RESUMO
Xenopus oocytes and oocyte nuclear extracts repair ultraviolet photoproducts on double-stranded (ds) DNA and replicate single-stranded (ss) to ds DNA. M13 ss DNA molecules containing cyclobutane pyrimidine dimers were maintained but not replicated in Xenopus oocytes yet were replicated in progesterone-matured oocytes. The replication arrest functioned only in cis. The replication arrest was alleviated by injection into oocytes of messenger RNAs encoding the prokaryotic mutagenesis proteins UmuD'C or MucA'B. These results may help explain how cells stabilize repair or replication events on DNA with unrepairable lesions.
Assuntos
Proteínas de Bactérias/fisiologia , Replicação do DNA , Proteínas de Escherichia coli , Escherichia coli/genética , Oócitos/metabolismo , Animais , Bacteriófago M13/genética , Bacteriófago phi X 174/genética , DNA/biossíntese , Dano ao DNA , Reparo do DNA , DNA de Cadeia Simples/biossíntese , DNA Polimerase Dirigida por DNA , Raios Ultravioleta , XenopusAssuntos
Núcleo Celular/metabolismo , Dano ao DNA , Reparo do DNA , DNA/genética , Oócitos/metabolismo , Dímeros de Pirimidina/metabolismo , Animais , Fracionamento Celular/métodos , DNA/isolamento & purificação , DNA/metabolismo , Replicação do DNA , DNA Viral/genética , Eletroforese em Gel de Ágar/métodos , Endodesoxirribonucleases/metabolismo , Feminino , Complexos Multienzimáticos/metabolismo , N-Glicosil Hidrolases/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Plasmídeos/efeitos da radiação , Raios Ultravioleta , Xenopus laevisRESUMO
Apoptosis is characterized by various cell morphological and biochemical features, one of which is the internucleosomal degradation of genomic DNA. The role of the human chromatin-bound Ca(2+)- and Mg(2+)-dependent endonuclease (CME) DNAS1L3 and its inhibition by poly(ADP-ribosyl)ation in the DNA degradation that accompanies apoptosis was investigated. The nuclear localization of this endonuclease is the unique feature that distinguishes it from other suggested apoptotic nucleases. Purified recombinant DNAS1L3 was shown to cleave nuclear DNA into both high molecular weight and oligonucleosomal fragments in vitro. Furthermore, exposure of mouse skin fibroblasts expressing DNAS1L3 to inducers of apoptosis resulted in oligonucleosomal DNA fragmentation, an effect not observed in cells not expressing this CME, as well as in a decrease in cell viability greater than that apparent in the control cells. Recombinant DNAS1L3 was modified by recombinant human poly(ADP-ribose) polymerase (PARP) in vitro, resulting in a loss of nuclease activity. The DNAS1L3 protein also underwent poly(ADP-ribosyl)ation in transfected mouse skin fibroblasts in response to inducers of apoptosis. The cleavage and inactivation of PARP by a caspase-3-like enzyme late in apoptosis were associated with a decrease in the extent of DNAS1L3 poly(ADP-ribosyl)ation, which likely releases DNAS1L3 from inhibition and allows it to catalyze the degradation of genomic DNA.
Assuntos
Apoptose/fisiologia , Fragmentação do DNA , Endodesoxirribonucleases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Cloreto de Cálcio/farmacologia , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Sobrevivência Celular , Endodesoxirribonucleases/antagonistas & inibidores , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Humanos , Cinética , Cloreto de Magnésio/farmacologia , Camundongos , Proteínas Recombinantes/metabolismo , Pele/enzimologiaRESUMO
Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.