Richard R. Sinden

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.

Biological implications of the DNA structures associated with disease-causing triplet repeats.

The discovery of expanding repeat tracts associated with human disease has revealed an unexpected characteristic of a simple DNA sequence in the context of the human genome. This instability and the propensity for massive expansion likely reflect a new mutational mechanism (or combination of processes) that is not a feature of simpler model genetic organisms. The molecular mechanisms responsible for the genetic instability observed in triplet-repeat–associated disorders likely involve the unique structural properties associated with simple triplet-repeat tracts. A greater understanding of both the triplet-repeat DNA structures and the molecular mechanisms responsible for spontaneous mutations will be required before we can understand which of the models discussed above are responsible for repeat instability leading to triplet-repeat–associated human diseases.

Triplet repeat DNA structures and human genetic disease: dynamic mutations from dynamic DNA.

Fourteen genetic neurodegenerative diseases and three fragile sites have been associated with the expansion of (CTG)n•(CAG)n, (CGG)n•(CCG)n, or (GAA)n•(TTC)n repeat tracts. Different models have been proposed for the expansion of triplet repeats, most of which presume the formation of alternative DNA structures in repeat tracts. One of the most likely structures, slipped strand DNA, may stably and reproducibly form within triplet repeat sequences. The propensity to form slipped strand DNA is proportional to the length and homogeneity of the repeat tract. The remarkable stability of slipped strand DNA may, in part, be due to loop-loop interactions facilitated by the sequence complementarity of the loops and the dynamic structure of three-way junctions formed at the loop-outs.

DNA structure, mutations, and human genetic disease.

The etiology of fragile X syndrome, myotonic dystrophy and Kennedys 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.

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

The onset and progress of Friedreichs 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.

Neurodegenerative diseases: Origins of instability

Errors in DNA replication are thought to underlie the lengthening of tracts of repeated DNA that occurs in some neurodegenerative diseases. But mechanisms for repairing damaged DNA may also be responsible.

DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli.

The influence of mutations in the 3′ to 5′ exonucleolytic proofreading ε-subunit of Escherichia coliDNA polymerase III on the genetic instabilities of the CGG·CCG and the CTG·CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49 ts and themutD5 mutations destabilize the CGG·CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG·CAG repeats were destabilized by thednaQ49 ts mutation by a process mediated by long hairpin loop structures (≥5 repeats). The mutD5 mutator strain stabilized the (CTG·CAG)175 tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in themutD5 strain, presumably due to the greater stability of long CTG·CAG tracts (>100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG·CAG and CGG·CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.

Stably maintained microdomain of localized unrestrained supercoiling at a Drosophila heat shock gene locus.

A psoralen crosslinking assay was utilized to detect localized, unrestrained DNA supercoiling (torsional tension) in vivo in Drosophila chromosomal regions subject to differential transcriptional activity. By comparing rates of crosslinking in intact cells with those in cells where potential tension in chromosomal domains was relaxed by DNA strand nicking, the contribution to psoralen accessibility caused by altered DNA‐protein interactions (e.g. nucleosomal perturbations) was distinguished from that due to the presence of unrestrained supercoiling in a region of interest. The heat shock protein 70 (hsp70) genes were wound with a significant level of superhelical tension that remained virtually unaltered whether or not the genes were transcriptionally activated by thermal elevation. Constitutively expressed 18S ribosomal RNA genes also exhibited unrestrained superhelical tension at a level comparable with that across hsp70. In contrast, flanking regions downstream of each of the divergent hsp70 genes at locus 87A7 exhibited substantially less tension. Thus the results point to the existence of stable, torsionally stressed topological domains within eukaryotic chromosomal DNA, suggesting that the relaxing action of topoisomerases is not ubiquitous throughout the nucleus but, in fact, is likely to be tightly regulated.

Targeted transposition by the V(D)J recombinase

ABSTRACT Cleavage by the V(D)J recombinase at a pair of recombination signal sequences creates two coding ends and two signal ends. The RAG proteins can integrate these signal ends, without sequence specificity, into an unrelated target DNA molecule. Here we demonstrate that such transposition events are greatly stimulated by—and specifically targeted to—hairpins and other distorted DNA structures. The mechanism of target selection by the RAG proteins thus appears to involve recognition of distorted DNA. These data also suggest a novel mechanism for the formation of alternative recombination products termed hybrid joints, in which a signal end is joined to a hairpin coding end. We suggest that hybrid joints may arise by transposition in vivo and propose a new model to account for some recurrent chromosome translocations found in human lymphomas. According to this model, transposition can join antigen receptor loci to partner sites that lack recombination signal sequence elements but bear particular structural features. The RAG proteins are capable of mediating all necessary breakage and joining events on both partner chromosomes; thus, the V(D)J recombinase may be far more culpable for oncogenic translocations than has been suspected.

DNA structural transitions within the PKD1 gene

Autosomal dominant polycystic kidney disease (ADPKD) affects over 500 000 Americans. Eighty-five percent of these patients have mutations in the PKD1 gene. The focal nature of cyst formation has recently been attributed to innate instability in the PKD1 gene. Intron 21 of this gene contains the largest polypurine. polypyrimidine tract (2.5 kb) identified to date in the human genome. Polypurine.polypyrimidine mirror repeats form intramolecular triplexes, which may predispose the gene to mutagenesis. A recombinant plasmid containing the entire PKD1 intron 21 was analyzed by two-dimensional gel electrophoresis and it exhibited sharp structural transitions under conditions of negative supercoiling and acidic pH. The superhelical density at which the transition occurred was linearly related to pH, consistent with formation of protonated DNA structures. P1 nuclease mapping studies of a plasmid containing the entire intron 21 identified four single-stranded regions where structural transitions occurred at low superhelical densities. Two-dimensional gel electrophoresis and chemical modification studies of the plasmid containing a 46 bp mirror repeat from one of the four regions demonstrated the formation of an H-y3 triplex structure. In summary, these experiments demonstrate that a 2500 bp polypurine.polypyrimidine tract within the PKD1 gene is capable of forming multiple non-B-DNA structures.