Microsatellites are abundant in vertebrate genomes, but their sequence representation and length distributions vary greatly within each family of repeats (e.g., tetranucleotides). Biophysical studies of 82 synthetic single-stranded oligonucleotides comprising all tetra- and trinucleotide repeats revealed an inverse correlation between the stability of folded-back hairpin and quadruplex structures and the sequence representation for repeats > or =30 bp in length in nine vertebrate genomes. Alternatively, the predicted energies of base-stacking interactions correlated directly with the longest length distributions in vertebrate genomes. Genome-wide analyses indicated that unstable sequences, such as CAG:CTG and CCG:CGG, were over-represented in coding regions and that micro/minisatellites were recruited in genes involved in transcription and signaling pathways, particularly in the nervous system. Microsatellite instability (MSI) is a hallmark of cancer, and length polymorphism within genes can confer susceptibility to inherited disease. Sequences that manifest the highest MSI values also displayed the strongest base-stacking interactions; analyses of 62 tri- and tetranucleotide repeat-containing genes associated with human genetic disease revealed enrichments similar to those noted for micro/minisatellite-containing genes. We conclude that DNA structure and base-stacking determined the number and length distributions of microsatellite repeats in vertebrate genomes over evolutionary time and that micro/minisatellites have been recruited to participate in both gene and protein function.

Friedreich’s ataxia is caused by the massive expansion of the GAA·TTC repeats in intron 1 of the frataxin gene. Long GAA·TTC repeats form very stable triplex and/or sticky DNA structures which cause the two repeat tracks to adhere to each other. These structures inhibit the transcription of the frataxin gene thus diminishing the production of the mitochondrial protein, frataxin. Recent studies revealed that sticky DNA, which is unique to FRDA, forms inside living cells and the in vitro association of the long GAA·TTC tracks generates two independent supercoiled domains. Interestingly, DNA sequence-specific polyamides alleviate transcription inhibition associated with these long GAA·TTC repeats. Hence, these initial investigations aimed at gene targeted therapies for FRDA appear promising. Sticky DNA: in vivo Formation in E. coli and in vitro Association of Two Long GAA•TTC Sequences Related to Friedreich’s Ataxia. Leslie S. Son, Albino Bacolla, and Robert D. Wells. J. Mol. Biol. 360, 267-284 (2006). DNA Sequence-Specific Polyamides Alleviate Transcription Inhibition Associated with Long GAA•TTC Repeats in Friedreich’s Ataxia. Ryan Burnett, Christian Melander, James W. Puckett, Leslie S. Son, Robert D. Wells, Peter B. Dervan, and Joel M. Gottesfeld. Proc. Natl. Acad. Sci. U.S.A. 103, 11497-11502 (2006). Structure-Dependent Recombination Hot Spot Activity of GAA•TTC Sequences from Intron 1 of the Friedreich’s Ataxia Gene. Marek Napierala, Ruhee Dere, Alexandre A. Vetcher, and Robert D. Wells. J. Biol. Chem. 279, 6444-6454 (2004). Title: The Distribution of Polypurine•Polypyrimidine Sequences in the Human Genome Authors: Bacolla A, Collins JR, Gold B, Chuzhanova N, Ming Y, Stephens RM, Stefanov S, Olsh A, Jakupciak JP, Dean M, Lempicki RA, Cooper DN, and Wells RD Institutions: 1 Institute of Biosciences and Technology, Houston TX; 2 Advanced Biomedical Computing Center, NCI-Frederick, MD; 3 Laboratory of Genomic Diversity, NCI-Frederick, MD; 4 Institute of Medical Genetics, Cardiff University, Cardiff, UK; 5 DNA Technology Group, NIST, Gaithersburg, MD; 6 Laboratory of Immunopathogenesis and Bioinformatics, SAIC-Frederick, Inc., Frederick, MD. Corresponding author email address: abacolla@ibt.tamhsc.edu Abstract: Expansion of a polymorphic GAA•TTC repeat in the first intron of the FXN gene severely compromises gene expression, leading to recessive Friedreich’s Ataxia (FA). The mechanism is believed to involve non-B DNA structural intermediates either specific for the expanded sequence or common to other polypurine•polypyrimidine (R•Y) tracts. We searched the human genome for long, uninterrupted, R•Y tracts and found 814 that equaled or exceeded 250 bases, the longest totaling 1,303 bases in the CENTA1 gene. Comparative searches in other mammalian and avian genomes also revealed large numbers of tracts. With respect to location, the 814 tracts populated all chromosomes but were significantly clustered in the pseudoautosomal region of sex chromosomes, which plays an indispensable role in male meiosis and chromosome pairing. Of the repetitive motifs with>30 nt, runs of A•T were the most common with a total of 16,679 copies, followed by GAAA•TTTC motifs with 3,217 copies. The FArelated GAA•TTC motif was only present in <400 copies. Comparisons with the chimpanzee genome revealed that the long R•Y tracts evolved at rates at least 30 times faster than genome average. These results strongly implicate the R•Y tracts in the generation of double-strand breaks, which in turn promote high recombination rates, and therefore mutation. We conclude that R•Y tracts with lengths in the range of the FAexpanded GAA•TTC repeats are not uncommon in the human genome; however, none matched the monotonous repetition of identical motifs that characterize the pathological FA-expanded sequence. Expansion of a polymorphic GAA•TTC repeat in the first intron of the FXN gene severely compromises gene expression, leading to recessive Friedreich’s Ataxia (FA). The mechanism is believed to involve non-B DNA structural intermediates either specific for the expanded sequence or common to other polypurine•polypyrimidine (R•Y) tracts. We searched the human genome for long, uninterrupted, R•Y tracts and found 814 that equaled or exceeded 250 bases, the longest totaling 1,303 bases in the CENTA1 gene. Comparative searches in other mammalian and avian genomes also revealed large numbers of tracts. With respect to location, the 814 tracts populated all chromosomes but were significantly clustered in the pseudoautosomal region of sex chromosomes, which plays an indispensable role in male meiosis and chromosome pairing. Of the repetitive motifs with>30 nt, runs of A•T were the most common with a total of 16,679 copies, followed by GAAA•TTTC motifs with 3,217 copies. The FArelated GAA•TTC motif was only present in <400 copies. Comparisons with the chimpanzee genome revealed that the long R•Y tracts evolved at rates at least 30 times faster than genome average. These results strongly implicate the R•Y tracts in the generation of double-strand breaks, which in turn promote high recombination rates, and therefore mutation. We conclude that R•Y tracts with lengths in the range of the FAexpanded GAA•TTC repeats are not uncommon in the human genome; however, none matched the monotonous repetition of identical motifs that characterize the pathological FA-expanded sequence. Bacolla A., et al. (2006). Long homopurine•homopyrimidine sequences are characteristic of genes expressed in brain and the pseudoautosomal region. Nucleic Acids Res. 34, 2663-2675. Title: Replication through GAA repeats in Cos-1 Cells Authors: Maria M. Krasilnikova, Sergei M. Mirkin Institutions: Pennsylvania State University, University of Illinois at Chicago Corresponding author email address: muk19@psu.edu Abstract: Long GAA repeat in the intron of frataxin gene is responsible for severe neurological disorder Friedreich’s ataxia. The propensity of GAA repeat to impede replication and transcription observed in a number of model systems was proposed as a mechanism of the disease. The detailed mechanisms of how replication and transcription are affected in humans are unknown. It was shown that GAA repeat can adopt triplex structure conformation in supercoiled plasmids. This peculiar DNA conformation is believed to cause problems for the replication and transcription machineries. However, it still has to be proven that this structure is the main cause of Friedreich’s ataxia. We have previously shown that replication through GAA repeats, located within the plasmid, is slowed down in yeast. We also pointed out the correlation between replication stalling and instability of the repeat. Now we studied the effects of GAA repeat on replication in mammalian cells. We observed a very strong effect of two (GAA)57 stretches located within the same plasmid on replication in mammalian cells. The orientation of two stretches turned out to be crucial for replication blockage: they should be in head to tail orientation in order to be able to block replication progression. We believe that the complex between two GAA stretches, so-called “sticky DNA” causes replication blockage. Since an extremely long GAA stretch can be viewed as two separate stretches joined together in head to tail orientation, this complex formation can potentially form within a single stretch, provided that it is long enough. This stricture can cause a replication stalling and subsequent instability for the long GAA stretches, characteristic for the full-scale disease. Long GAA repeat in the intron of frataxin gene is responsible for severe neurological disorder Friedreich’s ataxia. The propensity of GAA repeat to impede replication and transcription observed in a number of model systems was proposed as a mechanism of the disease. The detailed mechanisms of how replication and transcription are affected in humans are unknown. It was shown that GAA repeat can adopt triplex structure conformation in supercoiled plasmids. This peculiar DNA conformation is believed to cause problems for the replication and transcription machineries. However, it still has to be proven that this structure is the main cause of Friedreich’s ataxia. We have previously shown that replication through GAA repeats, located within the plasmid, is slowed down in yeast. We also pointed out the correlation between replication stalling and instability of the repeat. Now we studied the effects of GAA repeat on replication in mammalian cells. We observed a very strong effect of two (GAA)57 stretches located within the same plasmid on replication in mammalian cells. The orientation of two stretches turned out to be crucial for replication blockage: they should be in head to tail orientation in order to be able to block replication progression. We believe that the complex between two GAA stretches, so-called “sticky DNA” causes replication blockage. Since an extremely long GAA stretch can be viewed as two separate stretches joined together in head to tail orientation, this complex formation can potentially form within a single stretch, provided that it is long enough. This stricture can cause a replication stalling and subsequent instability for the long GAA stretches, characteristic for the full-scale disease. Title: DNA sequence-specific polyamides alleviate transcription inhibition associated with long GAA•TTC repeats in Friedreich’s ataxia Authors: Daniel A. Harki*, Ryan Burnett, Christian Melander, James W. Puckett*, Leslie S. Son, Robert D. Wells, Peter B. Dervan*, and Joel M. Gottesfeld Institutions: *Division of Chemistry and Chemical Engineering, California Institute of Technology; Department of Molecular Biology, The Scripps Research Institute; Center for Genome Research, Institute for Biosciences and Technology, Texas A&M University System Health Science Center Corresponding author email address: harki@caltech.edu Abstract: The hyperexpansion of GAA•TTC triplet repeats

Non-B DNA conformations adopted by certain types of DNA sequences promote genetic instabilities, especially gross rearrangements including translocations. We conclude the following: (a) slipped (hairpin) structures, cruciforms, triplexes, tetraplexes and i-motifs, and left-handed Z-DNA are formed in chromosomes and elicit profound genetic consequences via recombination-repair, (b) repeating sequences, probably in their non-B conformations, cause gross genomic rearrangements (translocations, deletions, insertions, inversions, and duplications), and (c) these rearrangements are the genetic basis for numerous human diseases including polycystic kidney disease, adrenoleukodystrophy, follicular lymphomas, and spermatogenic failure.

The expansions of long repeating tracts of CTG.CAG, CCTG.CAGG, and GAA.TTC are integral to the etiology of myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), and Friedreich's ataxia (FRDA). Essentially all studies on the molecular mechanisms of this expansion process invoke an important role for non-B DNA conformations which may be adopted by these repeat sequences. We have directly evaluated the role(s) of the repeating sequences per se, or of the non-B DNA conformations formed by these sequences, in the mutagenic process. Studies in Escherichia coli and three types of mammalian (COS-7, CV-1, and HEK-293) fibroblast-like cells revealed that conditions which promoted the formation of the non-B DNA structures enhanced the genetic instabilities, both within the repeat sequences and in the flanking sequences of up to approximately 4 kbp. The three strategies utilized included: the in vivo modulation of global negative supercoil density using topA and gyrB mutant E. coli strains; the in vivo cleavage of hairpin loops, which are an obligate consequence of slipped-strand structures, cruciforms, and intramolecular triplexes, by inactivation of the SbcC protein; and by genetic instability studies with plasmids containing long repeating sequence inserts that do, and do not, adopt non-B DNA structures in vitro. Hence, non-B DNA conformations are critical for these mutagenesis mechanisms.

This chapter focuses on gross rearrangements caused by long triplet and other repeat sequences. The most fascinating and unique feature of TRSs and other repeat sequences in DNA is their ability to adopt alternative conformations that differ dramatically from the commonly known, right-handed, antiparallel, double helix, generally referred to as B-DNA. The formation of non-B conformations in vivo is mostly based on the behavior of the DNA sequences in vitro, their relationships with DNA topology, and in certain cases antibody binding. Some of most relevant nonB-DNA are slipped (hairpin) structures, cruciforms, triplexes, tetraplexes and/-motifs, and left-handed Z-DNA are formed in chromosomes and elicit profound genetic consequences via recombination repair. On the other hand, repeating sequences, probably in their nonB conformations, cause gross genomic rearrangements such as deletions, insertions, inversions, translocations, and duplications. These rearrangements are the genetic basis for scores of human diseases, including polycystic kidney disease, adrenoleukodystrophy, follicular lymphomas, and spermatogenic failure.

The capacity of (CTG . CAG)(n) and (GAA . TTC)(n) repeat tracts in plasmids to induce mutations in DNA flanking regions was evaluated in Escherichia coli. Long repeats of these sequences are involved in the etiology of myotonic dystrophy type 1 and Friedreich's ataxia, respectively. Long (CTG . CAG)(n) (where n = 98 and 175) caused the deletion of most, or all, of the repeats and the flanking GFP gene. Deletions of 0.6 - 1.8 kbp were found as well as inversions. Shorter repeat tracts ( where n = 0 or 17) were essentially inert, as observed for the (GAA . TTC)(176)-containing plasmid. The orientation of the triplet repeat sequence (TRS) relative to the unidirectional origin of replication had a pronounced effect, signaling the participation of replication and/or repair systems. Also, when the TRS was transcribed, the level of deletions was greatly elevated. Under certain conditions, 30 - 50% of the products contained gross deletions. DNA sequence analyses of the breakpoint junctions in 47 deletions revealed the presence of 1-8-bp direct or inverted homologies in all cases. Also, the presence of non-B folded conformations (i.e. slipped structures, cruciforms, or triplexes) at or near the breakpoints was predicted in all cases. This genetic behavior, which was previously unrecognized for a TRS, may provide the basis for a new type of instability of the myotonic dystrophy protein kinase (DMPK) gene in patients with a full mutation.

Genomic rearrangements are a frequent source of instability, but the mechanisms involved are poorly understood. A 2.5-kbp poly(purine.pyrimidine) sequence from the human PKD1 gene, known to form non-B DNA structures, induced long deletions and other instabilities in plasmids that were mediated by mismatch repair and, in some cases, transcription. The breakpoints occurred at predicted non-B DNA structures. Distance measurements also indicated a significant proximity of alternating purine-pyrimidine and oligo(purine.pyrimidine) tracts to breakpoint junctions in 222 gross deletions and translocations, respectively, involved in human diseases. In 11 deletions analyzed, breakpoints were explicable by non-B DNA structure formation. We conclude that alternative DNA conformations trigger genomic rearrangements through recombination-repair activities.

Homologous recombination was shown to enable the expansion of CTG·CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG·CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG·CAG)n repeats apparently recombine with an ∼60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG·CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG·CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.

Large expansions of GAA.TTC repeats in the first intron of the frataxin (X25) gene are the principal mutation responsible for Friedreich's ataxia (FRDA). Sticky DNA, based on R.R.Y triplexes, was found at the expanded GAA.TTC repeats from FRDA patients. The (GAAGGA.TCCTTC)(65) repeat occurs in the same frataxin locus but is nonpathogenic and does not form sticky DNA. To elucidate the behavior of sticky DNA, we introduced various extents of GGA.TCC interruptions into the long GAA.TTC repeat. More than 20% of GGA.TCC interruptions abolished the formation of sticky DNA. However, the GAA.TTC repeats with less than 11% of GGA.TCC interruptions formed triplexes and/or sticky DNA similar to the uninterrupted repeat sequence. These triplexes showed different P1 nuclease sensitivities, and the GGA.TCC interruptions were slightly more sensitive than the surrounding GAA.TTC repeats. Furthermore, genetic instability investigations in Escherichia coli revealed that a small number (4%) of interruptions substantially stabilized the long GAA.TTC tracts. Furthermore, the greater the extent of interruptions of the GAA.TTC repeats, the less inhibition of in vitro transcription was observed, as expected, based on the capacity of interruptions to inhibit the formation of sticky DNA. We propose that the interruptions introduce base mismatches into the R.R.Y triplex, which explains the observed chemical and biological properties.

The 2.5-kilobase pair poly(purine pyrimidine) (poly(R.Y)) tract present in intron 21 of the polycystic kidney disease 1 (PKD1) gene has been proposed to contribute to the high mutation frequency of the gene. To evaluate this hypothesis, we investigated the growth rates of II Escherichia coli strains, with mutations in the nucleotide excision repair, SOS, and topoisomerase I and/or gyrase genes, harboring plasmids containing the full-length tract, six 5'-truncations of the tract, and a control plasmid (pSPL3), The full-length poly(R.Y) tract induced dramatic losses of cell viability during the first few hours of growth and lengthened the doubling times of the populations in strains with an inducible SOS response. The extent of cell loss was correlated with the length of the poly(R.Y) tract and the levels of negative supercoiling as modulated by the genotype of the strains or drugs that specifically inhibited DNA gyrase or bound to DNA directly, thereby affecting conformations at specific loci. We conclude that the unusual DNA conformations formed by the PKD1 poly(R.Y) tract under the influence of negative supercoiling induced the SOS response pathway, and they were recognized as lesions by the nucleotide excision repair system and were cleaved, causing delays in cell division and loss of the plasmid, These data support a role for this sequence in the mutation of the PKD1 gene by stimulating repair and/or recombination functions.

The properties of duplex CTG·CAG and CGG·CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 × 10−19 erg·cm for CTG and 1.27 × 10−19 erg·cm for CGG, ∼40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were ∼60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2.3 × 10−19 erg·cm and 10.4 base pairs (bp)/turn for CTG and 2.4 × 10−19 erg·cm and 10.3 bp/turn for CGG, respectively, all within the range for random B-DNA. Determination of the apparent helical repeat by the band shift assay indicated that the writhe of the repeats was different from that of random B-DNA. In addition, molecules of 224–245 bp in length (64–71 triplet repeats) were able to form topological isomers upon cyclization. The low bending moduli are consistent with predictions from crystallographic variations in slide, roll, and tilt. No unpaired bases or non-B-DNA structures could be detected by chemical and enzymatic probe analyses, two-dimensional agarose gel electrophoresis, and immunological studies. Hence, CTG and CGG are more flexible and highly writhed than random B-DNA and thus would be expected to act as sinks for the accumulation of superhelical density.

Induction of transcription into long CTG.CAG repeats contained on plasmids in Escherichia coli is shown to increase the frequency of deletions within the repeat sequences, This elevated genetic instability was detected because active transcription into the triplet repeat influenced the growth transitions of the host cell, allowing advantageous growth for cells harboring plasmids with deleted repeat sequences, The variety of deletion products observed in separate cultures suggests that transcription altered the metabolism of the DNA in a manner that produced random length changes in the repeat sequence, For cultures containing plasmids without active transcription into the triplet repeat, or those maintained in exponential growth, deletions occurred within the repeat at a lower frequency (5-20-fold lower), In these incubations the extent of deletions was proportional to the number of cell divisions and many repeat lengths were observed within each culture, suggesting that the decrease in average repeat length at long incubation times was due to multiple small deletions, These observations show that deletions within long CTG.CAG repeats contained on plasmids in E. coli occur via more than one pathway and their level of genetic instability is altered by the enzymatic processes occurring upon the DNA.

The properties of an intramolecular tripler formed in vitro at the 5'-flanking region of the human gamma-globin genes were studied by chemical and physical probes, Chemical modifications performed with osmium tetroxide, chloroacetaldehyde, and diethyl pyrocarbonate revealed the presence of non-paired nucleotides on the ''coding strand'' at positions -209 through -217, These reactivities were induced by negative supercoiling, low pH, and magnesium ions, Downstream point mutations associated with hereditary persistence of fetal hemoglobin (HPFH) altered the extent of the modifications and some of the patterns, Specifically, C-202 --> G and C-202 --> T significantly decreased the reactivities, whereas the patterns were increased and altered in the T-198 --> C, C-196 --> T and C-195 --> G caused local decreases in reactivity, Modifications at the upstream flanking duplex were modulated by the composition of the vector sequence, In summary, our data indicates the formation of an intramolecular tripler between nucleotides -209 to -217 of the ''non-coding strand'' and the downstream sequence containing the HPFH mutations, All of the HPFH point mutations altered the structure, More than one sequence alignment is possible for each of the triplexes, In addition, a consequence of some of the point mutations may be to facilitate slippage of the third strand relative to the Watson-Crick duplex.

The structural and energetic consequences of cytosine methylation in the 5-position on the supercoil-dependent B-Z equilibrium in alternating dC-dG sequences cloned into recombinant plasmids were investigated. The helical parameters determined with the band shift method for right-handed [10.7 base pairs (bp)/turn] and left-handed (12.8 bp/turn) 5MedC-dG inserts were different from the helical repeat values for unmethylated dC-dG inserts (10.5 bp/turn in the right-handed and 11.5 bp/turn in the left-handed form). We analyzed the thermodynamic parameters delta GBZ (free energy difference per base pair between right-handed and left-handed helix structure), delta Gjx (free energy for formation of one B-Z junction), and b (helix unwinding at a junction region) for varying lengths of dC-dG inserts by two-dimensional gel electrophoresis and application of a statistical mechanics model. A comparison of plasmids fully methylated in vitro with HhaI methylase and their unmethylated counterparts revealed that delta Gjx is not significantly changed by cytosine methylation. However, this base modification results in an approximate 3-fold decrease of delta GBZ and an approximate 2-fold decrease of the unwinding b at B-Z junction regions. Analysis of a pair of related plasmids, each containing two dC-dG blocks, revealed qualitatively different transition behaviors. When the two dC-dG blocks were separated by 95 bp of a mixed sequence, they underwent independent B to Z transitions with separate nucleation events and junction formations. When the two blocks were separated by only a 4 bp GATC sequence, only one nucleation event was necessary, and the Z-helix spread across the nonalternating GATC region.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosine methylation has energetic and structural influences on left-handed Z-DNA formation in supercoiled plasmids. The restriction and modification enzymes from Haemophilus haemolyticus (HhaI and M · HhaI) provide a system to locate and analyze small segments of Z-DNA in large supercoiled plasmids. An approach is outlined that uses M · HhaI as an in vivo conformational probe for the detection of unusual DNA structures in a living cell. Also, characteristic features of the M · HhaI gene and protein are discussed.

Right-handed B and left-handed Z conformations coexist in equilibrium in portions of plasmids in Escherichia coli. The equilibria are influenced by the length of the sequences that undergo the structural transitions and are perturbed by biological processes. The composite results of three types of determinations indicate a supercoil density of -0.025 in vivo. The coexistence of alternative DNA conformations in living cells implies the potential of these structures or their transitions for important functions in genetic regulatory processes.

A genetic-biochemical assay has been developed to investigate the in vivo existence and consequences of unusual DNA structures. Left-handed DNA was shown to exist in living Escherichia coli. The EcoRI methyltransferase gene (temperature-sensitive) was cloned to serve as a probe for perturbed GAATTC sites in vivo. This plasmid was cotransformed with different plasmids containing inserts that had varying capacities to form left-handed helices or cruciforms with a target EcoRI site in the center or at the ends of the inserts. Inhibition of methylation in vivo was found for the stable inserts with the longest left-handed helices. In vitro methylation with the purified M.EcoRI enzyme agreed with the in vivo results.