RESUMO
The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small ρ(-) mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ(-) cells and increased deletion mutagenesis at the ori5 region in ρ(+) cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.
Assuntos
Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA Mitocondrial/metabolismo , Exodesoxirribonucleases/metabolismo , Reparo de DNA por Recombinação , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Variações do Número de Cópias de DNA , DNA Mitocondrial/biossíntese , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Epistasia Genética , Exodesoxirribonucleases/genética , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/enzimologia , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
Din7 is a DNA damage-inducible mitochondrial nuclease that modulates the stability of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. How DIN7 gene expression is regulated, however, has remained largely unclear. Using promoter sequence alignment, we found a highly conserved 19-bp sequence in the promoter regions of DIN7 and NTG1, which encodes an oxidative stress-inducible base-excision-repair enzyme. Deletion of the 19-bp sequence markedly reduced the hydroxyurea (HU)-enhanced DIN7 promoter activity. In addition, nuclear fractions prepared from HU-treated cells were used in in vitro band shift assays to reveal the presence of currently unidentified trans-acting factor(s) that preferentially bound to the 19-bp region. These results suggest that the 19-bp sequence is a novel cis-acting element that is required for the regulation of DIN7 expression in response to HU-induced DNA damage.
Assuntos
Dano ao DNA/genética , Exodesoxirribonucleases/genética , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Bases , Sequência Conservada , DNA Fúngico/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Inibidores Enzimáticos/farmacologia , Exodesoxirribonucleases/metabolismo , Hidroxiureia/farmacologia , Modelos Genéticos , Dados de Sequência Molecular , N-Glicosil Hidrolases/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Yeast DIN7 is a DNA damage-inducible gene. Its expression is increased in the absence of Dun1, a DNA damage checkpoint kinase. We identified a DIN7 promoter region responsible for Dun1-mediated downregulation and found that DIN7 expression was not further increased in response to hydroxyurea in Deltadun1 cells. Thus DIN7 repression by Dun1 can be released upon DNA damage.