INTRODUCTION

Biologically unstable repeating CTG·CAG, CGG·CCG, and AAG·CTT triplet DNA sequences are involved in the etiology of several neurological diseases (reviewed in the accompanying paper (1)). To date, only these three types of repeating triplet sequences have been associated with human hereditary diseases; a total of 10 triplet repeats are possible. Other triplet repeats have been observed in human and mammalian microsatellites (2, 3, 4). Several long tracts of TRS have been identified in the human genome using the repeat expansion detection method (5), which is based on hybridization of short triplet repeat oligonucleotides, and fluoresence in situ hybridization (6). The only triplet repeat containing 100% A/T is TTA·TAA.

A survey of triplet repeats in the human genome revealed that TTA·TAA is the most abundant (by at least 4-fold) and is the most frequently polymorphic (4). Also, the TTA·TAA microsatellites are very abundant in introns but are uncommon in exons (3) and were implicated in regulation of transcription (7, 8). At least one protein binds to TTA·TAA microsatellites (9). A 270-bp¹ tract of this sequence is found upstream of a variant surface glycoprotein gene of Trypanosoma brucei (10). Since long AAG·CTT tracts have very recently been implicated as the cause of Friedreich's ataxia (11), perhaps some of the other seven triplet repeats will also be associated with other hereditary diseases.

Simple repeating DNA sequences such as homo-, di-, and other types of repeats adopt non-B DNA conformations in recombinant plasmids under appropriate conditions of negative supercoil density, ionic environments, protein binding, etc. (reviewed in the accompanying paper (1)). As part of a broader investigation (1, 12, 13, 14, 15, 16, 17, 18, 19)² on the biochemical and biological behaviors of all 10 triplet repeat sequences (TRS), we report that the TTA·TAA sequence has a propensity to adopt non-paired single-stranded regions under the influence of negative supercoil density and heat.

MATERIALS AND METHODS

pGE117.6, which contains (TTA·TAA)₉₀ derived from the variant surface glycoprotein gene of T. brucei (gift of Dr. David A. Campbell, University of California at Los Angeles) (10), was digested with SspI, and then the 1104 to 761 fragment (344 bp), which contained the TTA·TAA repeat and the neighboring (A + T)-rich sequences, was recloned into the HincII site of pUC19 to give pRW3143. pRW3148, pRW3146, and pRW3145 were constructed as described (19). Briefly, pRW3143 was digested with SacI and HindIII, and regions corresponding to fragments with shorter TTA·TAA repeats (invisible by staining with ethidium bromide) were eluted from a 1.8% agarose gel. These components were recloned into the SacI-HindIII site of pUC19. All plasmid DNAs were isolated from Escherichia coli HB101 by the alkaline lysis method as described (20). All DNA inserts were sequenced on both strands to determine the TTA·TAA repeat sizes of the plasmids.

Three chemical probes, OsO₄ (Aldrich), diethyl pyrocarbonate (DEPC) (Aldrich), and chloroacetaldehyde (CAA) (Fluka), were used to modify the TTA·TAA repeat containing plasmids as described previously (21, 22, 23). 3 µg of DNA was reacted with each reagent in 100-µl reaction volume under the conditions described in the figure legends. After the reactions, all samples were purified by gel filtration through Sephadex G-50 microcolumns equilibrated with H₂O (20) and precipitated with ethanol. Both strands of the modified plasmids were analyzed by primer extension (1, 13) or chemical degradation analyses (24).

After chemical probing, the DNA was analyzed by the procedure described previously (1, 13). The DNA was dissolved in 20 µl of a solution containing 0.2 M NaOH and 2 ng of 5-³²P-end-labeled primer. For primer extension analysis, two primers were used, namely M13/pUC forward sequencing primer 1211 (17-mer, New England Biolabs) and M13/pUC reverse sequencing primer 1201 (16-mer, New England Biolabs) for the bottom and top strands, respectively. The plasmid was heated for 90 s at 90 °C followed by incubation for 4 min at room temperature. The DNA was neutralized with 0.3 M sodium acetate (pH 5.2) and was precipitated with ethanol. The DNA was resuspended in 10 µl of a solution containing 40 mM Tris-Cl (pH 7.5), 50 mM NaCl, 20 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM 2-deoxynucleoside triphosphates, and 5 units of a modified T7 DNA polymerase (Sequenase Version 2.0, U. S. Biochemical Corp.), and incubated for 10 min at 37 °C. After termination of the reaction by the addition of 95% formamide and 20 mM EDTA, the DNA was fractionated on a 12% denaturing polyacrylamide gel, and the bands were visualized by autoradiography.

The modified DNA was divided into two samples, digested with either SacI and HindIII or EcoRI and PstI, and labeled with [-³²P]dATP using the large fragment of E. coli DNA polymerase I (Klenow fragment, U. S. Biochemical Corp.). After isolation from a polyacrylamide gel, the DNA fragments were treated with 1 M piperidine for 30 min at 90 °C, lyophilized, and applied to a 12% denaturing polyacrylamide gel in parallel with sequencing markers generated by the chemical degradation method (24). The bands were visualized by autoradiography.

Topoisomers of plasmids were prepared as described previously (25). Briefly, DNAs with various supercoil densities were generated by incubating 6 µg of DNA in 100 µl of a solution containing 10 mM Tris-Cl (pH 7.6), 50 mM KCl, 10 mM 2-mercaptoethanol, 1 mM EDTA, 0-11 µM ethidium bromide, and chicken erythrocyte topoisomerase (26) for 60 min at 37 °C. The ethidium bromide and topoisomerase were removed by two phenol extractions followed by two ether extractions, and the pooled DNAs were resuspended in H₂O after ethanol precipitation.

Two-dimensional gel electrophoresis was performed as described previously (27). Mixtures of topoisomer populations prepared as described above were subjected to first-dimension gel electrophoresis in 1.25% agarose at 3.3 V/cm at 25 °C in 0.5 × TBE buffer (pH 8.3) (0.5 × TBE, 45 mM Tris borate, 1 mM EDTA) or TAE buffer (pH 8.0) (TAE, 40 mM Tris acetate, 1 mM EDTA). The gel then was soaked in 0.5 × TBE buffer (pH 8.3) containing 0.7-20 µM chloroquine diphosphate (Sigma) for 3 h. Electrophoresis in the second dimension was carried out at a 90° angle to the first dimension at 3.3 V/cm at 25 °C in 0.5 × TBE buffer (pH 8.3) containing 0.7-20 µM chloroquine diphosphate.

RESULTS

Fig. 1 shows the cloned sequences in the recombinant plasmids and a list of the plasmids used in this study. Three plasmids with shorter TTA·TAA repeats were produced as described (19) from the original pRW3143, which contains (TTA·TAA)₉₀. As expected, only a part of the perfect (TTA·TAA)₉₀ repeat was deleted; inserts with 60, 30, and 18 repeats of TTA·TAA were produced. The plasmids were designated pRW3148, pRW3145, and pRW3146, respectively.

To investigate the formation of unusual DNA (non-B DNA) structures, such as Z-DNA, triplexes, and cruciforms, chemical probes have been used (28, 29) because they can specifically modify bases in single-stranded or conformationally perturbed DNA or B-Z junctions. The modified base(s) can be detected by cleavage of the modified sites with a specific reagent (i.e. hot piperidine) or the primer extension assay which detects the inhibition of elongation by DNA polymerase. In this study, we used OsO₄, DEPC, and CAA to evaluate the existence of non-B DNA structures in the TTA·TAA repeat sequences.

OsO₄ reacts at the C-5=C-6 double bond of pyrimidines (T  C) in the presence of tertiary amines, such as pyridine and 2,2-bipyridine, and is substantially more reactive to single-stranded DNA than to double-stranded DNA (21, 30, 31, 32). It has been used for the single-stranded regions of cruciforms (32, 33, 34) and triplexes (21, 35) and for the structural perturbations at B-Z junctions (36). For inverted repeat sequences neighbored by (A + T)-rich sequences, OsO₄ was used as a chemical probe to detect unpaired single-stranded DNA (32, 33, 37, 38, 39). Also, AT·AT repeat sequences were found to form cruciform structures by detection of single-stranded regions with OsO₄ (40, 41, 42). DEPC carboxyethylates purines (A > G) at the N-7 position by the opening of the imidazole ring in single-stranded DNA as in triplexes (21, 35) and cruciforms (34, 43, 44) or in a syn conformation as in Z-DNA (36, 45). On the other hand, CAA as well as bromoacetaldehyde reacts at the N-1 and the C-6 amino groups of adenines or the N-3 and the C-4 amino groups of cytosines to generate 1,N⁶-ethenoadenine or 3,N⁴-ethenocytosine derivatives at unpaired adenines or cytosines (46, 47), and has been used for single-stranded regions of cruciforms in inverted repeats containing (A + T)-rich sequences (48) and AT·AT repeat sequences (49), for triplexes (22, 23), and for B-Z junctions (50, 51).

Fig. 2 shows chemical reactivities with these three chemical probes (OsO₄, DEPC, and CAA) on the bottom strand of pRW3146, which contains 18 repeats of TTA·TAA, as analyzed by the primer extension assay. By this analysis, the modified bases, thymines by OsO₄ and adenines by DEPC or CAA, on the template strand were detected by the inhibition of elongation by the DNA polymerase (Sequenase) at the corresponding bases on the extended strand. Surprisingly, modifications were observed throughout the (TAA)₁₈ sequence by all three chemical probes as a result of inhibition of elongation by Sequenase on the top strand (TTA strand) (Fig. 2, lanes 1, 3, and 5). The (A + T)-rich sequences, which are located on the 3 side of (TTA)₁₈ and contain (TTA)₅ and (TTA)₂, also were modified by DEPC and CAA (Fig. 2, lanes 3 and 5) but not by OsO₄ (Fig. 2, lane 1). We also determined that the modification pattern of bromoacetaldehyde was identical to that of CAA (data not shown). These observations may result from the fact that DEPC was reacted at 20 °C for 30 min, a longer reaction time than with OsO₄, and CAA was used at 37 °C for 30 min, a higher temperature and a longer reaction time than with OsO₄. On the other hand, on the bottom strand (TAA strand), modifications by OsO₄, DEPC, and CAA gave the same pattern as those of the top strand (TTA strand) (data not shown), indicating the existence of unpaired regions in both strands of supercoiled pRW3146.

We investigated the effect of the length of the TTA·TAA repeats. Fig. 3 shows the OsO₄ modification patterns for different lengths of TTA·TAA repeat containing plasmids under the same conditions described above. From 18 up to 90 repeats of TTA·TAA, the modifications were evenly distributed throughout the TTA·TAA repeats on both strands (Fig. 3, A and B, lanes 1, 3, 5, and 7), indicating that unpaired regions exist in all of these lengths of TTA·TAA repeats.

Recently, we observed that CTG·CAG repeat sequences derived from the myotonic dystrophy gene and CGG·CCG from the fragile X gene, even without chemical modification, caused pausings of DNA polymerases, namely Sequenase, the Klenow fragment of E. coli DNA polymerase I, and human DNA polymerase  (13). This behavior was attributed to the formation of a new type of non-B conformation by this triplet repeat sequence. Parallel studies were performed on the TTA·TAA triplet repeats described herein. No pausing of Sequenase was found on both strands of all TTA·TAA inserted plasmids (Fig. 3, A and B, lanes 2, 4, 6, and 8), even with preincubation (at 37 or 50 °C for 10 min) before primer extension, a condition which strongly enhanced the pausings for CTG·CAG repeat sequences (13). No chemical probes were employed in this study. Also, there was no pausing with the Klenow fragment in the absence of chemical modifications, even under the preincubation conditions (data not shown). Hence, we conclude that TTA·TAA repeats do not adopt unusual DNA conformations that inhibit the progression of DNA polymerase, in contrast to the behavior with CTG·CAG and CGG·CCG repeats.

The chemical probing described above revealed that TTA·TAA repeat sequences formed unpaired regions at the supercoil density of 0.06. Some unusual DNA structures (left-handed Z-DNA, cruciforms, triplexes) are known to be induced by negative superhelical density in recombinant plasmids (21, 35, 52, 53, 54, 55). Accordingly, the effect of superhelical density on OsO₄ modification on these TTA·TAA sequences was investigated. Fig. 4 shows the quantitation of the amount of OsO₄ modification for pRW3146 as a function of average negative supercoil densities (). At low supercoil density (0.025), few OsO₄ modification sites were observed. In linearized pRW3146, no sites were observed by chemical degradation analyses (data not shown). As the torsional stress was increased, however, the degree of OsO₄ modification increased. The reactivity relative to supercoiling shown in Fig. 4 is broad compared with the all-or-none transitions found for triplexes or cruciforms. 80% of the transition took place over 0.07 density units, whereas only ~0.01 was required for the other conformational changes (35, 52, 56). Thus, the gradual transition (Fig. 4) indicates that a larger duplex region is unpaired as more supercoil density is added to the system.

Supercoil-dependent alternate structures of DNA, such as cruciforms, triplexes, and Z-DNA, have been detected by two-dimensional gel electrophoresis (38, 39, 53, 54, 55). The chemical probe analyses described above suggested that unpaired DNA regions exist throughout the TTA·TAA repeat and in a part of the (A + T)-rich neighboring sequence, depending on the conditions employed. We used two-dimensional agarose gel electrophoresis to further investigate the unwound DNA regions revealed by the chemical probing results. The topoisomer populations of pUC19, pRW3146, and pRW3145 were analyzed as shown in Fig. 5. pRW3146 and pRW3145 exhibited an initial relaxation of 3.5 and 5 supercoil turns, respectively, at topoisomer numbers 10 and 11. This corresponded to relaxation of 37 and 53 bp at supercoil densities of 0.037 and 0.041, similar to the superhelical density at which the initiation of OsO₄ modification occurred (Fig. 4 for pRW3146 and data not shown for pRW3145). The relaxation continued until topoisomer numbers 17 and 18 (superhelical density 0.061 and 0.063, respectively). The total relaxation for pRW3146 and pRW3145 was 10.5 and 12 supercoil turns corresponding to unpairing of 110 and 126 bp, respectively. These results indicate the relaxation of the entire region of the perfect TTA·TAA repeat (54 and 90 bp, respectively) and the neighboring (A + T)-rich 40-bp sequences. This observation was consistent with the results of the chemical probing in 0.5 × TBE buffer (pH 8.0) described below. No transition was observed for the control pUC19. The electrophoresis was repeated on the same samples in TAE buffer (pH 8.0). Interestingly, no transitions were observed. This result will be discussed in the next section.

Similar studies were performed on pRW3143 which contains 90 repeats of TTA·TAA with 20 µM chloroquine in the second dimension. Whereas the resolution of the gel did not permit an accurate determination of the total number of topoisomers relaxed, at least 26 were detected indicating an unpairing of at least 270 bp.

Temperature and salt concentration have been shown to affect the unpairing of (A + T)-rich sequences as well as AT·AT repeat sequences in a supercoiled molecule (37, 38, 39, 42, 54, 57, 58). (A + T)-rich sequences were observed to induce adjacent inverted repeats to form cruciform structures via the C-type extrusion formed by large-scale helix opening, and this was dependent on supercoil density, temperature, and salt concentration (37). Bowater and co-workers (39) observed that large-scale opening of (A + T)-rich regions within a supercoiled molecule was suppressed by salt. It is also known that AT·AT repeat sequences form cruciform structures at low temperature or in the presence of metal ions (Na⁺, Mg²⁺, K⁺, Ca²⁺, [(NH₃)Co]³⁺, etc.) (40, 42). Thus, we investigated the effect of temperature and metal ions for the TTA·TAA repeat sequence. On the presumption that T·T and A·A bp might be formed in addition to A·T pairs in the TTA·TAA sequence, a TTA·TAA repeat could form a cruciform structure.

Fig. 6 shows the effect of temperature on OsO₄ modification of supercoiled pRW3146. As the temperature was increased, OsO₄ modification intensified in the (TAA)₁₈ region. pRW3146 contains an (A + T)-rich region, 87.5% A/T content, adjacent to a perfect (TAA)₁₈. At 37 °C, the (A + T)-rich region was strongly modified. This modification pattern was identical to that found with CAA (Fig. 2, lane 5). However, at lower temperatures, 0 or 10 °C, there was little modification and the patterns did not reveal the presence of cruciforms.

The reversibility of the temperature effect was investigated. After preincubation at one temperature (i.e. 37, 20, 10, or 0 °C) for 20 min, the chemical probe reaction was conducted at a second temperature (i.e. 0, 10, 20, or 37 °C) for 10 min. The resulting OsO₄ modification pattern was the same as that found when the second temperature was used for both the preincubation and the reaction (data not shown). Thus, this result indicates that the helix opening in the (TAA·TTA)₁₈ repeat and the neighboring (A + T)-rich sequences is formed reversibly as a function of temperature.

The effect of metal ions on the OsO₄ modification patterns also was investigated for supercoiled pRW3146. In the case of NaCl up to 75 mM, the modification was observed throughout the (TAA·TTA)₁₈ tract at 20 °C, but at 150 mM NaCl, pH 7.4 and 20 °C, no modification was observed (data not shown). Under physiological conditions, at 150 mM NaCl, pH 7.4 and 37 °C, although the modification was observed throughout the (TAA·TTA)₁₈ region, the intensity of OsO₄ modification was much weaker than with no NaCl (data not shown). Incidentally, for the range 0.1-150 mM NaCl, no cruciform-like structures were observed. On the other hand, in the presence of 5 mM MgCl₂ or higher, no modification was observed at 20 °C, pH 7.4, while there was modification throughout the (TAA·TTA)₁₈ region up to 1 mM MgCl₂ (data not shown). Thus, metal ions (Na⁺, Mg²⁺), especially at higher concentration, inhibited helix opening.

As described above, when two-dimensional gel electrophoresis was performed in TAE buffer (pH 8.0) instead of 0.5 × TBE buffer (pH 8.3) in the first dimension, no transitions were observed, although a positive control, pRW1561, containing a potential Z-DNA sequence (55) exhibited the expected transitions in both buffers (data not shown). To further investigate this result, we evaluated the effect of buffer conditions (pH and salt concentration) on the OsO₄ modification of supercoiled pRW3146. There were distinct differences in the modification patterns as influenced by salt and pH. In 0.5 × TBE buffer (pH 8.0), the entire 94-bp (A + T)-rich regions (the (TTA·TAA)₁₈ repeat and the 40 bp of 87.5% A/T sequence on the 3 side) were modified (Fig. 7, A and B, lane 1), in agreement with the two-dimensional gel results. On the other hand, in TAE buffer (pH 8.0), the OsO₄ modification was observed throughout the (TTA·TAA)₁₈ repeat and the neighboring (TTA·TAA)₅ repeat sequences, although the intensity of the modification in the (TTA·TAA)₂ repeat region was much less than that in 0.5 × TBE buffer (pH 8.0). While this result seems inconsistent with that of the two-dimensional gel electrophoresis described above, it is known that OsO₄ reactions are more sensitive than the two-dimensional gel electrophoresis analysis because the gels measure an equilibrium state, whereas the OsO₄ reactions can detect a transitory relaxation. Since the ionic strength of the two buffers is similar, it appears that the borate ions favor the helix opening, whereas the acetate ions may preserve the hydrogen bonded B-DNA conformation.

The modification also was influenced by pH. The (TTA·TAA)₅ repeat sequence was modified more strongly at pH 8.0 than at pH 7.4 in TE buffer (TE, 50 mM Tris-Cl, 1 mM EDTA) (Fig. 7, A and B, lanes 3 and 5), whereas at acidic pH (pH 4.5) in TAE buffer (Fig. 7, A and B, lane 9) the modification pattern was similar to that at pH 7.4. Hence, these results indicate that the helix opening in the (TTA·TAA)₁₈ repeat and the neighboring (A + T)-rich sequences of pRW3146 are influenced not only by pH, salt, and metal ion concentration but also by the type of anion.

DISCUSSION

We demonstrate that a TTA·TAA triplet repeat sequence shows the unusual propensity of adopting a non-H bonded structure in plasmids under appropriate conditions of supercoiling, temperature, pH, salt, and metal ion concentrations. Compared with the other nine TRS, only this TRS exhibits this behavior. The SspI fragment of pGE117.6, which contains 90 repeats of TTA·TAA and the neighboring (A + T)-rich sequences, was inserted into pUC19 to give pRW3143. Kang et al. (19) constructed several lengths of CTG·CAG repeats in plasmids by deletion of the original 130-repeat sequence derived from the myotonic dystrophy gene (19), and similar procedures worked effectively for other triplet repeats (1, 14, 15). Using this method, we constructed three other plasmids with shorter TTA·TAA repeats (60, 30, and 18 triplets) from pRW3143 as a result of deletions of a portion of the (TTA·TAA)₉₀ tract. Attempts to obtain expansions (1, 14, 19) of (TTA·TAA)₉₀ were unsuccessful.

The opened helix structure was observed in all four inserts by chemical probe (OsO₄, DEPC, and CAA) and two-dimensional gel electrophoretic analyses. All thymines and adenines in both strands of the TTA·TAA repeats reacted with the reagents and were detected by the primer extension assay as a result of the inhibition of elongation by the DNA polymerase (Sequenase). This result shows that non-H bonded single-stranded regions exist throughout the repeats. These single-stranded regions may be disoriented random coils or may be highly ordered, stacked structures; our data do not enable more definitive conclusions from a structural standpoint.

Fig. 8 shows a model of the unpairing of the TTA·TAA repeats and the neighboring (A + T)-rich sequences in pRW3146. This model also pertains to other longer TTA·TAA repeats since the chemical reactivities by OsO₄ were similar to that of pRW3146. This indicates that the helix opening of the TTA·TAA repeats and the neighboring (A + T)-rich sequences is not influenced by the repeat lengths. The opening was dependent on negative supercoil density and was not observed for linear DNA. The unpairing occurred gradually over a broad range of supercoil density, especially as compared with that found for triplexes or cruciforms. Hence, the transition is not a cooperative, all-or-none process.

The unpairing of the neighboring (A + T)-rich sequence was sensitive to environmental conditions also. As the temperature was increased, the (A + T)-rich sequence was modified strongly by OsO₄. At 37 °C, the TTA·TAA repeats and the neighboring (A + T)-rich sequences were entirely opened, but both regions were not opened at 0 °C (Fig. 8). The other neighboring region (5 side of the TTA·TAA repeats) was not unpaired even at 37 °C. Hence, high A/T content sequences were easily opened. The unpaired structure formed reversibly as a function of temperature. This temperature-sensitive type of helix opening has been found in other (A + T)-rich sequences (37, 38, 39, 42, 54, 57). Different modification patterns were found between OsO₄ (reaction time of 10 min) and DEPC (reaction time of 30 min) at 20 °C, indicating that longer incubation times caused further unpairing.

In addition, the concentration of metal ions affected the helix opening. Na⁺ (150 mM) and Mg²⁺ (5 mM) inhibited the unpairing at 20 °C. Under physiological conditions (37 °C, 150 mM NaCl, pH 7.4), the intensity of the OsO₄ modification was low, suggesting that in vivo the opening may not be occurring. Bowater et al. (39) suggested that some proteins will be required to facilitate the helix unpairing in order to regulate gene expression in vivo. Umek and Kowalski (59) also suggested that an (A + T)-rich sequence destabilizes helical structure and promotes unwinding for the initiation of DNA replication. The original (TTA·TAA)₉₀ and the neighboring (A + T)-rich sequences are located in the region upstream of the transcription start site, suggesting that the region may play a role in transcriptional initiation. However, the biological function(s) of the TTA·TAA tracts is unknown.

Furthermore, the opening was influenced by pH and buffer conditions. OsO₄ modifications differed in the (TTA·TAA)₅ tract between pH 7.4 and 8.0 in TE buffer. There also were different modifications between pH 4.5 and 8.0 in TAE buffer. Two-dimensional agarose gel electrophoresis showed relaxations in 0.5 × TBE buffer (pH 8.3) but not in TAE (pH 8.0), although the ionic strength of the two buffers is similar. On the other hand, OsO₄ reactivities were observed in both buffers although there was a difference between the two buffers in the (TTA·TAA)₂ tract. This suggested that the OsO₄ reaction is more sensitive than the two-dimensional gel electrophoresis; the gels measure an equilibrium state between relaxed and unrelaxed molecules, whereas the OsO₄ reactions can detect a transient single-stranded region. This electrophoresis phenomenon is peculiar to the (A + T)-rich sequence in this study because plasmids containing a Z-DNA forming sequence showed relaxation in both buffers. These results also indicate that the borate ions favor the helix unpairing more than the acetate ions. Sodium acetate has been shown in previous work (60) on studies with left-handed Z-DNA to have an unconventional effect on DNA conformation.

Kang et al. (13) observed strong pausings of DNA polymerases (Sequenase, the Klenow fragment, and human DNA polymerase ) at specific locations within CTG·CAG and CGG·CCG repeats depending on the number of repeats and pretreatment of the duplex template, indicating that appropriate lengths of the repeats adopted a non-B DNA conformation(s) which caused the pausings. We tested the capacity of the TTA·TAA repeats to cause pausing in the primer extension assay; 90 repeats of TTA·TAA, as well as the shorter inserts (Fig. 1), did not show any pausings of the DNA polymerases as had been found for CTG·CAG and CGG·CCG repeats. By comparison, pausings had been observed for 80 repeats of CTG·CAG (13). This and other studies (1, 13, 15)² show that the TTA·TAA and the other nine repeats have quite different biochemical and genetic properties.

Although the mechanism of the expansion of the CTG·CAG, CGG·CCG, and AAG·CTT repeats is not yet known (1, 12, 14, 15, 19, 61), slippage-mediated DNA structures may cause expansion during DNA replication (12, 62, 63, 64). Schlötterer and Tautz (65) showed that the synthesis rate for the TTA·TAA repeat in vitro is the fastest of the 10 triplet repeats, indicating that slippage correlated with the A/T content of the sequences. These results, along with the case of base pair disruption, may explain why polymerase pausing was not found with the TTA·TAA repeat. Since the TTA·TAA repeat sequences have several physical, biochemical, and genetic properties that differ significantly from those observed for the other nine repeats (1, 12, 13, 14, 15, 16),² we conclude that this sequence may play different roles in gene regulation.