RESUMO
When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Cromatina/metabolismo , Dano ao DNA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Sequência de Aminoácidos , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/química , Cromatina/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteína de Replicação A/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Solubilidade/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Fatores de TempoRESUMO
When replication forks collapse, Rad3 phosphorylates the checkpoint-clamp protein Rad9 in a manner that depends on Thr 225, a residue within the PCNA-like domain. The physiological function of Thr 225-dependent Rad9 phosphorylation, however, remains elusive. Here, we show that Thr 225-dependent Rad9 phosphorylation by Rad3 regulates DNA repair pathways. A rad9(T225C) mutant induces a translesion synthesis (TLS)-dependent high spontaneous mutation rate and a hyper-recombination phenotype. Consistent with this, Rad9 coprecipitates with the post-replication repair protein Mms2. This interaction is dependent on Rad9 Thr 225 and is enhanced by DNA damage. Genetic analyses indicate that Thr 225-dependent Rad9 phosphorylation prevents inappropriate Rhp51-dependent recombination, potentially by redirecting the repair through a Pli1-mediated sumoylation pathway into the error-free branch of the Rhp6 repair pathway. Our findings reveal a new mechanism by which phosphorylation of Rad9 at Thr 225 regulates the choice of repair pathways for maintaining genomic integrity during the cell cycle.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/genética , Genes cdc/fisiologia , Instabilidade Genômica/genética , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2 , Dano ao DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Fosforilação , Proteínas Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais/genética , Treonina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
The last decade has seen appreciable advancements in efforts towards increased portability of lab-on-a-chip devices by substituting microfluidics with molecular motor-based transportation. As of now, first proof-of-principle devices have analyzed protein mixtures of low complexity, such as target protein molecules in buffer solutions optimized for molecular motor performance. However, in a diagnostic work-up, lab-on-a-chip devices need to be compatible with complex biological samples. While it has been shown that such samples do not interfere with crucial steps in molecular diagnostics (for example antibody-antigen recognition), their effect on molecular motors is unknown. This critical and long overlooked issue is addressed here. In particular, we studied the effects of blood, cell lysates and solutions containing genomic DNA extracts on actomyosin and kinesin-microtubule-based transport, the two biomolecular motor systems that are most promising for lab-on-a-chip applications. We found that motor function is well preserved at defined dilutions of most of the investigated biological samples and demonstrated a molecular motor-driven label-free blood type test. Our results support the feasibility of molecular-motor driven nanodevices for diagnostic point-of-care applications and also demonstrate important constraints imposed by sample composition and device design that apply both to kinesin-microtubule and actomyosin driven applications.