RESUMO
The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinase Dun1. These physical interactions encompass small subpopulations of MCM and RNR, are independent of the major subcellular locations of these two complexes, augment in response to DNA damage and, in the case of the Rnr2 and Rnr4 subunits of RNR, depend on Dun1. Partial disruption of the MCM/RNR interactions impairs the release of Rad52 -but not RPA-from the DNA repair centers despite the lesions are repaired, a phenotype that is associated with hypermutagenesis but not with alterations in the levels of dNTPs. These results suggest that a specifically regulated pool of MCM and RNR complexes plays non-canonical roles in genetic stability preventing persistent Rad52 centers and hypermutagenesis.
Assuntos
Proteínas de Ciclo Celular , Dano ao DNA , Reparo do DNA , Replicação do DNA , Instabilidade Genômica , Proteína Rad52 de Recombinação e Reparo de DNA , Ribonucleotídeo Redutases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicação do DNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Dano ao DNA/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Reparo do DNA/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Proteína de Replicação A/metabolismo , Proteína de Replicação A/genética , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismoRESUMO
DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage-induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57-dependent and -independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.