Trinucleotide repeat DNA structures: dynamic mutations from dynamic DNA.

Models for the disease-associated expansion of (CTG)n.(CAG)n, (CGG)n.(CCG)n, and (GAA)n.(TTC)n trinucleotide repeats involve alternative DNA structures formed during DNA replication, repair and recombination. These repeat sequences are inherently flexible and can form a variety of hairpins, intramolecular triplexes, quadruplexes, and slipped-strand structures that may be important intermediates and result in their genetic instability.

Structure and dynamics of three-way DNA junctions: atomic force microscopy studies.

We have used atomic force microscopy (AFM) to study the conformation of three-way DNA junctions, intermediates of DNA replication and recombination. Immobile three-way junctions with one hairpin arm (50, 27, 18 and 7 bp long) and two relatively long linear arms were obtained by annealing two partially homologous restriction fragments. Fragments containing inverted repeats of specific length formed hairpins after denaturation. Three-way junctions were obtained by annealing one strand of a fragment from a parental plasmid with one strand of an inverted repeat-containing fragment, purified from gels, and examined by AFM. The molecules are clearly seen as three-armed molecules with one short arm and two flexible long arms. The AFM analysis revealed two important features of three-way DNA junctions. First, three-way junctions are very dynamic structures. This conclusion is supported by a high variability of the inter-arm angle detected on dried samples. The mobility of the junctions was observed directly by imaging the samples in liquid (AFM in situ). Second, measurements of the angle between the arms led to the conclusion that three-way junctions are not flat, but rather pyramid-like. Non-flatness of the junction should be taken into account in analysis of the AFM data.

Length-dependent structure formation in Friedreich ataxia (GAA)n*(TTC)n repeats at neutral pH.

More than 15 human genetic diseases have been associated with the expansion of trinucleotide DNA repeats, which may involve the formation of non-duplex DNA structures. The slipped-strand nucleation of duplex DNA within GC-rich trinucleotide repeats may result in the changes of repeat length; however, such a mechanism seems less likely for the AT-rich (GAA)n*(TTC)n repeats. Using two-dimensional agarose gels, chemical probing and atomic force microscopy, we characterized the formation of non-B-DNA structures in the Friedreich ataxia-associated (GAA)n*(TTC)n repeats from the FRDA gene that were cloned with flanking genomic sequences into plasmids. For the normal genomic repeat length (n = 9) our data are consistent with the formation of a very stable protonated intramolecular triplex (H-DNA). Its stability at pH 7.4 is likely due to the high proportion of the T.A.T triads which form within the repeats as well as in the immediately adjacent AT-rich sequences with a homopurine. homopyrimidine bias. At the long normal repeat length (n = 23), a family of H-DNAs of slightly different sizes has been detected. At the premutation repeat length (n = 42) and higher negative supercoiling, the formation of a single H-DNA structure becomes less favorable and the data are consistent with the formation of a bi-triplex structure.

Hyperreactivity of B-Z junctions to 4,5',8-trimethylpsoralen photobinding assayed by an exonuclease III/photoreversal mapping procedure.

We have developed an exonuclease III/photoreversal procedure to map, with base-pair resolution, the bases that have photoreacted with 4,5',8-trimethylpsoralen (Me3-psoralen) forming either monoadducts or interstrand crosslinks in DNA. This assay allows quantification of relative rates of Me3-psoralen photobinding to bases in DNA at levels less than one crosslink per 8000 base-pairs. We demonstrate the applicability of the Me3-psoralen mapping procedure on the Z-forming sequence GAATT(CG)6-TA(CG)6AATTC. The results confirm our previous findings that Me3-psoralen forms crosslinks in the 5'TA within the (CG)6TA(CG)6 sequence when it exists in the B conformation but not when it exists in the Z conformation. In addition, with increasing superhelical density we observe at least a hundred-fold increased Me3-psoralen presumably represent B-Z junctions. The two presumed junctions respond differently with increasing negative superhelical tension, however, suggesting that the structures of these negative superhelical tension, however, suggesting that the structures of these junctions differ. This increased Me3-psoralen photoreactivity provides a positive signal for the presence of Z-DNA. The sequence and assay described here provide a "torsionally tuned probe" for determining the effective superhelical density of DNA in vivo.

CTG repeats associated with human genetic disease are inherently flexible.

The lengthening of tracts of CTG, CGG and GAA triplet repeats during progression of a pedigree has been associated with more than 12 human genetic diseases, including fragile X syndrome, myotonic dystrophy and Friedreich's ataxia. These repetitive sequence elements have the potential to form alternative DNA secondary structures that may contribute to their instability. The alternative DNA secondary structures may mediate errors during DNA replication, repair or recombination of the triplet repeat, leading to expansion. Here we show that DNA composed of pure CTG or CGG repeats exhibits anomalously fast mobility on polyacrylamide gels, confirming a previous observation for DNA containing CTG and CGG triplet repeats flanked by mixed sequence DNA. Moreover, we show that even short tracts of duplex CTG repeats have an unusual helix structure. CTG repeats reduce overall curvature associated with phased A-tract or GGCC curves, but alone they do not introduce curvature into DNA. The reduction in curvature of phased A-tracts by CTG repeats is similar to that afforded by an interspersed flexible region associated with a (TT).(TT) mispair. CTG-containing DNAs exhibit a rapid rate of cyclization, consistent with a flexible helix. These results suggest that tracts of (CTG).(CAG) repeats are inherently flexible. In addition, our results suggest that the unusual rapid electrophoretic mobility of CTG or CGG-containing DNA may be a consequence of an extended flexible DNA chain.

Leading strand specific spontaneous mutation corrects a quasipalindrome by an intermolecular strand switch mechanism.

Imperfect inverted repeats or quasipalindromes can undergo spontaneous, often complex mutational events that correct them to perfect palindromes. Two models that depend on the quasipalindrome providing a template for a specific mutational event have been described to explain this mutation: an intramolecular and an intermolecular strand switch model. A 17bp quasipalindrome containing a -1 deletion within the chloramphenicol acetyl transferase (CAT) gene in plasmid pJT7 undergoes a spontaneous +1 frameshift mutation that creates a perfect inverted repeat and a Cm(r) phenotype. By analyzing this mutation frequency in two plasmids that contain the CAT gene in either orientation with respect to the origin of replication, we show that the specific frameshift occurs preferentially in the leading strand during DNA replication. Due to the availability and proximity of the lagging strand template as a single strand during replication of the quasipalindrome in the leading but not lagging strand, we suggest that the specificity for the leading strand correction is due to a leading strand specific intermolecular strand switch rather than an intramolecular strand switch. To test this hypothesis, we have designed a genetic selection to detect a leading strand intermolecular strand switch. This selection utilizes asymmetric quasipalindromes, one of which contains two central stop codons. When cloned into the CAT gene in pJT7, reversion to Cm(r) requires inversion of the stop codons and addition of a +1 frameshift to correct the reading frame. The inversion of the central stop codons, which is predicted by an intermolecular but not an intramolecular strand switch, occurs concomitant with the specific correction of the original 17 bp quasipalindrome. Inversion of an asymmetric center can also be demonstrated when not under selective pressure using a quasipalindrome lacking central stop codons. These results are consistent with the correction of a quasipalindrome occurring predominantly by an intermolecular strand switch during replication of the leading strand.

Primer-template misalignments during leading strand DNA synthesis account for the most frequent spontaneous mutations in a quasipalindromic region in Escherichia coli.

Spontaneous mutant sequences which differ from the starting DNA sequence by the specific correction of quasipalindromic to perfect palindromic sequence are hallmarks of mutagenesis mediated by misalignments directed by palindromic complementarity. The mutant sequences are specifically predicted by templated, but ectopic, DNA polymerization on a misaligned DNA substrate. In a previous study, we characterized a spontaneous frameshift hotspot near a 17 bp quasipalindromic DNA sequence within the mutant chloramphenicol acetyl transferase (CAT) gene of plasmid pJT7. A one base-pair insertion hotspot, ectopically templated by misalignment mediated by palindromic complementarity, was shown to occur more frequently during synthesis of the leading than the lagging DNA strand. Here we analyze the misalignment mechanisms that can account for the DNA sequences of 123 additional spontaneous frameshift mutations (22 distinct genotypes) occurring in the same quasipalindromic DNA region in plasmids pJT7 and p7TJ (a pJT7 derivative with the CAT gene in the inverse orientation). Approximately 80% of the small frameshift mutants in each plasmid are predicted by palindromic misalignments of the leading strand. Smaller numbers of mutations are consistent with other DNA misalignments, including those predicted by simple slippage of the nascent DNA strand on its template. The results show that remarkable changes in the mutation spectra of a reporter gene may not be revealed by measurements of mutation frequency.

Unexpected formation of parallel duplex in GAA and TTC trinucleotide repeats of Friedreich's ataxia.

The onset and progress of Friedreich's ataxia (FRDA) is associated with the genetic instability of the (GAA).(TTC) trinucleotide repeats located within the frataxin gene. The instability of these repeats may involve the formation of an alternative DNA structure. Poly-purine (R)/poly-pyrimidine (Y) sequences typically form triplex DNA structures which may contribute to genetic instability. Conventional wisdom suggested that triplex structures formed by these poly-purine (R)/poly-pyrimidine (Y) sequences may contribute to their genetic instability. Here, we report the characterization of the single-stranded GAA and TTC sequences and their mixtures using NMR, UV-melting, and gel electrophoresis, as well as chemical and enzymatic probing methods. We show that the FRDA GAA/TTC, repeats are capable of forming various alternative structures. The most intriguing is the observation of a parallel (GAA).(TTC) duplex in equilibrium with the antiparallel Watson-Crick (GAA).(TTC) duplex. We also show that the GAA strands form self-assembled structures, whereas the TTC strands are essentially unstructured. Finally, we demonstrate that the FRDA repeats form only the YRY triplex (but not the RRY triplex) at neutral pH and the complete formation of the YRY triplex requires the ratio of GAA to TTC strand larger than 1:2. The structural features presented here and in other studies distinguish the FRDA (GAA)¿(TTC) repeats from the fragile X (CGG).CCG), myotonic dystrophy (CTG).(CAG) and the Huntington (CAG).(CTG) repeats.

The structure of intramolecular triplex DNA: atomic force microscopy study.

We applied atomic force microscopy (AFM) for direct imaging of intramolecular triplexes (H-DNA) formed by mirror-repeated purine-pyrimidine repeats and stabilized by negative DNA supercoiling. H-DNA appears in atomic force microscopy images as a clear protrusion with a different thickness than DNA duplex. Consistent with the existing models, H-DNA formation results in a kink in the double helix path. The kink forms an acute angle so that the flanking DNA regions are brought in close proximity. The mobility of flanking DNA arms is limited compared with that for cruciforms and three-way junctions. Structural properties of H-DNA may be important for promoter-enhancer interactions and other DNA transactions.

Torsionally tuned cruciform and Z-DNA probes for measuring unrestrained supercoiling at specific sites in DNA of living cells.

We describe the development and application of "torsionally tuned" Z-DNA and cruciform probes for analyzing the level of unrestrained supercoiling at specific sites in the DNA of living cells. This approach is applicable for the analysis of dynamic differences in supercoiled DNA in different parts of plasmid, bacterial, or eukaryotic chromosomes. Using a psoralen-based assay, we have shown that the Z-DNA forming sequence (CG)6TA(CG)6, cloned into plasmid pUC8, exists as Z-DNA in 30 to 40% of plasmid molecules in wild-type Escherichia coli. This level suggested an in vivo superhelical density of sigma = -0.034 at the site of insertion in the plasmid. A higher level of Z-DNA found in cells deficient in topoisomerase I (topA10) suggested an in vivo superhelical density of sigma = -0.048. We have constructed a set of torsionally tuned inverted repeated DNA molecules which require different superhelical densities for cruciform formation. Using these inverted repeats and a crosslink assay for cruciforms, we present quantitative evidence for the existence of cruciforms in living E. coli cells. Cruciform formation was dependent on DNA supercoiling in vivo and on the location of the inverted repeat within a plasmid. In topA10 cells cruciforms were detected in less than 0.5% of plasmids when cloned into two different transcriptional units: the lacZ and CAT genes. However, when cloned outside a transcriptional unit, cruciforms were found at levels up to 50% in topA10 cells. More cruciforms were found upstream than downstream from divergent promoters in pBR322. From analysis of the fraction of different inverted repeats existing as cruciforms in vivo and the levels of supercoiling required for cruciform formation in vitro, we estimate in vivo superhelical densities of sigma = -0.034 and -0.041 for the EcoRI site of pUC8-based plasmids in wild-type and topA10 cells, respectively.

A cruciform structural transition provides a molecular switch for chromosome structure and dynamics.

The interaction between specific sites along a DNA molecule is often crucial for the regulation of genetic processes. However, mechanisms regulating the interaction of specific sites are unknown. We have used atomic force microscopy to demonstrate that the structural transition between cruciform conformations can act as a molecular switch to facilitate or prevent communication between distant regions in DNA. Cruciform structures exist in vivo and they are critically involved in the initiation of replication and the regulation of gene expression in different organisms. Therefore, structural transitions of the cruciform may play a key role in these processes.

Relationship between Escherichia coli growth and deletions of CTG.CAG triplet repeats in plasmids.

Instabilities that are intrinsic to natural repetitive DNA sequences produce high frequencies of length changes in vivo. Triplet repeats cloned in plasmids in Escherichia coli undergo expansions and deletions, and this instability is affected by multiple factors. We show that CTG-CAG repeats in plasmids can influence the growth of E. coli, which affects the observed stabilities. At extended growth periods, the observed frequencies of deletions were dramatically increased if the cells passed through stationary phase before subculturing. Deletions were particularly pronounced for a plasmid containing the longest repeat, 525 bp in total, with the CTG sequence as the lagging strand template for replication. Measurements of cell growth showed that the lag phase associated with E. coli growth was increased for cultures containing plasmids with long CTG-CAG repeats, particularly when the CTG-containing strand was the lagging template. High frequencies of deletions were observed because of a growth advantage of cells containing plasmids with deleted triplet repeats. Incubation conditions that reduced the bacterial growth-rate produced a decreased extent of deletions, presumably because they alleviated the growth advantage of cells harboring plasmids with deleted triplet repeats. The experimental observations were simulated by a model in which shorter triplet repeats provided a growth advantage due to a shorter lag phase. We demonstrate that the accumulation of deletions within repeating sequences during growth of E. coli can be prevented, and discuss these findings in relation to the studies of repetitive DNA sequences. These are the first observations to show a direct influence between a plasmid-based DNA sequence or structure and factors controlling bacterial growth.

Structure of branched DNA molecules: gel retardation and atomic force microscopy studies.

DNA heteroduplexes as models for slipped strand DNA have been analyzed by polyacrylamide gel migration and atomic force microscopy (AFM). All heteroduplexes containing one hairpin or loop have reduced electrophoretic mobilities compared with that expected for their molecular weights. The retarded gel mobility correlates with the formation of a sharp kink detected by AFM. Increasing the hairpin length from 7 bp to 50 bp results in a monotonous decrease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no dependence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analysis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobility is due to an inherent curvature in one of the long arms. This is confirmed by the end-to-end distance measurements from AFM images. In addition, significant variation of the end-to-end distances is consistent with a dynamic structure of heteroduplexes at the three-way junction. Double heteroduplexes containing one hairpin in each of the complementary strands also separate in a gel as two isomers. Their appearance in AFM showed a complicated pattern of flat representations of the three-dimensional structure and may indicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart.

Structure and dynamics of supercoil-stabilized DNA cruciforms.

Understanding DNA function requires knowledge of the structure of local, sequence-dependent conformations that can be dramatically different from the B-form helix. One alternative DNA conformation is the cruciform, which has been shown to have a critical role in the initiation of DNA replication and the regulation of transcription in certain systems. In addition, cruciforms provide a model system for structural studies of Holliday junctions, intermediates in homologous DNA recombination. Cruciforms are not thermodynamically stable in linear DNA due to branch point migration, which makes their study using many biophysical techniques problematic. Atomic Force Microscopy (AFM) was applied to visualize cruciforms in negatively supercoiled plasmid DNA. Cruciforms are seen as clear-cut extrusions on the DNA filament with the lengths of the arms consistent with the size of the hairpins expected from a 106 bp inverted repeat. The cruciform exists in two different conformations, an extended one with the angle of ca. 180 degrees between the hairpin arms and a compact, X-type conformation, with acute angles between the hairpin arms and the main DNA strands. The ratio of molecules with the different conformations of cruciforms depends on ionic conditions. In the presence of high salt or Mg cations, a compact, X-type conformation is highly preferable. Remarkably, the X-conformation was highly mobile allowing the cruciform arms to adopt a parallel orientation. The structure observed is consistent with a model of the Holliday junction with a parallel orientation of the exchanging strands.

The influence of primary and secondary DNA structure in deletion and duplication between direct repeats in Escherichia coli.

We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events.

On the deletion of inverted repeated DNA in Escherichia coli: effects of length, thermal stability, and cruciform formation in vivo.

We have studied the deletion of inverted repeats cloned into the EcoRI site within the CAT gene of plasmid pBR325. A cloned inverted repeat constitutes a palindrome that includes both EcoRI sites flanking the insert. In addition, the two EcoRI sites represent direct repeats flanking a region of palindromic symmetry. A current model for deletion between direct repeats involves the formation of DNA secondary structure which may stabilize the misalignment between the direct repeats during DNA replication. Our results are consistent with this model. We have analyzed deletion frequencies for several series of inverted repeats, ranging from 42 to 106 bp, that were designed to form cruciforms at low temperatures and at low superhelical densities. We demonstrate that length, thermal stability of base pairing in the hairpin stem, and ease of cruciform formation affect the frequency of deletion. In general, longer palindromes are less stable than shorter ones. The deletion frequency may be dependent on the thermal stability of base pairing involving approximately 16-20 bp from the base of the hairpin stem. The formation of cruciforms in vivo leads to a significant increase in the deletion frequency. A kinetic model is presented to describe the relationship between the physical-chemical properties of DNA structure and the deletion of inverted repeats in living cells.

DNA structure, mutations, and human genetic disease.

The etiology of fragile X syndrome, myotonic dystrophy and Kennedy's disease has been attributed to the massive expansion of triplet repeat DNA sequences. This review details the relationships between the structural diversity of DNA, its secondary structure or DNA-directed mutagenesis, and the expansion of triplet repeats.

Analysis of trimethylpsoralen photoreactivity to Z-DNA provides a general in vivo assay for Z-DNA: analysis of the hypersensitivity of (GT)n B-Z junctions.

We have described an exonuclease III/photoreversal procedure to map, with base pair resolution, the bases which have photoreacted with 4,5',8-trimethylpsoralen (Me3-psoralen) forming either monoadducts or interstrand cross-links in DNA (20). This assay allows quantitation of relative rates of Me3-psoralen photobinding to bases in DNA at levels as low as one cross-link per 8,000 base pairs. This assay should be useful for a wide variety of applications of Me3-psoralen photobinding to DNA. Here, we demonstrate the applicability of the Me3-psoralen exo III assay for analysis of the conformation of the Z forming sequences (GT)12ATGT and GAATTC(TG)6TA(TG)6. We have shown previously that Me3-psoralen forms crosslinks in the 5'TA within the (CG)6TA(CG)6 sequence when it exists in the B conformation but not when it exists in the Z conformation (34). More recently we have confirmed this result with the exo III assay and have shown at least a hundred fold increase in Me3-psoralen photoreactivity at the 5'AT sequence within the EcoR I sites (GAATTC) which presumably represent B-Z junctions flanking (CG)6TA(CG)6 (20). Here we demonstrate both the characteristic decrease in psoralen photobinding to 5'TAs within (GT)12ATGT and (TG)6TA(TG)6 and the hyperreactivity of B-Z junctions. These characteristic properties of Me3-psoralen photobinding provide an assay for Z-DNA that is applicable in vivo. The general applicability of this approach for assaying Z-DNA in vivo is discussed.

DNA-directed mutations. Leading and lagging strand specificity.

The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.