Flexible DNA: Genetically Unstable CTG·CAG and CGG·CCG from Human Hereditary Neuromuscular Disease Genes

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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...

Section: Resultsmentioning

confidence: 99%

Section: Contribution Of Writhe and Twist To The Linking Numbermentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Gellibolian

Bacolla

Wells

1997

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“…The plasmid was 2705 bp in length with a total of 338 CG steps (on average, 1 every 8 bp). (CGG⅐CCG) 73 was an XbaI-BamHI restriction fragment from plasmid pRW3691 and contained a total of 156 CG steps (including 10 from the triplet repeat flanking sequences) (47). d(I-C⅐I-C) ϳ7000 (48) give a stock solution of 500 M CG steps.…”

Section: Dnmt1 and S-(5ј-adenosyl)-l-[methyl-mentioning

confidence: 99%

Recombinant Human DNA (Cytosine-5) Methyltransferase

Bacolla¹,

Pradhan²,

Roberts³

et al. 1999

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115

Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA and S-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA and S-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG⅐CCG) 12 , (m 5 CGG⅐CCG) 12 , and (CGG⅐CCG) 73 but were not linear for (CGG⅐Cm 5 CG) 12 . Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at <20 M AdoMet. Addition of the product (methylated DNA) to unmethylated plasmid DNA increased V max(app) resulting in mixed stimulation and inhibition. Velocity equations indicated a two-step mechanism as follows: first, activation of DNMT1 by methylated DNA that bound to an allosteric site, and second, the addition of AdoMet and DNA to the catalytic site. The preference of DNMT1 for hemimethylated DNA may be the result of positive cooperativity of AdoMet binding mediated by allosteric activation by the methylated CG steps. We propose that this activation plays a role in vivo in the regulation of maintenance methylation.The genome of most organisms contains modified nucleotides including N 6 -methyladenine, N 4 -methylcytosine, and C 5 -methylcytosine (m 5 C) 1 (1-3). However, both the biological significance and the types of DNA methylation differ greatly between prokaryotes and eukaryotes. In prokaryotes, most modified bases participate in restriction-modification, a defense mechanism that protects the host from heterologous phage infection (4). In addition, N 6 -methyladenine plays a role in the initiation of DNA replication (5) and in post-replicative methyl-directed mismatch repair (6).In higher eukaryotes, DNA methylation is confined to m 5 C and is implicated in the regulation of development, genomic imprinting (7, 8), X chromosome inactivation, gene expression (9), and retrotransposon inactivation (10 -12). In mammals, the patterns of methylation are inherited from both parental genomes but are erased and reconstructed (de novo methylation) in somatic cells following implantation (13,14). Such patterns are then copied and maintained by hemimethylation of the daughter strands during semiconservative DNA synthesis in S phase (maintenance methylation) (15, 16). However, the mechanisms by which both de novo and maintenance methylation occur remain to be elucidated.Several DNA methyltransferases have been isolated from human and mouse (17-20), but it is not clear whether the de novo and maintenance methylations are carried out by separate proteins in vivo or whether both activities are shared by one or more enzymes (21-26). Nevertheless, a role for in vivo methylation has been established for isoforms of the human DNMT1 gene (27, 28) whose product, DNMT1, me...

“…(Promega) and were described in detail (12,33,34). The repeat tracts in pRW3546 (12), pRW3691 (34), and pRW3246 (33) are located downstream of the functional lacZ promoter, thus allowing for the direct control of the transcription level by isopropyl-␤-D-thiogalactopyranoside (IPTG) induction and lacI Q repressor inhibition.…”

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confidence: 99%

Increased Negative Superhelical Density in Vivo Enhances the Genetic Instability of Triplet Repeat Sequences

Напиерала

Bacolla

Wells

2005

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The influence of negative superhelical density on the genetic instabilities of long GAA⅐TTC, CGG⅐CCG, and CTG⅐CAG repeat sequences was studied in vivo in topologically constrained plasmids in Escherichia coli. These repeat tracts are involved in the etiologies of Friedreich ataxia, fragile X syndrome, and myotonic dystrophy type 1, respectively. The capacity of these DNA tracts to undergo deletions-expansions was explored with three genetic-biochemical approaches including first, the utilization of topoisomerase I and/or DNA gyrase mutants, second, the specific inhibition of DNA gyrase by novobiocin, and third, the genetic removal of the HU protein, thus lowering the negative supercoil density (؊). All three strategies revealed that higher ؊ in vivo enhanced the formation of deleted repeat sequences. The effects were most pronounced for the Friedreich ataxia and the fragile X triplet repeat sequences. Higher levels of ؊ stabilize non-B DNA conformations (i.e. triplexes, sticky DNA, flexible and writhed DNA, slipped structures) at appropriate repeat tracts; also, numerous prior genetic instability investigations invoke a role for these structures in promoting the slippage of the DNA complementary strands. Thus, we propose that the in vivo modulation of the DNA structure, localized to the repeat tracts, is responsible for these behaviors. Presuming that these interrelationships are also found in humans, dynamic alterations in the chromosomal nuclear matrix may modulate the ؊ of certain DNA regions and, thus, stabilize/destabilize certain non-B conformations which regulate the genetic expansions-deletions responsible for the diseases.Genetic instability of microsatellite sequences have been widely observed throughout genomes of all organisms studied (1-3). In the majority of cases this phenomenon occurs without phenotypical consequences, but in some circumstances instability (mostly expansions of the repeat tracts) results in the development of disease (1, 4, 5). Expansions of tri-, tetra-and pentanucleotide microsatellites are related to the etiology of more than 20 neurological diseases including myotonic dystrophy types I and II, fragile X syndrome, Friedreich ataxia, and spinocerebellar ataxias (1,4,5).Studies on the mechanisms of trinucleotide repeat sequence (TRS) 2 expansions have revealed that replication, recombination, and repair, probably acting in concert, are responsible for the instabilities of the repetitive tracts (1, 4 -7). Several cis elements as well as trans-acting factors influencing genetic instabilities were discovered (8 -11). The sequences of the repeats, their length, and the presence of polymorphisms (interruptions) in the repeating tract are among the most important determinants of the extent of their instability (10,(12)(13)(14)(15). In addition, the orientation of the repeats relative to the origin of replication, their distance from the origin, transcription through the repeats, and DNA methylation status are factors (9, 16 -21).Expansions of the repeats in affected individuals occur ...