Triplet Repeat Instability and DNA Topology: An Expansion Model Based on Statistical Mechanics

Abstract

The variance of writhe, the contribution of writhe to supercoiling, and the free energies of supercoiling were calculated for (CTG⅐CAG) n and (CGG⅐CCG) n triplet repeat sequences (TRS) by statistical mechanics from the bending and torsional moduli previously determined. Expansions of these sequences are inherited by nonmendelian transmission and are linked with several hereditary neuromuscular diseases. The variance of writhe was greater for the TRS than for random B-DNA. For random B-DNA, (CGG) n , and (CTG) n , the contribution of writhe to supercoiling was 70, 78, and 79%, whereas the free energy of supercoiling at a length of 10 kilobase pairs was 1040⅐RT, 760⅐RT, and 685⅐RT, respectively. These data indicate that the TRS are preferential sites for the partitioning of supercoiling. Calculations of the differences in free energy of supercoiling between the TRS and random B-DNA revealed a local minimum at ϳ520 base pairs. Human medical genetic studies have shown that individuals carrying up to 180 -200 copies of TRS (540 -600 base pairs, premutations) in the fragile X or myotonic dystrophy gene loci are usually asymptomatic, whereas large expansions (>200 repeats, full mutations), which lead to disease, are observed in their offspring. Therefore, the length corresponding to the local minimum in free energy of supercoiling correlates with the genetic breakpoint between premutation and full mutation. We propose that (a) TRS instability is mediated by DNA mispairing caused by the accumulation of supercoiling within the repeats, and (b) the expansions that take place at the premutation to full mutation threshold are associated with increased mispairing caused by the optimal partitioning of writhe within the TRS at this length.Several human loci associated with neurodegenerative disorders have been shown to carry a new form of mutation, i.e. the expansion of a DNA triplet repeat sequence (TRS) 1 with composition (CTG⅐CAG) n , (CGG⅐CCG) n , or (GAA⅐TTC) n , referred to as (CTG) n , (CGG) n , and (GAA) n , respectively (reviewed in Refs. 1-4). These diseases fall into two categories (1).The first, which includes spinal and bulbar muscular atrophy, Huntington's disease, spinocerebellar ataxia type 1, dentatorubral-pallidoluysian atrophy, and Machado-Joseph disease, is characterized by small expansions of a (CTG) n repeat from the ϳ10 -40 in the normal population to ϳ40 -120 units in diseased individuals that encode a polyglutamine tract in the corresponding gene products. This mutation may impart a gain of function to the mature polypeptides that is deleterious to neuronal activity (1-3).The second, which includes the myotonic dystrophy (dystrophia myotonica (DM)) and the fragile X and E (FRAXA and FRAXE) genes, is characterized by much larger expansions of either a (CTG) n or (CGG) n repeat, respectively, in the untranslated region of the genes. The mechanisms by which these expansions lead to disease are not fully understood (5, 6). The number of repeats is polymorphic in the normal population and ranges from 6 to 5...

Genetic Instabilities in (CTG·CAG) Repeats Occur by Recombination

“…CTG⅐CAG seems to have special properties for recombination. Whereas the reason for this behavior is uncertain, prior investigations of nucleosome assembly (44), expansion by replication (29), conformational flexibility and writhing (26,38), capacity for adopting hairpin loops (23, 28 -32, 52), and susceptibility for double-strand breaks in vivo (18) revealed its unorthodox character. A prior review has summarized the molecular similarities between studies in humans and E. coli related to hereditary neurological diseases (45).…”

“…The box at the top of the figure represents a 300-bp sequence of (CTG⅐CAG) 100 within pRW-4404. The CTG⅐CAG tract adopts slipped structures by misalignment of the complementary strands by slippage (38,45,52). The staggered, single-stranded loops may rehybridize and, depending on the alignment of the loops, generate three forms of intrahelical pseudoknots: theta shape, figure eight, and bow-shaped structures.…”

“…Also, this length is in good agreement with prior determinations on the required extent of sequence identity (50 -75 bp) for homologous recombination in E. coli (36,37), especially considering that substantially different types of sequences were studied in rather disparate systems. Longer CTG⅐CAG tracts that are flexible and writhed (26) have a propensity to slip (38,52) which may initiate recombination. Prior work (39 -43) revealed the recombinogenic properties of simple, direct repeat sequences.…”

Molecular Basis for Expression of Common and Rare Fragile Sites

“…AT-dinucleotide runs were experimentally shown to be more flexible than random DNA (6) and thus can act as sinks for the superhelical density generated ahead of the replication fork in its progress. Accumulating superhelical density can hinder efficient topoisomerase activity and decrease the processivity of the polymerase complex (2,14). This perturbation is expected to be enhanced in the presence of low levels of aphidicolin, which inhibits the activity of polymerases ␣ and ␦.…”

Recombinant Human DNA (Cytosine-5) Methyltransferase

“…Because the bending restoring force, but not the torsional modulus, for CGG⅐CCG DNA is ϳ40% lower than for random DNA (34), we hypothesized that an expanded CGG⅐CCG tract would represent a genomic site for the preferential partitioning of negative supercoiling (37), which is known to unwind stretches of DNA (38). The free energy of supercoiling associated with unwinding would consequently generate alternative DNA structures such as slipped structures, which could increase the rates of the methylation reaction.…”