RESUMO
In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa-Atr-Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects.
Assuntos
Dano ao DNA , Reparo de Erro de Pareamento de DNA , Animais , Apoptose , Linhagem Celular , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias/fisiologia , Epistasia Genética , Humanos , Camundongos da Linhagem 129 , Proteína 2 Homóloga a MutS/fisiologia , MutagêneseRESUMO
To investigate involvement of DNA mismatch repair in the response to short-wave ultraviolet (UVC) light, we compared UVC-induced mutant frequencies and mutational spectra at the Hprt gene between wild type and mismatch-repair-deficient mouse embryonic stem (ES) cells. Whereas mismatch repair gene status did not significantly affect survival of these cells after UVC irradiation, UVC induced substantially more mutations in ES cells that lack the MutSalpha mismatch-recognizing heterodimer than in wild type ES cells. The global UVC-induced mutational spectra at Hprt and the distribution of most spectral mutational hotspots were found to be similar in mismatch-repair-deficient and wild type cells. However, at one predominant spectral hot spot for mutagenesis in wild type cells, the UVC-induced mutation frequency was not affected by the mismatch repair status. Together these data reveal a major role of mismatch repair in controlling mutagenesis induced by UVC light and may suggest the sequence context-dependent direct mismatch repair of misincorporations opposite UVC-induced pyrimidine dimers.
Assuntos
Reparo de Erro de Pareamento de DNA , Mutagênese , Raios Ultravioleta , Animais , Apoptose , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/efeitos da radiação , Hipoxantina Fosforribosiltransferase/genética , Camundongos , Modelos Genéticos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Mutação PuntualRESUMO
In Catharanthus roseus cell suspensions, expression of several terpenoid indole alkaloid (TIA) biosynthetic genes, including those encoding strictosidine synthase and tryptophan decarboxylase, is coordinately induced by fungal elicitors such as yeast extract (YE). This induction is mediated by several signaling steps including the biosynthesis of jasmonic acid, and the activation of the jasmonic acid-responsive ORCA transcription factors. We investigated a possible role of reactive oxygen species (ROS) as a second messenger in this system. YE was shown to activate the production of ROS, which was dependent on protein phosphorylation and calcium influx. However, ROS generation was neither necessary for the induction of genes involved in TIA biosynthesis by YE nor by itself sufficient to induce these genes. Therefore, we conclude that activation of the oxidative burst by YE occurs independently of the activation of genes involved in TIA biosynthesis.
Assuntos
Catharanthus/genética , Meios de Cultura/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Alcaloides de Triptamina e Secologanina/metabolismo , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Acetilcisteína/farmacologia , Ácido Ascórbico/farmacologia , Northern Blotting , Cálcio/metabolismo , Catharanthus/citologia , Catharanthus/metabolismo , Células Cultivadas , Meios de Cultura/química , Gadolínio/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Lantânio/farmacologia , Modelos Biológicos , Nifedipino/farmacologia , Fosforilação/efeitos dos fármacos , RNA de Plantas/genética , RNA de Plantas/metabolismo , Leveduras/químicaRESUMO
In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus.
