RESUMO
Common fragile sites are specific loci that form gaps and constrictions on metaphase chromosomes exposed to replication stress, which slows DNA replication. These sites have a role in chromosomal rearrangements in tumors; however, the molecular mechanism of their expression is unclear. Here we show that replication stress leads to focus formation of Rad51 and phosphorylated DNA-PKcs, key components of the homologous recombination (HR) and nonhomologous end-joining (NHEJ), double-strand break (DSB) repair pathways, respectively. Down-regulation of Rad51, DNA-PKcs, or Ligase IV, an additional component of the NHEJ repair pathway, leads to a significant increase in fragile site expression under replication stress. Replication stress also results in focus formation of the DSB markers, MDC1 and gammaH2AX. These foci colocalized with those of Rad51 and phospho-DNA-PKcs. Furthermore, gammaH2AX and phospho-DNA-PKcs foci were localized at expressed fragile sites on metaphase chromosomes. These findings suggest that DSBs are formed at common fragile sites as a result of replication perturbation. The repair of these breaks by both HR and NHEJ pathways is essential for chromosomal stability at these sites.
Assuntos
Sítios Frágeis do Cromossomo/genética , Reparo do DNA/fisiologia , Recombinação Genética/fisiologia , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sítios Frágeis do Cromossomo/fisiologia , DNA Ligase Dependente de ATP , DNA Ligases/fisiologia , Proteína Quinase Ativada por DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Células HeLa , Histonas/fisiologia , Humanos , Metáfase/fisiologia , Proteínas Nucleares/fisiologia , Fosforilação , Rad51 Recombinase/fisiologia , Transativadores/fisiologiaRESUMO
A significant fraction of disease-causing mutations affects pre-mRNA splicing. These mutations can generate both aberrant and correct transcripts, the level of which varies among different patients. An inverse correlation was found between this level and disease severity, suggesting a role for splicing regulation as a genetic modifier. Overexpression of splicing factors increased the level of correctly spliced RNA, transcribed from minigenes carrying disease-causing splicing mutations. However, whether this increase could restore the protein function was unknown. Here, we demonstrate that overexpression of Htra2-beta1 and SC35 increases the level of normal cystic fibrosis transmembrane conductance regulator (CFTR) transcripts in cystic-fibrosis-derived epithelial cells carrying the 3849+10 kb C --> T splicing mutation. This led to activation of the CFTR channel and restoration of its function. Restoration was also obtained by sodium butyrate, a histone deacetylase inhibitor, known to upregulate the expression of splicing factors. These results highlight the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.
Assuntos
Processamento Alternativo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Linhagem Celular , Éxons , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Inibidores de Histona Desacetilases , Humanos , Proteínas Mitocondriais , Mutação , RNA/química , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Oxibato de Sódio/química , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. The molecular basis of rare fragile sites is associated with expanded repeats capable of adopting unusual non-B DNA structures that can perturb DNA replication. The molecular basis of common fragile sites was unknown. Fragile sites from R-bands are enriched in flexible sequences relative to nonfragile regions from the same chromosomal bands. Here we cloned FRA7E, a common fragile site mapped to a G-band, and revealed a significant difference between its flexibility and that of nonfragile regions mapped to G-bands, similar to the pattern found in R-bands. Thus, in the entire genome, flexible sequences might play a role in the mechanism of fragility. The flexible sequences are composed of interrupted runs of AT-dinucleotides, which have the potential to form secondary structures and hence can affect replication. These sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B. We further demonstrate that the normal alleles of FRA16B and FRA10B span the same genomic regions as the common fragile sites FRA16C and FRA10E. Our results suggest that a shared molecular basis, conferred by sequences with a potential to form secondary structures that can perturb replication, may underlie the fragility of rare fragile sites harboring AT-rich minisatellite repeats and aphidicolin-induced common fragile sites.
