RESUMO
The disease-associated expansion of (CTG)*(CAG) repeats is likely to involve slipped-strand DNAs. There are two types of slipped DNAs (S-DNAs): slipped homoduplex S-DNAs are formed between two strands having the same number of repeats; and heteroduplex slipped intermediates (SI-DNAs) are formed between two strands having different numbers of repeats. We present the first characterization of S-DNAs formed by disease-relevant lengths of (CTG)*(CAG) repeats which contained all predicted components including slipped-out repeats and slip-out junctions, where two arms of the three-way junction were composed of complementary paired repeats. In S-DNAs multiple short slip-outs of CTG or CAG repeats occurred throughout the repeat tract. Strikingly, in SI-DNAs most of the excess repeats slipped-out at preferred locations along the fully base-paired Watson-Crick duplex, forming defined three-way slip-out junctions. Unexpectedly, slipped-out CAG and slipped-out CTG repeats were predominantly in the random-coil and hairpin conformations, respectively. Both the junctions and the slip-outs could be recognized by DNA metabolizing proteins: only the strand with the excess repeats was hypersensitive to cleavage by the junction-specific T7 endonuclease I, while slipped-out CAG was preferentially bound by single-strand binding protein. An excellent correlation was observed for the size of the slip-outs in S-DNAs and SI-DNAs with the size of the tract length changes observed in quiescent and proliferating tissues of affected patients-suggesting that S-DNAs and SI-DNAs are mutagenic intermediates in those tissues, occurring during error-prone DNA metabolism and replication fork errors.
Assuntos
DNA/química , Sequências Repetitivas de Ácido Nucleico , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Humanos , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/metabolismo , Ácidos Nucleicos Heteroduplexes/ultraestrutura , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismoRESUMO
The genetic stability of tandemly repeated DNAs is affected by repeat sequence, tract length, tract purity, and replication direction. Alterations in DNA methylation status are thought to influence many processes of mutagenesis. By use of bacterial and primate cell systems, we have determined the effect of CpG methylation on the genetic stability of cloned di-, tri-, penta- and minisatellite repeated DNA sequences. Depending on the repeat sequence, methylation can significantly enhance or reduce its genetic stability. This effect was evident when repeat tracts were replicated from either direction. Unexpectedly, methylation of adjacent sequences altered the stability of contiguous repeat sequences void of methylatable sites. Of the seven repeat sequences investigated, methylation stabilized five, destabilized one, and had no effect on another. Thus, although methylation generally stabilized repeat tracts, its influence depended on the sequence of the repeat. The current results lend support to the notion that the biological consequences of CpG methylation may be affected through local alterations of DNA structure as well as through direct protein-DNA interactions. In vivo CpG methylation in bacteria may have technical applications for the isolation and stable propagation of DNA sequences that have been recalcitrant to isolation and/or analyses because of their extreme instability.
Assuntos
Clonagem Molecular , Ilhas de CpG/genética , Metilação de DNA , Repetições de Dinucleotídeos/genética , Expansão das Repetições de Trinucleotídeos/genética , Anormalidades Múltiplas/enzimologia , Anormalidades Múltiplas/genética , Animais , Células COS , Linhagem Celular , Centrômero/enzimologia , Centrômero/genética , Centrômero/patologia , Chlorocebus aethiops , Clonagem Molecular/métodos , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/deficiência , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , DNA-Citosina Metilases/biossíntese , DNA-Citosina Metilases/genética , DNA-Citosina Metilases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Face/anormalidades , Guanina/metabolismo , Humanos , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Plasmídeos/biossíntese , Plasmídeos/genética , Plasmídeos/metabolismo , Síndrome , TransfecçãoRESUMO
The mechanism of disease-associated trinucleotide repeat instability involves cis-acting factors (cis-elements) in the vicinity of the repeat, but the nature of these elements is unknown. One cis-element may be the location of the replication origin relative to the repeat. We have used an SV40 DNA replication system to investigate the effect of the location of replication initiation on (CTG)(n)*(CAG)(n) stability in primate cells. Depending on the distance between the SV40 replication origin and the repeat tract, templates with 79 repeats yield predominantly expansions or predominantly deletions or remain intact. All templates with 17 repeats are stable. Thus, cis-elements that affect the sites of Okazaki fragment initiation relative to the repeat are crucial determinants of instability. This model system recapitulates the bias for expansions observed in many of the diseases associated with trinucleotide repeats. Our results might explain the variable amounts of CTG/CAG instability that are observed in different chromosomal contexts.