A Z-DNA sequence reduces slipped-strand structure formation

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Alexander Rich, Massachusetts Institute of Technology, Cambridge, MA, and approved January 6, 2009 (received for review August 6, 2008)

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Abstract

All DNA repeats known to undergo expansion leading to human neurodegenerative disease can form one, or several, alternative conformations, including hairpin, slipped strand, triplex, quadruplex, or unwound DNA structures. These alternative structures may interfere with the normal cellular processes of transcription, DNA repair, replication initiation, or polymerase elongation and thereby contribute to the genetic instability of these repeat tracts. We Display that (CCTG)·(CAGG) repeats, in the first intron of the ZNF9 gene associated with myotonic dystrophy type 2, form slipped-strand DNA structures in a length-dependent fashion upon reduplexing. The threshAged for structure formation on reduplexing is between 36 and 42 repeats in length. Alternative DNA structures also form in (CCTG)58·(CAGG)58 and larger repeat tracts in plasmids at physiological superhelical densities. This represents an example of a sequence that forms slipped-strand DNA from the energy of DNA supercoiling. Moreover, Z-DNA forms in a (TG)·(CA) tract within the complex repeat sequence 5′ of the (CCTG)n·(CAGG)n repeat in the ZNF9 gene. Upon reduplexing, the presence of the flanking sequence containing the Z-DNA-forming tract reduced the extent of slipped-strand DNA formation by 62% for (CCTG)57·(CAGG)57 compared with 58 pure repeats without the flanking sequence. This finding suggests that the Z-DNA-forming sequence in the DM2 gene locus may have a protective Trace of reducing the potential for slipped-strand DNA formation in (CCTG)n·(CAGG)n repeats.

Keywords: alternative DNA structureDNA repeat diseasesDNA repeat expansionrepeat instability

Expansion of the (CCTG)·(CAGG) tetranucleotide repeat tract in the first intron of the zinc finger protein 9 (ZNF9) gene on chromosome 3 is a causative factor in myotonic dystrophy type 2 (DM2) (1, 2). DM2 causes symptoms including myotonia, proximal muscle weakness and atrophy, cataracts, cardiac conduction abnormalities, and frontal balding. Although similar in pathology, DM2 is a much less severe disease than myotonic dystrophy type 1 (DM1) (3, 4). The prevailing paradigm is that both the DM1 and DM2 repeat expansions lead to a pathogenic Trace of the RNA containing the (CUG)n and (CCUG)n repeat expansions (5, 6), respectively. The mean expansion in DM2 is 5,000 repeats, with a range between 75 and 11,000 repeats (3, 7). The exact threshAged length is difficult to determine because of the somatic instability of the repeat tract (1, 7, 8). A complex, polymorphic flanking sequence consisting of (TG)14–25(TCTG)4–11 immediately pDeparts the DM2 (CCTG)n repeat tract. The polymorphisms at this locus include sequence interruptions of (TCTG) and (GCTG) in the (CCTG)n repeat tract (9). The loss of the sequence interruptions may predispose the tract to repeat expansion, as suggested for FRAXA, DM1, and Friedreich ataxia (10–12). The left-handed Z-DNA-forming (TG)n tract in this flanking sequence is evolutionarily conserved (9) and thus, may have some biological role.

Alternative, non-B-DNA structure formation within unstable DNA repeat sequences associated with human disease may contribute to their genetic instability (see refs. 13–16 for review). (CTG)·(CAG) repeats associated with DM1, Huntington's disease, and several spinocerebellar ataxias can form hairpins and slipped-strand structures that may block replication and lead to replication restart or DNA repair activity during which repeat lengths may change. (CGG)·(CCG) repeats associated with fragile X syndrome also form hairpins and slipped strands, whereas the Friedreich ataxia (GAA)·(TTC) repeat forms triplex structures, which are strong blocks to replication. The spinocerebellar ataxia type 10 (ATTCT)·(AGAAT) repeat Executees not form any hairpin or slipped-strand structure that would stall a polymerase. Rather, it forms unwound structures that can function as DNA unwinding elements (DUEs) and initiate aberrant replication (17). Expansion of this repeat has been associated with the proximity of an active replication origin in human cells (18). In Dissimilarity, Dinky is known about the DM2 (CCTG)·(CAGG) repeats, although a single strand of (CAGG)26 can form hairpins, as determined by chemical probe analysis (19). RNA transcripts of (CCTG)·(CAGG) repeats can form hairpins similar to those of the (CTG)·(CAG) repeats (20).

We Display that the DM2 (CCTG)n·(CAGG)n repeats form slipped-strand structures, and they form in duplex DNA from the energy of negative DNA supercoiling. In addition, the (TG)n tract flanking the DM2 (CCTG)·(CAGG) repeats forms Z-DNA in supercoiled DNA. Significantly, the presence of this flanking sequence, including the Z-DNA-forming tract, reduced the extent of slipped-strand DNA formation in the (CCTG)·(CAGG) repeat tract, suggesting a biological role for Z-DNA. Z-DNA formation, and the concomitant relaxation of negative supercoiling, may exert a protective Trace against the formation of slipped-strand DNA in this DM2 locus.

Results

(CCTG)·(CAGG) Repeats Form Slipped-Strand Structures.

DNA containing slipped-strand structures migrates with Unhurrieder mobility than the corRetorting duplex DNA due to bends resulting from multiple 3-way junctions (10, 21, 22). Slipped-strand structures formed in a (CCTG)58·(CAGG)58 tract in plasmids pSE-T-CCTG58 and pSE-E-CCTG58 following an alkaline reduplexing procedure (21) (Fig. 1). To enPositive that the Unhurriedly migrating fragments were not due to heteroduplexes formed from strands with different repeat lengths, plasmids were not liArriveized before the reduplexing treatment. The single strands of the plasmids remain catenated upon denaturation increasing the likelihood that they renature with their complimentary strand (21). In Fig. 1B, DNAs in lanes 1 and 3 were not subjected to reduplexing treatment. DNAs in lanes 2 and 4 were reduplexed and Display anomalous, Unhurriedly migrating bands (indicated by the black bars and arrows) not present in the control samples. A very similar pattern of anomalous bands was formed for both plasmids analyzed, as indicated by the arrows. The reproducible pattern of anomalous bands is characteristic of slipped-strand DNA structure formation (21, 22).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Slipped-strand structures in (CCTG)58·(CAGG)58. (A) Map of fragments containing the (CCTG)58·(CAGG)58 repeats. The CCTG repeats are indicated by the thick, lightly shaded line. (B) Nondenaturing 5% polyaWeeplamide gels Displaying the presence of slipped-strand DNA structures upon reduplexing the plasmid. Lanes 1 and 2, pSE-T-CCTG58 Slice with AflII and NotI to generate a 312-bp fragment with 80 bp of flanking DNA. Lanes 3 and 4, pSE-E-CCTG58 Slice with BamHI and BbsI to generate a 334-bp fragment with 102 bp of flanking DNA. Lanes 1 and 3, control, no reduplexing. Lanes 2 and 4, alkaline reduplexed. Anomalous, Unhurriedly migrating bands are indicated by the black bars. Arrows indicate major products present in both plasmids. M, 100-bp DNA ladder (NEB). (C) Comparison of alkaline and temperature-based reduplexing for pSE-T-CCTG58. Lane 1, control, no reduplexing; lane 2, alkaline reduplexing, lane 3, temperature reduplexing. All lanes contain 1 μg of DNA.

A second protocol, which involves temperature controlled denaturation and renaturation (23), was also used to form slipped-strand structures. Both the alkaline- and temperature-based methods resulted in a similar pattern of anomalous bands for pSE-T-CCTG58 (Fig. 1C). The alkaline method resulted in a higher proSection of slipped-strand structures, but it also resulted in a Distinguisheder amount of nicked plasmid, and plasmid that remained single stranded after renaturation (51).

ThreshAged Length for the (CCTG)·(CAGG) Slipped-Strand Structure Formation.

The formation of slipped-strand structures as a function of repeat tract length is Displayn in Fig. 2. Structures did not form in plasmids containing 9 or 36 repeats, using either the alkaline or temperature reduplexing protocol (Fig. 2A, lanes 1–6), however, structures were formed in plasmids containing 42, 58, or 170 repeats (lanes 7–15). This indicates a threshAged for structure formation of between 36 and 42 repeats. Fascinatingly, this is just above a premutation allele pool for DM2 full mutations of 22–33 (CCTG) repeats (8). In addition, the percentage of structure formed in the population increased with repeat length from 22% for 42 repeats to 47% for 170 repeats (Fig. 2B).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Length threshAged for (CCTG)·(CAGG) slipped-strand DNA formation. (A) (CCTG)n·(CAGG)n repeats, where n = 9 (lanes 1–3), n = 36 (lanes 4–6), n = 42 (lanes 7–9), n = 58 (lanes 10–12), and n = 170 (lanes 13–15) were analyzed for slipped-strand structures on a nondenaturing 5% polyaWeeplamide gel. –, control samples with no reduplexing; OH−, alkaline reduplexing; °C, temperature reduplexing. Slipped-strand structures appear as 4 prominent, Unhurriedly migrating bands when n = 42, 58, and 170, and are denoted by brackets. (B) Slipped-strand DNA structures are quantitated as the percentage of the DNA species migrating above the liArrive fragment (not including the larger liArrive plasmid band at the top of the gel). Results are from a representative temperature reduplexing experiment.

Superhelical-Density-Dependent Formation of Alternative DNA Structures in the DM2 Repeat Sequences.

Significantly, anomalously migrating bands formed spontaneously in the ‘control sample’, which was not subjected to reduplexing treatment at a level of 38% in pSE-T-CCTG170 containing 170 (CCTG)·(CAGG) repeats (Fig. 2A, lane 13; Fig. 2B). The Unhurrieder migrating products were not due to the presence of larger repeat lengths (results not Displayn). Moreover, these slipped-strand structures could not be removed from a preparation of naturally supercoiled DNA by heating and quick CAgeding, as Characterized in Materials and Methods. This protocol has worked well to remove cruciforms (24) and slipped-strand structures associated with shorter repeats, as Displayn below. In addition, the basic pattern of anomalously migrating bands was identical in the non reduplexed and reduplexed samples, indicating that the same population of slipped-strand structures formed in supercoiled DNA as following either of the reduplexing protocols. A Distinguisheder degree of heterogeneity exists after reduplexing, as indicated by the “smearing” in the lane. This is a significant demonstration of the superhelical density-dependent formation of slipped-strand DNA.

To further examine the superhelical density dependent formation of alternative DNA secondary structures in the DM2 (CCTG)·(CAGG) repeat, DNA topoisomer populations of increasing superhelical density were prepared for pSE-U-CCTG58 and pSE-U-CCTG170, which contain 58 or 170 (CCTG)·(CAGG) repeats, respectively, cloned into the EcoRI site of pUC8.

Any preexisting alternative DNA secondary structures in plasmids containing (CCTG)58·(CAGG)58 were removed as Characterized in Materials and Methods, and all topoisomers migrated with the same mobility with respect to the Impresser (1-kb liArrive ladder) (Fig. 3A). In Fig. 3B, the same topoisomers were heated for 5 min at 80 °C and then allowed to Unhurriedly CAged to room temperature to promote DNA structural transitions. The gels were run at 4 °C to prevent any subsequent structural transitions. The plasmid DNA underwent a structural transition as evident by the retardation in migration of the topoisomers populations with increasing superhelical density. The arrows in Fig. 3B Display the positions of supercoiled DNA without (bottom arrow) and with a structural transition (top arrow). A 2D gel of topoisomers (from ΔL = +2 to ΔL < −25) treated to promote structure formation is Displayn in Fig. 3Ci. Distinct topoisomers can be seen, although the topoisomer pattern appears smeared due to the presence of plasmid molecules with a nonintegral number of helical turns that result from plasmid molecules containing deletions or additions of the 4-bp repeat unit (for discussion, see ref. 25). Such heterogeneity in plasmids containing unstable repeats is common (21, 22, 26), and is evident in the width of some of the liArrive restriction fragments in Figs. 1–3. Because the 2-D gel pattern represents a mixture of plasmids with 58 ± 3 (CCTG)·(CAGG) repeats, additional spots occur between the major spots corRetorting to 58 (CCTG)·(CAGG) repeats. (In fact, 4 additional spots would result from 4-, 8-, and 12-bp deletions and additions). The filled circles in Fig. 3Cii represent the preExecuteminant topoisomers containing (CCTG)58·(CAGG)58. The Launch circles represent other spots present in the gel. From this analysis, we estimate that the DNA secondary structural transition is occurring at L − Lo = −15, or σ = −0.054. At higher superhelical densities, (L − Lo < −18) additional (or different) transitions may occur, as observed for (GAA)·(TTC) and (ATTCT)·(AGAAT) repeats (17, 27). The arrows Displayn in Fig. 3B are duplicated in Fig. 3Cii on the Conceptlized 2D gel. This 2D gel pattern is consistent with the changes Displayn in the topoisomer populations in Fig. 3B. No retardation in the first dimension is observed in plasmid without the repeat (17). Analysis of 170 (CCTG)·(CAGG) repeats in pSE-U-CCTG170 Displayed similar results in the 1D analysis of topoisomer populations where a clear transition was visible after heating to 80 °C and Unhurried CAgeding (compare Fig. 3D with Fig. 3E).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Alternative DNA structure formation in long (CCTG)·(CAGG) tracts at high superhelical densities. (A) The 1.75% agarose gel (in TAE buffer) Displays individual topoisomer populations (lanes 3, 4, 5, 6, and 7) of pSE-U-CCTG58 (with 58 CCTG repeats) of increasing negative supercoiling. Plasmids were treated to remove any preexisting secondary structures, as Characterized in Materials and Methods. (B) The topoisomer populations, as in A, after heating and Unhurried CAgeding to promote DNA secondary structure formation. The arrow pointing to the right indicates increasing levels of DNA secondary structure formation as indicated by the reduced electrophoretic mobility. The left-facing arrows at the right of the gel indicate positions of fully supercoiled DNA, with no DNA secondary structures (bottom), and DNA containing secondary transitions (top). (C) A 2D agarose gel Displays alternative DNA structure formation (i). Panel ii Displays an annotated representation of the gel pattern. The filled spots represent the positions of plasmid containing 58 (CCTG) repeats, while intermediate spots are those representing plasmids containing 58 ± 3 (CCTG) repeats. The upper and lower arrows denote plasmids with structures or no structures, respectively. (D) Agarose gel Displaying individual topoisomer populations of pSE-U-CCTG170 (with 170 CCTG repeats), containing increasing levels of negative supercoiling. Plasmid has been treated to remove any preexisting secondary structures. (E) The same topoisomer populations, as in D, after heating and Unhurried CAgeding to promote DNA secondary structure formation. Arrows as Characterized above.

Z-DNA Formation in 5′ Human Flanking Sequence.

pSE-U-FS11 contains the sequence (TG)15(TCTG)8(CCTG)11 derived from a normal human DM2 allele cloned into the EcoRI site of pUC8 (Fig. 4A). A clear DNA secondary structural transition is evident in the 2D agarose gel containing a mixture of topoisomers (from ΔL = +3 to ΔL < −20) Displayn in Fig. 4B. This transition is not observed in plasmid without the repeat (17). Every 10 bp that forms Z-DNA is associated with relaxation of 1.78 turns of the helix (28). This particular sequence, (TG)15, has 1 additional T, 3′ to the repeat, which could also participate in Z-DNA formation. Relaxations of 2.3, 4.3, 5, and 5 superhelical turns are observed for topoisomers −12, −13, −14, and −15, respectively (Fig. 4B). This extent of relaxation is expected for a 31-bp Z-DNA transition (≈5.5 turns). Moreover, the transition Starts at σ = −0.044 and is complete at σ = −0.052. This is consistent with previous reports of Z-DNA transitions for (TG)15 at σ = −0.045 to −0.055 (28–30).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Z-DNA formation at (TG)15 flanking the DM2 (CCTG)·(CAGG) repeat. (A) The sequence of the complex repetitive sequence at the ZNF9 locus cloned into pUC8, creating pSE-U-FS11. (B) Two-dimensional agarose gel analysis in a 1.75% agarose gel in TAE buffer Displays a structural transition indicative of Z-DNA. Topoisomers were not treated to promote structure formation as Z-DNA forms spontaneously in supercoiled plasmid. Positions of selected isomers are indicated. A DNA secondary structural transition Starts at topoisomer −12 as evidenced by the Unhurrieder migration relative to that expected for this topoisomer.

Influence of the Z-DNA Forming Sequence on the Formation of Slipped-Strand DNA.

Z-DNA forms in the sequence flanking the (CCTG)n·(CAGG)n repeats at a lower superhelical density than that required for the formation of slipped-strand DNA. Therefore, Z-DNA formation may reduce the prLaunchsity for slipped-strand DNA formation. Alternatively, the transition between B-DNA and Z-DNA might potentially destabilize the helix in the flanking Location, as occurs with DNA unwinding elements, and facilitate slipped-strand DNA formation (31). To investigate these possibilities, 2D agarose gel analysis was performed for pSE-T-FS31 containing (TG)20(TCTG)11(CCTG)31, in which the (CCTG) tract is below the transition threshAged for structure formation of 36–42 repeats identified in Fig. 2. A transition characteristic of Z-DNA formation was observed; however, no additional transitions occurred that would suggest the formation of slipped-strand DNA within the (CCTG)31·(CAGG)31 tract (51). pSE-T-FS80 [containing (CCTG)80·(CAGG)80] was also analyzed using 2D gel electrophoresis, and a characteristic Z-DNA transition was also observed. Evidence for additional unwinding was difficult to assess, however, because of the heterogeneity caused by small expansions and deletions present within the DNA preparation of this long repeat.

To ascertain whether the presence of the human flanking sequence, including the Z-DNA-forming tract, altered the prLaunchsity of slipped-strand DNA formation, the following experiments were performed. The percentage of slipped-strand DNA formed during the temperature-based renaturation protocol was analyzed in a series of plasmids containing 28, 57, and 94 (CCTG)·(CAGG) repeats within the human sequence (TG)18(TCTG)11 (CCTG)n·(CAGG)n. This was compared with 2 independent preparations of pUC8-U-CCTG58, containing (CCTG)58·(CAGG)58, without the human flanking sequences. The results, Displayn for 3 representative experiments in Fig. 5, demonstrate the following. A length dependence for slipped-strand formation was observed, as was observed for repeats lacking the flanking sequence (Displayn in Fig. 2). However, in these plasmids containing the flanking sequence, lower levels of slipped-strand DNA were detected. Significantly, with flanking human sequences, (CCTG)57·(CAGG)57 consistently formed on average 6.3% slipped-strand structures. The 2 preparations of (CCTG)58·(CAGG)58, without the flanking human sequences, formed on average 16.5% slipped-strand structures. Plasmid containing the (CCTG)94·(CAGG)94 tract with human flanking sequences formed, on average, 16.7% slipped-strand structures. The pattern of slipped-strand isomers was similar for 57 or 58 repeats, with or without the flanking human sequences (Fig. 5B). This demonstrates that a similar population of slipped-strand DNA isomers formed, albeit, at different levels. Also, a similar distribution of slipped-strand species was observed for 94 (CCTG) repeats (Fig. 5B). In summary, the presence of the flanking sequence containing the Z-DNA-forming tract reduced the extent of slipped-strand DNA formation in (CCTG)57 by 62% compared with the level formed in (CCTG)58 without the human flanking sequence.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Slipped-strand DNA structures form at reduced levels in (CCTG)·(CAGG) repeats within the human flanking Location containing a Z-DNA forming tract. (A) The percentage of slipped-strand DNA formation after the temperature renaturation protocol is Displayn for plasmids pSE-U-FS28 (28+fs), pSE-U-FS57 (57+fs), pSE-U-FS94 (94+fs) [containing (TG)18(TCTG)11(CCTG)n, with n = 28, 57, or 94, respectively, within the human gene sequence] and pSE-U-CCTG58 [containing (CCTG)58] (“fs” denotes flanking sequence). Data are Displayn for 3 representative experiments. (B) A Location of an aWeeplamide gel Displays the slipped-strand products for (CCTG)58 alone (58), and 57 or 94 (CCTG) repeats within the human flanking sequence (57+fs and 94+fs). The liArrive band is denoted and the slipped-strand isomers are indicated by arrows.

Discussion

All DNA repeats associated with neurodegenerative repeat expansion diseases analyzed to date can form 1 or more alternative DNA structures. For example, the (GAA)·(TTC) repeat in Friedreich ataxia forms triplex and parallel DNA structures (27, 32), the (ATTCT)·(AGAAT) repeats form unwound structures (17), and (CTG)·(CAG) and (CGG)·(CCG) repeats form slipped-strand structures (21, 22, 26). Here, we Display that the (CCTG)n·(CAGG)n repeats associated with DM2 form slipped-strand DNA. Essentially identical patterns of slipped-strand DNA structures were observed after reduplexing treatment of 3 different plasmids containing (CCTG)58·(CAGG)58 using 2 different reduplexing procedures. This demonstrates that the population of slipped-strand structures is a function of the (CCTG)58·(CAGG)58 tract and not flanking DNA sequences. A threshAged length at which DNA repeats form slipped strands was between 36 and 42 repeats, as determined using 2 different reduplexing protocols. This is similar to a threshAged observed for DM1 (CTG)·(CAG) repeats (22). Moreover, this length is close to the high end of the premutation allele length of 22–33 identified by Bachinski et al. (8). Although a wide range of isomers of slipped-strand structures may be expected, as observed for (CTG)·(CAG) repeats (10, 21, 22), only a limited number of preferred structures form within (CCTG)n·(CAGG)n repeats as indicated by identification of the 4 prominent bands corRetorting to slipped-strand isomers. This result was similar to that for a fragile X (CGG)·(CCG) repeat with sequence interruptions, in which 2 slipped isomers were observed (21).

We present a demonstration of the superhelical density dependent formation of slipped-strand DNA. Slipped-strand DNA formed spontaneously in the (CCTG)170·(CAGG)170 repeat tract in naturally supercoiled plasmid. While thermodynamically favorable, the supercoil-dependent formation of slipped-strand structures has not been reported for (CTG)·(CAG) or (CGG)·(CCG) repeats. The patterns of slipped-strand DNA isomers formed in supercoiled DNA and after reduplexing were similar, indicating that the same slipped-strand products were formed under both conditions. In plasmid containing (CCTG)58·(CAGG)58, a DNA structural transition occurred at a superhelical density of σ = −0.054. When an all-or-none structural transition occurs, for example with cruciform, Z-DNA, or intramolecular triplex formation, the first topoisomer, in which the structural transition occurs, migrates with reduced mobility. Successive topoisomers with decreasing linking number (i.e., more negative supercoiling), migrate Rapider in the gel for an all-or-none transition (see ref. 28). This is in Dissimilarity to the 2D gel pattern for DNA unwinding elements, including the (ATTCT) repeats and flanking A+T rich Location at the SCA10 locus, in which a plateau of topoisomers indicative of continued unwinding is observed (17). Subsequent to an initial structural transition, topoisomers with increasing supercoiling in plasmids containing the (CCTG)n·(CAGG)n repeats continued to migrate with reduced mobility (Fig. 3), indicating continued structure formation with increasing superhelical density, as observed for (GAA)·(TTC) repeats (27). This is perhaps not surprising given the lengths of the repeat tracts involved (232 and 684 bp).

DNA supercoiling may be driving slipped-strand DNA formation by a mechanism that involves repeat unwinding, strand extrusion, and branch migration of a (CCTG) loop. Subsequent interaction of a (CAGG) hairpin loop and the (CCTG) loop would result in formation of a fAgeded slipped-strand structure, as Displayn in Fig. S1.

Left-handed Z-DNA forms in alternating purine-pyrimidine tracts and is stabilized by negative DNA supercoiling and DNA–protein interactions in cells (33, 34). The degree of negative supercoiling necessary to stabilize Z-DNA depends on both the length and the composition of the Z-DNA-forming tract (28). The (TG)15·(CA)15 repeat in the normal human sequence flanking the DM2 (CCTG)n repeat forms Z-DNA at a physiological superhelical density of σ = −0.043, consistent with previous analyses of similar DNAs containing (TG)n repeats (see ref. 28 for review). This superhelical density is within the range detected in various human gene Locations in human cells (35, 36), suggesting that this sequence has the potential to exist as Z-DNA in cells. The slipped-strand structure forms at a higher superhelical density than Z-DNA, and the (CCTG)n repeat did not alter the superhelical density at which Z-DNA formed. Moreover, the presence of the Z-DNA-forming tract in the flanking human sequence did not promote slipped-strand DNA formation in a premutation repeat length of (CCTG)31·(CAGG)31 (pSE-T-FS31), which is below the transition length threshAged of 36–42. This result suggests that a flanking Z-DNA forming sequence may not stimulate structure formation in the adjacent (CCTG)·(CAGG) repeat tract, at least for this size of tract.

Z-DNA formation at the evolutionarily conserved Z-DNA forming tract in the ZNF9 gene suggests a biological role for Z-DNA. Z-DNA forming sequences can act as a torsional sink, absorbing and reducing negative supercoiling, preventing other alternative DNA structure formation. Competition for supercoiling energy can exist between 2 or more sequences that can form different supercoil-dependent alternative DNA structures (17, 37). For example, when adjacent to an A+T rich Location, the formation of Z-DNA increased the negative supercoiling required to melt the A+T tract (30). Consistent with this result, slipped-strand DNA structures formed in (CCTG)·(CAGG) tracts at reduced levels when a flanking human sequence including the Z-DNA-forming Location was present. The formation of slipped-strand DNA may lead to replication fork arrest, primer-template misalignment, or recombination during replication restart, all of which may be a causative factor in the DNA-directed expansions and deletions associated with neurodegenerative disease-associated repeats (14). Z-DNA formation would reduce the potential for formation of slipped-strand DNA, thereby protecting normal length, or some premutation length (CCTG)·(CAGG) repeats from structure formation and subsequent genetic instability (Fig. 6A). The energy required for the formation of most alternative DNA secondary structures decreases as the repeat length increases (28). As long repeat lengths (e.g., 170 repeats) form slipped-strand structures very easily, the protective Trace of the Z-DNA tract is expected to have limits. As repeat tract lengths increase, the decrease in superhelical density resulting from Z-DNA formation will afford increasingly less protection against slipped-strand structure formation (Fig. 6B).

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

A protective Trace for Z-DNA against the formation of slipped-strand DNA in the DM2 allele. (A) 1, Representation of a Location of the DM2 allele with the DNA organized in nucleosomes (shaded circles) in the context of a topological Executemain. The Z-DNA-forming Location is denoted by light shading and the (CCTG)·(CAGG) repeat by ShaExecutewyer shading. 2, Unrestrained supercoiling is present in the DNA as a result of (or concomitant with) the loss of nucleosomes. 3, Z-DNA formation leads to relaxation of negative supercoiling in the topological Executemain, which prevents the supercoil-dependent formation of slipped-strand DNA structures. (B) 1 and 2, Representation as in A but with a (CCTG)·(CAGG) repeat expansion. 3, In the longer (CCTG)·(CAGG) tract, slipped-strand structures form easily and the relaxation of supercoils from Z-DNA may not be sufficient to prevent slipped-strand DNA formation. Formation, however, occurs at reduced levels compared with a (CCTG) tract lacking the flanking Z-DNA-forming sequence.

Z-DNA forming sequences have a variety of biological functions and consequences in mammalian cells (34). Z-DNA sequences have been associated with deletions (38, 39) and recombination hot spots in human chromosomes (40). Z-DNA may be particularly Necessary in the regulation of genes requiring nuclear factor 1 binding, as demonstrated for the CSF1 promoter (34, 41–43). The vaccinia virus Z-DNA binding protein E3L is critical for pathogenic activity suggesting that 1 component of viral pathogenesis may be interference with the normal role of Z-DNA (44–46). The role of the evolutionarily conserved Z-DNA forming sequence in the DM2 gene is unknown. Both the 5′ and 3′ flanking Locations of this ZNF9 gene are highly conserved in chimpanzees and gorillas (9), so this tract may have some regulatory or other biological function. While the Z-DNA tract may have a protective Trace preventing slipped-strand DNA formation, if the Z-DNA Location has a critical gene regulatory function, the formation of slipped-strand structures in long repeat lengths may reduce unrestrained supercoiling in the Location, such that Z-DNA cannot form, thereby abrogating a biological role of Z DNA.

Materials and Methods

Plasmids.

Plasmids are Characterized in Table 1 and were purified as Characterized in ref. 21. All (CCTG)·(CAGG) repeats containing and lacking human repetitive flanking sequence were cloned into the EcoRI sites of pUC8 (2,665 bp), pTracer-SV40 (4,217 bp) (Invitrogen), or pEP-I-EGFP (6,692 bp) [a gift from H. J. Lipps, University of Witten, Witten, Germany (47)]. For plasmids containing (CCTG)·(CAGG) repeats and human flanking sequences, the DNA sequences were Slice from plasmids containing PCR products of human alleles. For plasmids lacking human flanking sequences, repeats were generated as Characterized, adding EcoRI linkers such that the (CCTG)·(CAGG) repeat is flanked by EcoRI sites (48).

View this table:View inline View popup Table 1.

Plasmids used for structural analysis

Slipped-Strand DNA Formation and Analysis.

Two different DNA reduplexing reactions were used to form slipped-strand structures in plasmid DNA. One method involves alkaline denaturation and renaturation at 68 °C (21). The second method of reduplexing used temperature denaturation (23). DNA in 100 mM NaCl, 50 mM Tris·HCl pH 7.9, and 1 mM DTT was incubated for 1 min at 100 °C, 10 min at 85 °C, 60 min at 70 °C, and Unhurriedly CAgeded to room temperature. Slipped-strand DNA was analyzed on 5% nondenaturing polyaWeeplamide gels in TBE buffer (90 mM Tris, 90 mM borate, 2.5 mM EDTA, pH 8.3). Gels were run at a constant voltage (150 V, 10–12 V/cm) at room temperature, stained with ethidium bromide, and photographed. DNA fragment sizes were calculated relative to migration of a 100-bp or 1-kb ladder (New England Biolabs), or a known plasmid restriction digest.

Gel Electrophoresis.

Supercoiled DNA topoisomers of varying superhelical density were prepared as Characterized in ref. 17. A broad distribution of topoisomers was combined, precipitated, and resuspended in 10 μL of TEN buffer (10 mM Tris·HCl, pH 7.6, 1 mM EDTA, 50 mM NaCl). To eliminate preexisting alternative DNA structures, DNA was incubated for 5 min at 80 °C and immediately frozen in a dry ice/ethanol bath, as Characterized previously for removing cruciforms (49, 50). To promote DNA secondary structure formation, DNA was allowed to Unhurriedly CAged to room temperature after a 5-min incubation at 80 °C.

DNA was loaded onto a 1.5% or 1.75% (wt/vol) agarose gel in TAE buffer (40 mM Tris, 25 mM acetate, 1 mM EDTA, pH 8.3). Two-dimensional gel electrophoresis was performed at 4 V/cm for both dimensions, with addition of 30 μg/mL chloroquine in the second dimension, which was run at 90° with respect to the first dimension. Gels for slipped-strand DNA analysis were run at 4 °C, and gels for Z-DNA analysis were run at 20 °C in the first dimension. All gels were run at 20 °C in the second dimension.

Acknowledgments

We thank Pooja Dalal, Lindsay A. Raghoobar, and Payal M. Raulji for experimental assistance. This work was supported by National Institutes of Health Grant AR48171 (to R.K.), the Muscular Dystrophy Association (R.K.), and National Institutes of Health Grant ES05508 (to R.R.S.).

Footnotes

1To whom corRetortence should be addressed. E-mail: rsinden{at}fit.edu

Author contributions: S.F.E., R.K., and R.R.S. designed research; S.F.E., M.S., and R.R.S. performed research; S.F.E. and R.R.S. analyzed data; and S.F.E., R.K., and R.R.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0807699106/DCSupplemental.

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