RESUMO
PURPOSE: To test the hypothesis that differences in DNA double-strand breaks (DSB) repair fidelity underlies differences in radiosensitivity. MATERIALS AND METHODS: A primary fibroblast culture (C42) derived from a pediatric cancer patient treated with reduced radiation doses consequent to a family history of radiosensitivity reminiscent of chromosomal fragility syndrome, was compared to a normal control (C29). DNA DSB rejoining and repair fidelity were studied by Southern blotting and hybridization to specific fragments: Alu repetitive sequence representing the overall DSB rejoining capacity in the genome and a 3.2 Mbp NotI restriction fragment on chromosome 21 for DSB repair fidelity. RESULTS: Although both assays showed statistically significant difference (p ≤ 0.05) between the two cell strains in residual misrepaired (un-or mis-rejoined) DSB (24 h after 30 or 80 Gy), the residual damage was lower in the Alu enriched genome assay compared to NotI assay (0.01-0.07 and 0.10-0.37, respectively). CONCLUSIONS: These results suggest that, in comparison to classic DSB repair experiment, an assay of measuring DNA DSB repair fidelity can provide better resolution and a more accurate estimate of misrepair of radiation-induced DNA damage, which underlies genomic instability and increased radiosensitivity.
Assuntos
Transtornos Cromossômicos/genética , Fragilidade Cromossômica/genética , Fragilidade Cromossômica/efeitos da radiação , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Tolerância a Radiação/genética , Pareamento Incorreto de Bases/genética , Pareamento Incorreto de Bases/efeitos da radiação , Bioensaio/métodos , Pré-Escolar , Feminino , HumanosRESUMO
Ultraviolet (UV) irradiation is not known to induce chromosomal fragmentation in sublethal doses, and yet UV irradiation causes genetic instability and cancer, suggesting that chromosomes are fragmented. Here we show that UV irradiation induces fragmentation in sublethal doses, but the broken chromosomes are repaired or degraded by RecBCD; therefore, to observe full fragmentation, RecBCD enzyme needs to be inactivated. Using quantitative pulsed field gel electrophoresis and sensitive DNA synthesis measurements, we investigated the mechanisms of UV radiation-induced chromosomal fragmentation in recBC mutants, comparing five existing models of DNA damage-induced fragmentation. We found that fragmentation depends on active DNA synthesis before, but not after, UV irradiation. At low UV irradiation doses, fragmentation does not need excision repair or daughter strand gap repair. Fragmentation absolutely depends on both RecA-catalyzed homologous strand exchange and RuvABC-catalyzed Holliday junction resolution. Thus, chromosomes fragment when replication forks stall at UV lesions and regress, generating Holliday junctions. Remarkably, cells specifically utilize fork breakage to rescue stalled replication and avoid lethality.
Assuntos
Proteínas de Bactérias/genética , DNA Helicases/genética , Replicação do DNA/efeitos da radiação , Endodesoxirribonucleases/genética , Proteínas de Escherichia coli/genética , Recombinases Rec A/genética , Raios Ultravioleta/efeitos adversos , Fragilidade Cromossômica/genética , Fragilidade Cromossômica/efeitos da radiação , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/efeitos da radiação , Quebras de DNA/efeitos da radiação , Replicação do DNA/genética , DNA Cruciforme/genética , DNA Cruciforme/efeitos da radiação , Escherichia coli K12/genética , Escherichia coli K12/efeitos da radiaçãoRESUMO
Two UV-hypersensitive animal cell mutants defective in postreplication recovery (daughter strand synthesis) display quite different patterns of induced sister-chromatid exchange (SCE). One, an SV40-transformed Indian muntjac cell (SVM), shows extremely high frequencies of SCE after UV; induced exchanges can be measured after UV doses as low as 0.01 J/m2. This cell also displays exaggerated levels of induced and spontaneous chromosome aberrations. By contrast SCE rates in the Chinese hamster cell mutant, UV-1, are essentially normal. In both SVM and UV-1, however, there is a clear correlation between the cell density and spontaneous frequencies of SCE, a feature which could be related to the observed density-dependent rate of DNA maturation.
