2003

J. Biol. Chem., Vol. 279, Issue 8, 6434-6443, February 20, 2004
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Sticky DNA Formation in Vivo Alters the Plasmid Dimer/Monomer Ratio*

Alexandre A. Vetcher{ddagger} and Robert D. Wells§

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

Received for publication, August 28, 2003 , and in revised form, November 17, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Our discovery that plasmids containing the Friedreich's ataxia (FRDA) expanded GAA·TTC sequence, which forms sticky DNA, are prone to form dimers compared with monomers in vivo is the basis of an intracellular assay in Escherichia coli for this unusual DNA conformation. Sticky DNA is a single long GAA·GAA·TTC triplex formed in plasmids harboring a pair of long GAA·TTC repeat tracts in the direct repeat orientation. This requirement is fulfilled by either plasmid dimers of DNAs with a single trinucleotide repeat sequence tract or by monomeric DNAs containing a pair of direct repeat GAA·TTC sequences. DNAs harboring a single GAA·TTC repeat are unable to form this type of triplex conformation. An excellent correlation was observed between the ability of a plasmid to adopt the sticky triplex conformation as assayed in vitro and its propensity to form plasmid dimers relative to monomers in vivo. The variables measured that strongly influenced these measurements are as follows: length of the GAA·TTC insert; the extent of periodic interruptions within the repeat sequence; the orientation of the repeat inserts; and the in vivo negative supercoil density. Nitrogen mustard cross-linking studies on a family of GAA·TTC-containing plasmids showed the presence of sticky DNA in vivo and, thus, serves as an important bridge between the in vitro and in vivo determinations. Biochemical genetic studies on FRDA containing DNAs grown in recA or nucleotide excision repair or ruv-deficient cells showed that the in vivo properties of sticky DNA play an important role in the monomer-dimer-sticky DNA intracellular intercon-versions. Thus, the sticky DNA triplex exists and functions in living cells, strengthening the likelihood of its role in the etiology of FRDA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Friedreich's ataxia (FRDA),1 an autosomal recessive neuro-degenerative disease, is the most common inherited ataxia (1). The clinical and molecular biology features of FRDA have been reviewed (1–5). The FRDA gene (X25) contains seven exons and encodes a 210-amino acid protein named frataxin (1). Ninety eight percent of FRDA patients have an expanded GAA·TTC repeat in the first intron of the frataxin gene, and 2% have point mutations. FRDA is a typical triplet repeat disease (6) because an expansion of the repeat is found in patients; normal alleles have 6–34 repeats of GAA·TTC, but the FRDA patient alleles have 66–1700 repeats (1–3, 6). Individuals with longer GAA·TTC repeats (reviewed in Ref. 1) have an earlier age of onset and more severe disease manifestations.

FRDA is a loss of function disease because patients with two expanded alleles have reduced levels of frataxin. Also, a reduction in the amount of the frataxin protein, a mitochondrial protein involved in iron metabolism, is because of a diminution in the abundance of the frataxin mRNA (1). This inhibition of transcription is caused by the capacity of long GAA·TTC repeats to form triplexes (three-stranded DNA structures) that are known to effect this inhibition (5, 7–12). FRDA is the only triplet repeat disease that has a recessive inheritance pattern and is caused by the expansion of a GAA·TTC repeat (6).

Sticky DNA is a long triplex consisting of two strands of GAA repeats along with one TTC repeat tract (GAA·GAA·TTC) (8–10, 13, 14). This R·R·Y triplex is formed at neutral pH and requires divalent metal ions as well as negative supercoiling in recombinant plasmids grown in Escherichia coli. Hoogsteen base-pairing stabilizes the GAA·GAA interactions, whereas the GAA·TTC pairing is Watson-Crick. Two long tracts of (GAA·TTC)n (where n = 59–270) in the direct repeat orientation in a single DNA molecule are required for the formation of sticky DNA (13, 14); if the tracts are in the indirect repeat orientation, sticky DNA cannot be formed (13, 14). The requirement for two blocks of long GAA·TTC repeats can be fulfilled by either having two tracts in one recombinant plasmid or by biological dimers of the plasmids, each parent monomer containing a single repeat tract. The intermolecular formation of sticky DNA from two independent plasmid molecules, each harboring a single long GAA·TTC tract, has never been observed (13, 14). The novel feature of sticky DNA is the association into a triplex of two long R·Y tracts from distant regions of a DNA molecule; hence, upon its linearization, an X-shaped structure (8, 13) is formed with the crossover point at the repeat sequences. After the sticky DNA conformation has been formed, it is remarkably stable and requires the removal of divalent metal ions, with EDTA, as well as heat (70 °C) to revert the conformation to the duplex.

Several lines of evidence support the concept that simple triplexes exist in vivo including the following: oligonucleotidetargeted mutagenesis at R·Y tracts in mammalian cells (15–18); triplex-mediated inhibition of replication and transcription (19, 20); transcription-dependent recombination induced by intramolecular triplex formation in E. coli (21); chemical modification studies in situ (22, 23); psoralen photobinding in living cells (24, 25); and deletion analyses on poly(R·Y) sequences as a function of transcription and negative supercoil densities in vivo (26). Because triplexes are known to exist and to inhibit transcription in vitro and in vivo (reviewed in Ref. 27), and because the triplex-forming GAA·TTC repeats have an integral role in the pathology of FRDA (1–5), we attempted to directly investigate these interrelationships. During the course of our plasmid investigations, we realized that DNAs with long and/or pure tracts of GAA·TTC that had the highest propensity to form sticky DNA also showed the greatest tendency to form plasmid dimer isoforms compared with monomeric forms (D/M ratio) during cell growth.

Here we have evaluated the influence of TRS length, composition, and orientation as well as the effect of negative supercoil density and mutant host cells on the D/M ratio in E. coli. Furthermore, nitrogen mustard cross-linking studies directly demonstrate the formation of sticky DNA in vivo, thus validating the dimer to monomer ratio data as a measure of sticky DNA in living cells. The accompanying article (28) also documents the in vivo existence and properties of sticky DNA as well as the structure-dependent recombination hot spot activity of GAA·TTC tracts.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells—The genotypes for the E. coli strains used in this study are described in Table I. Strains HB101 and RR1 were obtained from Invitrogen; SURE was from Stratagene; KMBL1001 and its derivatives were from Dr. Nora Goosen (Leiden Institute of Chemistry, The Netherlands); and MG1665 and its derivatives were from Dr. R. G. Lloyd (Division of Genetics QMC, Nottingham, UK).


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  		TABLE I
E. coli strains used in this study

 
Preparation of Plasmids—Plasmids containing a single TRS tract (pSPL3, pGEM-3Zf(-), and pUC19 derivatives), which were used in these experiments, are shown in Table II and were described previously. Construction of the plasmids with two (GAA·TTC)150 tracts, which are pSPL3 derivatives, is described below.


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  		TABLE II
Plasmids used in this study The three vectors are all derivatives of ColE1.

 
To obtain analytical amounts of the monomeric and dimeric plasmid isoforms, E. coli cells were transformed using the CaCl2 transformation method (29) and grown in 10 ml of LB media containing 100 µg/ml ampicillin. Supercoiled plasmids were purified by the Wizard Plus Miniprep DNA Purification procedure (Promega Corp.) according to the manufacturer's recommendations.

To purify DNA monomers and dimers, 1–10 µg of the isolated plasmids, which contain monomers, dimers, and higher oligomers, in 60 µl of TE buffer containing 10% sucrose and 0.01% bromphenol blue (loading buffer) was loaded onto a 1% agarose gel. Electrophoresis was conducted in 40 mM Tris acetate (pH 8.3), 1 mM EDTA buffer (TAE) at 5 V/cm until the bromphenol blue reached a distance of 6 cm. Gels were stained by EtBr as described elsewhere (29). To prevent DNA damage during UV irradiation, aluminum foil was placed under the gel as a screen while excising the band. The bands corresponding to the monomeric and dimeric isoforms of the plasmids were excised from the agarose gels. DNAs were eluted from the gels using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's recommendations.

Cloning of a Pair of (GAA·TTC)150 Tracts into pSPL3—A fragment containing the (GAA·TTC)150 tract was prepared from pRW3822 (8) by XbaI digestion (New England Biolabs) followed by separation of the (GAA·TTC)150-containing 1.4-kb fragment by 1% agarose gel electrophoresis. The vector was prepared by digesting pRW3822 with NheI followed by phenol-chloroform extraction. The sticky-end ligation of the mixture of the two DNAs and all subsequent cloning steps were performed as described earlier (30). All plasmids were fully characterized by restriction mapping to verify the orientation and length of the cloned TRS. Thus, pRW4201 contains two (GAA·TTC)150 tracts in the direct repeat orientation, and pRW4202 contains the two (GAA·TTC)150 repeats in the inverted repeat orientation (Fig. 1). The distance between these two (GAA·TTC)150 inserts in pRW4201 and pRW4202 is 1,912 and 1,665 bp, respectively.



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  		FIG. 1.
Plasmids containing two (GAA·TTC)150 tracts, containing direct or inverted repeats, used to determine the influence of the insert orientations on RB formation. The lengths of the plasmids are 8511 bp.

 
Conditions of Bacterial Growth for Recultivation Assay—For the recultivation studies, supercoiled monomeric or dimeric forms of the plasmids were purified from 1% agarose gels and used as the starting materials. E. coli cells were transformed with 50 ng of the monomeric or dimeric forms of the plasmids using the CaCl2 transformation method (29) and grown in 10 ml of LB media containing 100 µg/ml ampicillin at 37 °C at a shaking rate of 250 rpm. Populations of ~1000 cells were used to initiate the culture. When the cultures reached an absorbance (600 nm) of 0.9 ± 0.05 unit (12–14 h) (i.e. step 1), an aliquot was inoculated into 10 ml of LB containing 100 µg/ml ampicillin as before at a final dilution of 1 x 10-7 and grown to the same OD (i.e. step 2), etc. (31). Supercoiled plasmids were purified from these cells by the Promega Wizard Plus Miniprep DNA Purification procedure according to the manufacturer's recommendations. Thus, the cultures were maintained in log phase growth by repeated recultivations (shown in the legends to the figures as the number of recultivations).

The relative amount of the plasmid dimer to monomer (D/M) reached a plateau after the second recultivation step (Fig. 2). Thus, most observations were recorded at the second recultivation step (as in Figs. 3 and 4 and Tables III, IV, V). In all cases where the D/M ratios were measured at the first recultivation step, they had intermediate values between 0 (pure monomer in the starting material) and the second step value (hereafter referred to as D/M from recultivation studies). In all cases, data from at least three parallel experiments were averaged; the S.D. for these data is ±10%.



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  		FIG. 2.
Effect of the presence of the (GAA·TTC)150 tract on the D/M ratio during recultivation in E. coli SURE cells. A, pSPL3 and its derivative harboring a (GAA·TTC)150 tract (pRW3822) were propagated in E. coli SURE cells in log phase for four stepwise recultivations as described under "Experimental Procedures." The plasmid DNAs were isolated and analyzed in a 1% agarose gel with subsequent EtBr staining as described under "Experimental Procedures." The bands above the dimers are higher oligomeric plasmid forms (trimers, tetramers, etc.) and were not considered in calculating the D/M molar ratios. That a small increase in the D/M is observed for the vector (B, open circles) is as expected because purified monomers were used for the transformations. Hence, this small increase represents the customary equilibrium between monomers and dimers (62). B, the influence of the (GAA·TTC)150 tract on the D/M ratio. Open circles designate pSPL3, and filled circles represent its derivative harboring the (GAA·TTC)150 tract (pRW3822). The procedure for quantitation of the gels is described under "Experimental Procedures." The error bars show the standard deviations, which are obviously much larger for the smaller numbers of recultivations. D, dimers; M, monomers.

 



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  		FIG. 3.
Relationship between the D/M ratio and RB formation for the plasmids harboring different lengths and composition of the TRS insert. Plasmid monomeric forms harboring the GAA·TTC tracts listed in Table II were used to transform E. coli SURE cells as described under "Experimental Procedures." The D/M ratios after the second step of recultivation were used. A, filled diamonds designate pSPL3 derivatives; open diamonds represent pUC19 derivatives. For pRW3806 (a pUC19 derivative harboring the (GAA·TTC)150 tract), the rate of deletions was so high that at least 90% of the starting material was lost after the first step of recultivation. Thus, to obtain data for the (GAA·TTC)150 tract, a pGEM-3Zf(-) derivative (pRW4204) was used. Open circles represent the data reported earlier (8) for D/M ratios (as described under "Experimental Procedures"). To estimate the correlation of the D/M ratios with RB formation, the reported data (8) for RB formation (filled circles connected by the line) were plotted on the same graph. B and C, relationship between the D/M ratio and RB formation for the plasmids harboring different amounts of substitution of GAA·TTC with GGA·TCC. Open circles represent the data reported earlier (10) for the D/M ratios using the standard procedure (as described under "Experimental Procedures") for the pSPL3 derivatives as well as for the pCR3.1 derivatives harboring inserts in both possible orientations. To reveal the correlation of the D/M ratio with RB formation, the reported data (10) for RB formation (filled circles connected by the line) were plotted on the same graph. Thus, the open and filled circles each represent three sets of data.

 



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  		FIG. 4.
In vivo cross-linking of sticky DNA. E. coli HB101 cells were transformed with the purified dimeric forms of several plasmids (Table II) harboring GAA·TTC inserts with different sequence features and, therefore, with different abilities to form sticky DNA (8, 10, 13). Aliquots of transformed cells were cross-linked with 50 µM HN2 in vivo followed by purification of the plasmids ("Experimental Procedures"). To reveal the extent of in vivo cross-linking, plasmids were cleaved with EcoNI and then heated at 70 °C for 1 min in the presence of 50 mM EDTA ("Experimental Procedures"). In parallel experiments, the same operations were done with non-modified DNAs. For the results in the absence of HN2 (upper half of the figure), no new bands were observed when the exposure time was increased at least 10-fold. LM, linear monomer; LD, linear dimer; RB, retarded band.

 


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  		TABLE III
Independence of D/M ratio on the length of CTG[cmpt]CAG repeats Plasmid monomeric forms harboring non-GAA·TTC tracts (Table II) were used to transform E. coli SURE cells as described under "Experimental Procedures." D/M ratios after the second step of recultivation were used. pUC19 and pGEM-3Zf(-) were used as standards for the vector and for a plasmid with the same origin and length as the TRS containing DNAs, respectively. The S.D. is ±10%. NA indicates not applicable.

 


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  		TABLE IV
The effect of negative supercoil density in vivo on D/M ratio Plasmid monomeric forms harboring GAA·TTC tracts (Table II) were used to transform E. coli KMBL1001 cells as described under "Experimental Procedures." To study the influence of negative supercoiling on the D/M ratio, aliquots of cells were grown in the presence or absence of novobiocin as described under "Experimental Procedures." D/M ratios after the second step of recultivation were used. The S.D. is ±10%.

 


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  		TABLE V
Effect of mutations in genetic systems that modulate DNA metabolism on D/M ratios Plasmid monomeric forms of pSPL3 and its derivative (pRW3822), harboring (GAA·TTC)150, were used to transform two parental strains of E. coli as well as recA and uvr mutants as described under "Experimental Procedures." D/M ratios after the second step of recultivation were used. The data for KMBL1001 were presented previously (Table IV), and similar results were found for the parental strain (MG1665) for the ruv mutants. The standard deviation is ±10%.

 
For the studies on the effect of negative supercoiling of the plasmids on the dimer to monomer ratio in vivo, an aliquot of the transformed E. coli KMBL1001 cells was grown in LB medium containing 100 µg/ml ampicillin and 5 µM novobiocin (32). Prior investigations from this lab optimized the conditions for novobiocin in this strain (32). All other procedures were as described above.

D/M from Prior Data—We wished to compare these recultivation results described above with the previous data (8) for the plasmids harboring (GAA·TTC)n with different lengths as well as for the plasmids with periodic substitutions of GAA·TTC with GGA·TCC repeats (10). These data are from standard plasmid preparations, not from recultivation studies. In both cases, the procedure for the purification of the plasmids after harvesting the E. coli SURE cells was described earlier (8, 10). For pRW3823 harboring (GAA·TTC)270, at least half of the TRS was deleted (see Fig. 1A in Ref. 8); thus, it was not possible to attribute the D/M ratio to a GAA·TTC length. To quantitate the photos of the EtBr-stained gels presented previously (8, 10) to get the D/M ratios, the Gel Doc 2000 system from Quantity One software (Bio-Rad) for Windows 98 was used. The standard deviation for these data is ±10%.

Effect of Vector Length on D/M—We studied the possible effect of the length of vectors on the D/M ratio to determine that the observed effects were due to the TRS and not to some property of the plasmids that were lengthened by the presence of the inserts. To evaluate this question, we conducted two-step recultivation experiments with a set of seven vectors with the ColEI type origin and deleted rop gene covering the length interval from 1990 to 7375 bp. The plasmids are as follows: pRW790 (1990 bp) (33) and pRW4888 (2330 bp)2 (both of these plasmids are deletion mutants of pBR322 with the deleted rop and tetracycline resistance genes, i.e. from 44 to 2435 bp and from 30 to 2061 bp, respectively); pUC19 (2686 bp) (34); pGEM-3Zf(-) (3199 bp) (Promega, Corp.); pCR3.1 (5044 bp) (Invitrogen); pSPL3 (6031 bp) (Invitrogen); and pTWIN1 (7375 bp) (New England Biolabs). E. coli SURE cells were transformed with monomers of these plasmids. A sigmoidal relationship of the D/M ratio with the length of the plasmid was found; if the plasmid is shorter than 2.5 kb, no dimers were found (i.e. D/M = 0), but if the plasmid is longer than 5 kb, the D/M ratio was 0.5 ± 0.05. The standard deviation for these data is ±10%. Hence, we conclude that under the conditions of our two-step recultivation experiments, the D/M ratio for the vector cannot exceed 0.5 ± 0.05. Therefore, the observed increment (Figs. 2, 3, 4) of up to 3 times this value must be attributed to the presence of the TRS.

Nitrogen Mustard Cross-linking Procedures—Prior studies (Refs. 35 and 36 and reviewed in Ref. 37) revealed that treatment of duplex DNA by nitrogen mustard (HN2) (mechlorethamine·HCl (Sigma)) for 100 min at 37 °C (pH 7.5) (2.4-fold molar excess of HN2 over the DNA phosphates) caused more than half of the guanines to be modified, and at least one-third of guanines were cross-linked through inter-strand bridges. Because sticky DNA contains an antiparallel R·R·Y triplex in its core, we hypothesized that it might be possible to covalently cross-link this conformation between the N-7 positions of the Gs in the R·R strands. Because the distance between the N-7 positions of the two Gs in the R·R strands of the antiparallel triplex are 10% shorter than the distance occupied by the HN2 molecule, we felt that this cross-linking might be successful. Alternatively, if the R·R strands were in the very unlikely parallel orientation (reviewed in Ref. 38), this distance would be 10% longer than the HN2 molecule (calculated from Ref. 38), and thus the cross-linking would be disfavored.

Sticky DNA was cross-linked both in vitro and in vivo (in situ) with 50 mM HN2 (prepared fresh before each set of reactions) in Me2SO. All cross-linking experiments were conducted in 40 mM sodium cacodylate (pH 7.5) (cross-linking buffer). For the in vitro experiments, 1 µg of pRW3822 was cleaved with AseI or EcoRI or EcoNI and was treated with 50 µM HN2 at 37 °C for 30–240 min in a 50-µl reaction volume. The restriction enzyme and the cross-linking reactions were conducted in the presence of 10 mM MgCl2. Addition of GMP to a final concentration of 1 mM and subsequent ethanol precipitation stopped the reactions. The maximum retention of RB (described below) after heat treatment in EDTA occurred at 120 min of incubation. Thus, we conducted all in vivo (in situ) cross-linking reactions for 2 h. Changing the sequence of linearization and cross-linking steps (for in vitro experiments) had no effect on the yield of RB retained when DNA was cross-linked in the presence of 10 mM MgCl2.

For in vivo (in situ) experiments, E. coli HB101 cells were transformed with 50 ng of the dimeric forms of a plasmid (Table II) (pRW3822, pMP178, pRW3821, pMP142, pRW4222, pRW2113, and pMP193) by using the CaCl2 transformation method (29) and grown in 10 ml of LB media containing 100 µg/ml ampicillin at 37 °C at a shaking rate of 250 rpm. When the cultures reached an absorbance (600 nm) of 0.6 ± 0.05 unit (overnight), the bacteria were pelleted and resuspended in 3 ml of cross-linking buffer. Aliquots were incubated at 37 °C for 2 h with 5, 50, or 250 µM HN2. Subsequent plating of the incubation mixtures allowed an estimate of the viability of the cells after HN2 treatment. After 2 h of incubation, the residual viability for 50 µM HN2 was 70% relative to the omission of the HN2, whereas for 250 µM HN2, it was 0.2%. Thus, the data for cross-linking with 50 µM HN2 for 2 h are referred to as sticky DNA cross-linked in vivo. After 2 h of incubation, plasmids were isolated by the Promega Wizard Plus Miniprep DNA Purification procedure. These treated plasmids were then used for subsequent investigation of the resistance of sticky DNA cross-linked in vivo against heat treatment in the presence of EDTA (described below).

Detection of RB Formation after HN2 Cross-linking—The presence of sticky DNA in a DNA preparation is determined routinely by the detection of a substantially retarded band (RB) on agarose gel electrophoresis after plasmid linearization. Detection of RB formation was performed as described (8) with some modifications. Cleavage of plasmid DNAs by appropriate restriction enzymes was conducted in NEB-uffer 1 (New England Biolabs Inc.) (10 mM bis-Tris/propane HCl, 10 mM MgCl2, 1 mM dithiothreitol (pH 7.0 at 25 °C)) supplemented with 100 µg/ml bovine serum albumin if recommended by the manufacturer. 5 units of the restriction enzyme per 0.5 µg of DNA was used in a total reaction volume of 12 µl. Incubation times at 37 °C were 200 min or overnight; no differences were detected. Chilling to room temperature and addition of the loading buffer, as described above, stopped the reactions.

RB is converted completely to the linear monomeric form after incubation of the DNA for 10 min at 80 °C in 50 mM EDTA as described earlier (8, 10, 13, 14). Thus, the heat sensitivity of RB in the absence of divalent metal ions allows us to distinguish between RB and other possible structures. The present studies revealed that 10 min is sufficient under these conditions to dissociate RB but they also hydrolyze the covalent cross-linking bonds at the sticky region. To avoid hydrolysis of these bonds, the incubation time as well as the temperature was decreased. Experiments at various incubation times, 0.25, 0.5, 1, 2, and 5 min at 70 °C, were conducted; 60 s of incubation is sufficient to dissociate the non-cross-linked RB, but the RB after cross-linking was stable.

The DNAs were 32P-labeled using the E. coli DNA polymerase I Klenow fragment end-labeling procedure (29) before loading onto the gel. Electrophoretic separations were conducted in a 0.7% agarose gel in TAE buffer at 5 V/cm with a distance of bromphenol blue migration of at least 6 cm. The agarose gels were dried at 65 °C overnight and exposed to Hyperfilm MP (Amersham Biosciences). To quantitate the autoradiographs, the Gel Doc 2000 system from Quantity One software (Bio-Rad) for Windows 98 was used. The standard deviation for RB formation is estimated as ±10%.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Plasmid Dimers Are Favored Over Monomers for Recombinant Molecules Containing Long Tracts of GAA·TTC—During routine DNA isolation studies, we noted that plasmids with the greatest propensity to form in vitro the substantially RB, which is indicative of the stable sticky DNA structure, favored the plasmid dimeric forms compared with the monomer isoforms. Fig. 2 shows the propensity of pRW3822, which contains 150 repeats of GAA·TTC, to form dimers compared with the vector (pSPL3), which lacks the FRDA TRS. pSPL3 and pRW3822 were propagated in E. coli SURE cells in log phase in stepwise recultivations as described previously (10, 31, 39). Fig. 2A shows the agarose gel analyses of the plasmid isoforms, and Fig. 2B shows the relative amount of plasmid dimers to monomers as a function of the number of generations of cell growth. For the vector, approximately twice as much monomer as dimer on a molar basis was found, as expected (see "Experimental Procedures"). However, for pRW3822 containing 150 repeats of GAA·TTC, ~1.5 times more dimer than monomer was observed. Thus, the presence of the TRS leads to an increase of D/M by ~3-fold.

Further studies were conducted on the relationship between the ratio of dimers to monomers with families of plasmids containing different lengths of GAA·TTC (from 0 to 270 repeats) in both pUC19 and pSPL3 vectors. The monomeric forms of these DNAs were transformed into E. coli SURE cells, and the D/M data were compared with similar prior results (8) as well as data for the formation of the RB which is indicative of sticky DNA formation (8) (Fig. 3A). Interestingly, we found for all four sets of data that the ability of a plasmid to exhibit RB formation in vitro is closely correlated with its ability to accumulate dimers in vivo. Parallel studies were also conducted with pUC19 derivatives harboring (CTG·CAG)n inserts (where n = 36 or 80) (Table III). These control investigations showed no enhancement of accumulation of dimers compared with monomers for either the pUC19 or pGEM-3Zf(-) vectors nor for the plasmids containing the myotonic dystrophy sequences. Thus, the enhancement of dimer formation occurs specifically with plasmids containing the FRDA mutation (GAA·TTC) with lengths that are sufficient to exhibit sticky DNA formation. Studies were conducted with the myotonic dystrophy sequence up to 80 repeats in length because this number of repeats is sufficient with the FRDA tract to form RB and to favor dimer formation (Fig. 3A) and because longer CTG·CAG tracts are quite unstable in E. coli SURE.

Dependence of D/M on the Percentage of Substitution of GAA·TTC with GGA·TCC—Experiments were also conducted with a family of plasmids containing GAA·TTC repeats ~130 in number (10); the plasmids contained a systematic variation of the extent of substitution of the homogeneous GAA·TTC sequence with GGA·TCC interruptions (Fig. 3, B and C). Prior studies (10) showed that more than 20% substitution of the GAA·TTC sequence with interruptions by the GGA·TCC sequence abolished the formation of sticky DNA. However, the GAA·TTC repeats with less than 11% of the GGA·TCC interruptions formed triplexes (sticky DNA) similar to the uninterrupted repeat sequence.

Fig. 3B shows the excellent correlation between the capacity of this family of interrupted molecules to form the retarded band, which is indicative of sticky DNA, and the propensity to form plasmid dimer instead of monomer isoforms. E. coli SURE cells were transformed with the monomeric forms of the pSPL3 derivatives harboring GAA·TTC repeats with different amounts of substitution by GGA·TCC (Fig. 3C). The recultivation data to give D/M ratios for these plasmids were compared with the D/M data from prior investigations as well as the data for RB formation (10) for both the pCR3.1 derivatives harboring inserts in both possible orientations and for the pSPL3 derivatives. This excellent correlation with all seven sets of data (Fig. 3B) along with the effect of repeat chain length (Fig. 3A) leads to the conclusion that the ability of a plasmid to form RB, which is detected in vitro, is closely related to the accumulation of plasmid dimers in vivo.

Effect of the Orientation of Two Inserts on D/M—Prior investigations (13) showed that sticky DNA is formed only when two tracts of sufficiently long GAA·TTC are present in a single plasmid molecule and are in the direct repeat orientation. This situation is fulfilled either by a biological dimer of a plasmid generated in vivo, which in the monomeric form contains a single long tract of GAA·TTC, or by a plasmid specially constructed in vitro to contain two blocks of GAA·TTC in the direct repeat orientation. Thus, we were interested to determine the effect of TRS orientation on the propensity to form dimers compared with monomers in vivo.

The E. coli SURE strain was transformed with monomeric forms of pSPL3 derivatives harboring two tracts of (GAA·TTC)150 in the direct (pRW4201) and the indirect (inverted) orientation (pRW4202) (Table II and Fig. 1B). The recultivation experiment demonstrates that the plasmid with repeats in the direct repeat orientation (pRW4201) had a D/M ratio (0.31) approximating the values found for the vectors. Because this plasmid can form sticky DNA intramolecularly, it has no need to form the biological dimers in order for two R·Y tracts to associate and form the sticky conformation, and thus, the formation of dimers was not favored (see "Discussion"). Monomers of pRW4201 can form RB (data not shown), thus obviating the necessity of forming biological dimers to produce sticky DNA.

However, for pRW4202 which contains the same inserts but in the indirect (inverted) orientation, the D/M ratio (1.43) is similar to that found for plasmids harboring a single copy of (GAA·TTC)150 (Figs. 1 and 2). The reason for this behavior is that pRW4202 must form biological dimers in order to adopt the sticky DNA conformation because the inverted repeat orientation of the TRS tracts precludes the formation of sticky DNA intramolecularly for monomers (13). Hence, the only way pRW4202 can form the sticky structure is to dimerize in vivo which would then generate a DNA with four TRS tracts (two pairs of direct repeats). The enhancement in the formation of biological dimers in this case is consistent with the hypothesis that they are favored over the monomeric isoforms due to sticky DNA formation.

In summary, these results provide an important internal control for the studies shown in Figs. 2 and 3 and strongly support the interrelationship between the in vivo formation of sticky DNA and the propensity of certain plasmids to favor the formation of the plasmid dimeric isoforms compared with the monomeric species.

Effect of Negative Supercoil Density on Sticky DNA Formation and D/M Ratio in Vivo—Sticky DNA formation depends on the negative supercoil density of plasmids harboring GAA·TTC repeats (8, 13). Thus, we conducted studies in E. coli by reducing the negative supercoil density that should reduce the propensity of the TRS to form sticky DNA and hence the D/M ratio. Novobiocin was chosen as the agent to reduce the negative supercoil density in vivo. Novobiocin inhibits the assembly of DNA gyrase (40, 41) by interfering with its ATP binding capacity. This prohibits the association of two GyrAB dimers to form the biologically active tetrameric GyrA2B2 form. Thus, the presence of novobiocin tends to achieve a relaxation of plasmid DNAs in vivo (32) and will thus destabilize (reduce) the formation of sticky DNA.

To reduce the negative supercoil density in vivo, E. coli KMBL1001 cells were transformed with monomeric forms of pSPL3 derivatives harboring (GAA·TTC)n (where n = 75 or 150) (Table IV). A portion of the transformed cells was grown in the presence of sufficient novobiocin (5 µM) to decrease the in vivo negative supercoil density (32). Table IV shows that in the presence of novobiocin where the supercoil density should be reduced to the point where a triplex should not be formed (32), the ratio of D/M for the GAA·TTC-containing plasmids was the same as for the vector. Alternatively, in the absence of novobiocin, pMP178 had an elevated D/M ratio consistent with the data shown in Fig. 2, and the same behavior was observed for pRW3822. Note that the D/M values in KMBL1001 cells are similar to the data in SURE cells shown in Figs. 2 and 3. Thus, the presence of novobiocin inhibits the formation of active DNA gyrase and thereby reduces the in vivo negative supercoil density and, hence, the propensity to form sticky DNA. The result is the reduced tendency to form plasmid dimers in vivo. Accordingly, the conclusions of these experiments are in agreement with those derived from the data in Figs. 2 and 3.

In Vivo Cross-linking of Sticky DNA—Nitrogen mustard (HN2) cross-linking experiments were conducted in vivo to cross-link the long GAA·GAA·TTC triplex that is responsible for the sticky DNA behavior. E. coli HB101 cells were transformed with the appropriate plasmids (Fig. 4) and treated as described under "Experimental Procedures" with 50 µM HN2; under these conditions, 70% of the cells were viable. Hence, we believe that these conditions accurately reflect the in vivo plasmid status. Studies were conducted with seven recombinant plasmids containing different lengths and types of GAA·TTC inserts that had different capacities to form sticky DNA (8–10, 13). pRW3822 contains 150 uninterrupted GAA·TTC repeats and had the greatest propensity to form the sticky triplex conformation. pRW4222 contains systematic substitutions of the GAA·TTC repeats to the extent of 15 of 136 repeats (11%) and also effectively forms sticky DNA (8, 13). pRW2113 contains a higher extent (20%) of substitution of the GAA·TTC repeat tract with GGA·TCC interruptions (26 of 131 repeats) (Fig. 3C). For studies with plasmid dimers (13), this sequence also formed sticky DNA but to a lesser extent. Alternatively, for studies with a mixture of plasmid isoforms (10), a very low yield of RB was observed (see "Discussion"). pMP193 contains the repeating hexanucleotide sequence GAAGGA·TCCTTC that is unable to adopt the X-shaped sticky conformation (4, 8–10, 13, 14). pMP178, pRW3821, and pMP142 contain 75, 59, and 33 GAA·TTC repeats, respectively. The first two inserts can weakly form sticky DNA, but the 33 repeat sequence in pMP142 is sufficiently short that the X-shaped structure has not been observed (8, 13).

Plasmid-transformed HB101 cells were treated with 50 µM HN2 (see "Experimental Procedures"). After isolation of the plasmids, the DNAs were cleaved with EcoNI, and then portions of the preparations were heated at 70 °C for 1 min in the presence of 50 mM EDTA. These conditions were sufficient to convert any sticky DNA structure that has not been cross-linked to the non-X-shaped sticky form (see "Experimental Procedures"). EcoNI was chosen because the difference in migration between the linear dimer and the RB for these pSPL3 derivatives is sufficient to eliminate ambiguities (8).

The top 14 lanes on the agarose gel electrophoregrams shown in Fig. 4 reveal that sticky DNA is formed as predicted (see previous paragraph), whereas after treatment with EDTA and heat, the sticky DNA was abolished in all seven cases (top right quadrant of Fig. 4).

Alternatively, after cross-linking by HN2 (lower 14 lanes in Fig. 4), sticky DNA is formed (lower left quadrant) as expected from studies without cross-linking (upper left quadrant). Thus, approximately the same yield of sticky DNA was observed with and without cross-linking. However, after treatment with HN2 as well as treatment of the EcoNI linearized DNA with EDTA and heating at 70 °C, substantial RB was observed (lower right quadrant) as well as the linear dimer. Thus, under these conditions, the formation of linear dimer may be taken as a confirmation of HN2 modification of the G residues, which causes an inhibition of the EcoNI restriction enzyme to recognize and cleave its target site. The residual RB that was resistant after heat treatment in 50 mM EDTA appeared only in the cases where we observed RB without HN2 treatment (Fig. 4, upper left quadrant). Thus, these results indicate that the HN2 cross-linking takes place at the crossover junction in the sticky DNA conformation in vivo.

In summary, these nitrogen mustard cross-linking studies in living cells confirm the presence of sticky DNA in vivo and validate the intracellular dimer to monomer ratio data as a measure of sticky DNA.

Effect of E. coli Mutations in DNA Metabolizing Systems on D/M Ratios—Studies were conducted to evaluate the effect of mutations in the recA, nucleotide excision repair, and ruv genes on the propensity to form dimers versus monomers. The enhancement of the D/M ratio reflects the interaction of the pair of (GAA·TTC)150 tracts in pRW3822 dimers to adopt the sticky DNA conformation. RecA, Uvr, and Ruv proteins are involved in DNA metabolism and its homeostasis (reviewed in Ref. 42). The RecA protein is involved in recombinational DNA repair and is an ATPase that forms helical filaments and promotes homologous pairing and strand exchange (42, 43). The UvrA, UvrB, UvrC, and UvrD proteins belong to the DNA excision repair system (42, 44–48). Before this process begins, two UvrA subunits combine into an active UvrA2 dimer. This dimer then assembles with UvrB in an ATP-dependent reaction, forming the active UvrA2UvrB trimer, which finally recognizes and binds to the damaged DNA region. Subsequent release of UvrA2 from the complex recruits the UvrC endonuclease. This leads to 3'- and 5'-incisions and finally, under the action of UvrD, to the release of the damaged region. The Ruv proteins play their role in the migration and resolution of Holliday junctions and, therefore, are also believed to be involved in the monomerization of dimers and higher oligomers through the homologous recombination pathway (42, 49–51). During this process, RuvA binds RuvB which then recognizes and binds specifically to a Holliday junction to form a RuvARuvB·-(Holliday junction) complex. Then, the RuvC endonuclease binds and resolves the Holliday junction.

Monomeric forms of the vector pSPL3 and its derivative pRW3822, which harbors (GAA·TTC)150, were used to transform the three parental strains of E. coli (RR1, KMBL1001, and MG1665) as well as the recA, uvr, and ruv mutants, respectively (Table I). Table V shows that dramatic differences were observed for the dimer to monomer ratios between the recombinant plasmid containing the FRDA TRS and the vector (pSPL3). For the parental strains RR1, KMBL1001, and MG1665 (Table V), substantial differences in D/M (~3-fold) were found between the pSPL3 vector and the FRDA containing pRW3822, as expected. However, for the recA- E. coli HB101, which is isogenic with RR1, no significant dimer formation was observed. Instead, only monomers were observed. Thus, the D/M ratios are shown as 0.

Fig. 5 shows the possible metabolic pathways between monomers, dimers, and the sticky DNA structure. The pathways are drawn as one-step reactions for the sake of simplicity and because the possible involvement of intermediates in these equilibria is uncertain. The result of the experiments with the recA-deficient strain can be explained by the reduced recombination dramatically decreasing the k1 value (Fig. 5). Parallel experiments were conducted with the recA- E. coli HB101 with the dimeric forms of pSPL3 and pRW3822. No monomers were observed, even up to the third step of recultivation (data not shown). Instead, only dimers were observed for both DNAs. These results can be explained by the decrement of k2 (Fig. 5), which is due to the mutation in recA. Any catalyst should accelerate the rates of both the forward and reverse reactions to an equal extent (first law of thermodynamics). Thus, RecA must increase both the forward (k1) and reverse (k2) rate constants due to its role in homologous recombination.



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  		FIG. 5.
Schematic representation of the equilibria between biological monomers, dimers, and sticky DNA in vivo. k1 and k2 are the rate constants for the forward and reverse processes of the monomer to dimer conversion, respectively; k3 and k4 are the rate constants for the conversion of the dimer to sticky DNA and its slow reverse reaction, respectively. For the sake of simplicity, the pathways outside of these equilibria (such as plasmid loss (degradation), recombination, further oligomerization, etc.) are not shown.

 
For the studies with the mutant Uvr proteins (Table V), mutations in the uvrA or the uvrB genes led to D/M ratios approximating those found for the vector. In contrast, mutations in uvrC and uvrD allowed the cell to maintain the D/M ratio close to the values found for the parental strain (KMBL1001) (Table V). These data indicate that the significant increment in the D/M ratios observed for pRW3822 when compared with pSPL3 in the parental strain was due to the activity of UvrA and UvrB. We propose that the recognition of DNA distortions at the sticky DNA structure by the UvrA2UvrB complex plays a significant role in the enhancement of the D/M ratio in vivo by accelerating k1 and/or k3.

For the preliminary experiments performed with strains with mutations in the ruv genes, all four mutations (ruvA-, ruvB-, ruvC-, and ruvABC-) led to the loss of pRW3822 because no plasmids were detectable by EtBr staining (data not shown). Because these three proteins (RuvA, -B, and -C) have a role in the migration and resolution of Holliday junctions, they may be involved in the conversion of dimers and higher oligomers back to monomers through homologous recombination. Thus, the loss of plasmids harboring (GAA·TTC)150 but not the vector may be due to problems with the formation of monomers from higher oligomers and sticky DNA. Sticky DNA, once formed, is extremely stable (8–10, 13, 14), and thus k4 may be very small which can reduce the value of k2.

In summary, the data from all three genetic systems (recA, uvr, and ruv) show a dramatic difference in the behaviors with the plasmid harboring (GAA·TTC)150 compared with the vector and thus is attributed to the formation of sticky DNA in vivo.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Triplexes and sticky DNA at long tracts of GAA·TTC inhibit transcription (5, 7, 9–12, 52, 53); also, this sequence forms sticky DNA in E. coli with the repeating hexanucleotide sequence GAAGGA·TCCTTC (14) that also occurs in intron 1 of the FRDA gene (4). Thus, we wished to investigate the occurrence of this non-B DNA structure in living E. coli and to evaluate its biological behaviors.

The determination of non-B DNA conformations inside living cells has been an extremely difficult endeavor (reviewed in Refs. 38, 54–60). Nonetheless, a number of compelling experiments show that three-stranded DNA structures exist in vivo in prokaryotic as well as eukaryotic cells (reviewed in the Introduction). However, sticky DNA is not an orthodox triplex because it is formed between two long GAA·TTC tracts originally located distally in a plasmid. This feature distinguishes sticky DNA (Fig. 5, bottom) from a simple folded back intramolecular triplex (Fig. 5, top left) and serves as the "glue" for the adherence of the two distant regions of the DNA (13, 14). Thus, new methodologies are required for this highly unusual conformation. During the course of our investigations on the properties of sticky DNA (8, 10, 13, 14), we discovered serendipitously that plasmids containing the GAA·TTC sequences most prone to form the sticky conformation (as monitored in vitro) also revealed a pronounced tendency to accumulate plasmid dimers compared with monomers in vivo. This observation has been exploited as an assay for sticky DNA in living E. coli cells.

The following four factors were investigated that influenced the yield of sticky DNA (detected as RB) as measured in vitro and the dimer to monomer ratio in vivo: the length of the GAA·TTC tracts (from 0 to 150 repeats); the extent of substitution of the homogenous GAA·TTC sequence (130–150 repeats in length) with GGA·TCC interruptions at regular intervals (from 0 to 50% of interruptions); the in vivo level of negative supercoil density as modulated by growth in the presence of novobiocin; and the orientation of two (GAA·TTC)150 tracts in one plasmid. In all cases, a close correlation was observed between the ability of a sequence to form RB as measured in vitro and the D/M ratio in vivo. Nitrogen mustard cross-linking studies revealed the presence of sticky DNA in living cells, which provides a direct conceptual bridge between the dimer to monomer ratio data found in vivo with the formation of RB as monitored in vitro. In summary, our data shows that sticky DNA exists and functions in living E. coli.

Fig. 5 presents a hypothetical overview of the equilibria in vivo between plasmid monomers, dimers, and sticky DNA. We assume that the formation of sticky DNA from the dimer generates an inert reservoir of molecules that do not participate in the other plasmid interconversions because the rate of reversion of the formation of sticky DNA (i.e. k4) is much smaller than any of the other rate constants. This behavior is due to the extreme stability of sticky DNA (8–10, 13, 14). Sticky DNA may be formed by the plectonemic slithering of the dimer molecules. Interactions between the two TRS direct repeat sequences may form a synapse for triplex formation; after this nucleation step, the conversion of the two long GAA·TTC tracts into the single long triplex (sticky conformation) probably ensues by a zipper mechanism driven by the energy of negative supercoiling. Proteins such as RecA (42, 43) and Ruv (Refs. 49–51 and reviewed in Ref. 42) are involved in Holliday junction creation and resolution, respectively. Furthermore, the Uvr system (42, 44–48, 61) may cleave DNA by recognition of non-B DNA conformations (32). Accordingly, studies were conducted with host cells containing mutations in recA, uvr, and ruv (Table V). We found that a mutation in recA leads to inhibition of both the forward and reverse reactions between monomers and dimers (k1 and k2 in Fig. 5). Thus, when transformation studies were performed with dimers or with monomers, no dramatic differences in behavior were observed for the plasmid and the vector. The influence of UvrA, UvrB, UvrC, and UvrD proteins on the dimer to monomer ratios found for the GAA·TTC-containing plasmid and its vector enables the conclusion that the NER system plays a role in the conversion of two monomers into a dimer (k1, Fig. 5). A significant increment in the D/M ratios was observed for the plasmid compared with the vector in the parental strain and the uvrC and uvrD mutants. However, for the uvrA and uvrB mutants, the D/M ratios for both DNAs had approximately the same value. We propose that the recognition of DNA distortions in the sticky DNA leads to the enhancement of the D/M ratio in vivo by accelerating k3. These data are consistent with previous observations on the interactions of the NER system with undamaged DNA (63) and with triplexes (64–66) as well as with the effect of the methyl-directed mismatch repair and NER systems on the behavior of plasmids harboring different types of triplexes (32).

The Ruv proteins participate in Holliday junction resolution (42, 49–51) and, therefore, may be involved in the dimer to monomer conversion. Our experiments with E. coli ruvA, ruvB, and ruvC mutations demonstrate a high propensity of loss of the GAA·TTC-containing plasmid compared with the vector. We attribute this loss to the ability of the dimer to form sticky DNA which leads to stepwise oligomerization of the plasmid caused by sticky DNA formation. Because these oligomers cannot be resolved stepwise back to the monomers, this leads to the eventual loss of plasmid during cell division as predicted by the "dimer catastrophe" model (62). Accordingly, sticky DNA may play a role in the process of segregation of lower oligomers after intramolecular homologous recombination of higher plasmid oligomers. Therefore, these data demonstrate the involvement of Ruv proteins in branch migration of Holliday junctions during homologous recombination of sticky DNA conversion to dimers (k4) and back to monomers (k2).

These results have important implications for our understanding of the molecular etiology of FRDA. Because sticky DNA exists in E. coli and has marked biological consequences, it is possible that a similar behavior will occur in eukaryotic cells, including humans. Prior studies in vitro,in E. coli, and in COS-7 monkey cells have documented the inhibitory effects of sticky DNA in transcription (5, 9, 10) that agree with studies showing the reduction in the abundance of the X25 mRNA in FRDA patients (1–3). Furthermore, the presence of long GAA·TTC tracts as well as the repeating hexanucleotide sequence (GAAGGA·TCCTTC) in intron 1 of the FRDA gene (4) and their known capacity to form sticky DNA in E. coli (14) makes plausible the existence of this conformation in patient cells.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants NS37554, ES11347, and GM52982, the Robert A. Welch Foundation, and the Friedreich's Ataxia Research Alliance. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Dept. of Molecular and Cell Biology, the University of Texas at Dallas, Mail Station F03.1, Richardson, TX 75083. Back

§ To whom correspondence should be addressed: Center for Genome Research, Institute of Biosciences and Technology, Texas A&M University, Texas Medical Center, 2121 W. Holcombe Blvd., Houston, TX 77030-3303. Tel.: 713-677-7651; Fax: 713-677-7689; E-mail: rwells{at}ibt.tamu.edu.

1 The abbreviations used are: FRDA, Friedreich's ataxia; TRS, trinucleotide repeat sequence(s); bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; RB, retarded band; NER, nucleotide excision repair. Back

2 M. Napierala and R. D. Wells, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. N. Sakamoto for the preparation and characterization of pRW4201 and pRW4202, Drs. N. Goosen and R. G. Lloyd for bacterial strains, and Drs. A. Bacolla, A. Jaworski, and M. Napierala for helpful discussions.



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