RESUMEN
7,8-Dihydro-8-oxo-deoxyguanosine (8oxodG) is a highly premutagenic DNA lesion due to its ability to mispair with adenine. Schizosaccharomyces pombe lacks homologs for relevant enzymes that repair 8oxodG, which suggests that this lesion could be persistent and must be tolerated. Here we show that SpPol4, the unique PolX in fission yeast, incorporates ATP opposite 8oxodG almost exclusively when all nucleotides (ribos and deoxys) are provided at physiological concentrations. Remarkably, this SpPol4-specific reaction could also occur during the NHEJ of DSBs. In cell extracts, misincorporation of ATP opposite 8oxodG was shown to be SpPol4-specific, although RNase H2 efficiently recognized the 8oxodG:AMP mispair to remove AMP and trigger error-free incorporation of dCTP. These data are the first evidence that ribonucleotides can be used safely for 8oxodG tolerance, suggesting that insertion of the highly abundant ATP substrate could be beneficial to promote efficient and error-free repair of 8oxodG-associated DSBs. Moreover, we demonstrate that purified SpPol4 uses 8oxo-dGTP and 8oxo-GTP as substrates for DNA polymerization, although with poor efficiency compared to the incorporation of undamaged nucleotides opposite either 8oxodG or undamaged templates. This suggests that SpPol4 is specialized in tolerating 8oxodG as a DNA template, without contributing significantly to the accumulation of this lesion in the DNA.
Asunto(s)
Adenosina Trifosfato/metabolismo , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxiguanosina/análogos & derivados , Proteínas de Schizosaccharomyces pombe/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Disparidad de Par Base , Reparación del ADN por Unión de Extremidades , Nucleótidos de Desoxiadenina/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Desoxiguanosina/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Ribonucleasa H/metabolismo , Schizosaccharomyces/enzimologíaRESUMEN
Human family X polymerases contribute both to genomic stability and variability through their specialized functions in DNA repair. Polµ participates in the repair of spontaneous double strand breaks (DSB) by non homologous end-joining (NHEJ), and also in the V(D)J recombination process after programmed DSBs. Polµ plays this dual role due to its template-dependent and terminal transferase (template-independent) polymerization activities. In this study we evaluated if Polµ could be regulated by Cdk phosphorylation along the cell cycle. In vitro kinase assays showed that the S phase-associated Cdk2/cyclin A complex was able to phosphorylate Polµ. We identified Ser12, Thr21 (located in the BRCT domain) and Ser372 (located in loop1) as the target residues. Mutation of these residues to alanine indicated that Ser372 is the main phosphorylation site. Mobilization of loop1, which mediates DNA end micro-synapsis, is crucial both for terminal transferase and NHEJ. Interestingly, the phospho-mimicking S372E mutation specifically impaired these activities. Our evidences suggest that Polµ could be regulated in vivo by phosphorylation of the BRCT domain (Ser12/Thr21) and of Ser372, affecting the function of loop1. Consequently, Polµ's most distinctive activities would be turned off at specific cell-cycle phases (S and G2), when these promiscuous functions might be harmful to the cell.
Asunto(s)
Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Ciclo Celular , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Quinasa 2 Dependiente de la Ciclina/genética , Daño del ADN , Reparación del ADN , ADN Polimerasa Dirigida por ADN/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Inestabilidad Genómica , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Alineación de SecuenciaRESUMEN
Human DNA polymerases mu (Polµ) and lambda (Polλ) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3' single-stranded overhangs and trans-polymerization using one 3' end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence ('brooch'), located at the N-terminal boundary of the Polß-like polymerase core, and formed by Tyr(141), Ala(142), Cys(143), Gln(144) and Arg(145) in Polµ, and by Trp(239), Val(240), Cys(241), Ala(242) and Gln(243) in Polλ. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Polµ. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Polµ and polymerase lambda to perform non-homologous end joining reactions in vitro.
Asunto(s)
ADN Polimerasa beta/química , ADN Polimerasa Dirigida por ADN/química , ADN/metabolismo , Secuencia de Aminoácidos , Quinasas Ciclina-Dependientes/metabolismo , Reparación del ADN por Unión de Extremidades , ADN Polimerasa beta/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Human DNA polymerase mu (Polµ), a family X member involved in DNA repair, has both template-directed and terminal transferase (template-independent) activities. In addition to their ability to incorporate untemplated nucleotides, another similarity between Polµ and terminal deoxynucleotidyl transferase (TdT) is their promiscuity in using ribonucleotides (NTPs), whose physiological significance is presently unknown. As shown here, Polµ can use NTPs instead of deoxynucleotides (dNTPs) during non-homologous end joining (NHEJ) of non-complementary ends, a Polµ-specific task. Moreover, a physiological concentration of Mn(2+) ions did benefit Polµ-mediated NHEJ by improving the efficiency and accuracy of nucleotide insertion. Analysis of different mutations in the 'steric gate' of the active site indicated that Polµ is taking advantage of an open active site, valid for selecting alternative activating metal ions and nucleotides as substrates. This versatility would allow ad hoc selection of the most appropriate nucleotide/metal ion combination for individual NHEJ events to gain efficiency without a cost in terms of fidelity, thus widening the spectrum of available solutions to position a discontinuous template strand in proper register for connection.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Polimerasa Dirigida por ADN/metabolismo , Manganeso/farmacología , Ribonucleótidos/metabolismo , Dominio Catalítico , Cationes , ADN Polimerasa beta/metabolismo , ADN Polimerasa Dirigida por ADN/química , Células HeLa , Humanos , Manganeso/química , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
The Schizosaccharomyces pombe Flp1p serine-threonine phosphatase is required for the degradation of the mitotic inducer Cdc25p at the end of mitosis. Cdc25p degradation prevents Cdc2p-tyrosine 15 dephosphorylation and, thus, contributes to the timely inactivation of mitotic CDK-associated kinase activity. Both RING- and HECT-type protein-ubiquitin ligases are involved in Cdc25p destabilization. Flp1p function is required for Cdc25p ubiquitination via anaphase-promoting complex/cyclosome or APC/C (RING-type) and the absence of Pub1p (HECT-type) stabilizes the mitotic inducer. In the present report, we study the functional relationship of Flp1p with Pub1p and Pub2p HECT-type-protein ubiquitin ligases. We show that Flp1p is required for the rapid degradation of Cdc25p while Pub1p is responsible for the long-term destabilization of the mitotic inducer. Accordingly, flp1 and pub1 mutants have a strong genetic interaction, correlating defects in the coordination of mitosis and cytokinesis with the stabilization of hyperactive Cdc25p. However, we also show that Flp1 and Pub2p proteins functionally interact in vivo suggesting that both proteins belong to the same regulatory network in S. pombe cells. Thus Flp1p appears to have an important role in integrating HECT- and RING-type ubiquitin ligases in cell cycle control.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Proteínas de Ciclo Celular/genética , Citocinesis/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/genética , Genes cdc/fisiología , Mitosis/genética , Proteínas Tirosina Fosfatasas/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Ubiquitinación/genética , ras-GRF1/genética , ras-GRF1/metabolismoRESUMEN
Human Cdc14A is an evolutionary conserved dual-specificity protein phosphatase that reverses the modifications effected by cyclin-dependent kinases and plays an important role in centrosome duplication and mitotic regulation. Few substrates of Cdc14A have been identified, some of them with homologues in yeast that, in turn, are substrates of the Saccharomyces cerevisiae Cdc14 homologue, a protein phosphatase essential for yeast cell viability owing its role in mitotic exit regulation. Identification of the physiological substrates of human Cdc14A is an immediate goal in order to elucidate which cellular processes it regulates. Here, we show that human Cdc14A can dephosphorylate Cdc25A in vitro. Specifically, the Cdk1/Cyclin-B1-dependent phosphate groups on Ser115 and Ser320 of Cdc25A were found to be removed by Cdc14A. Cdc25A is an important cell cycle-regulatory protein involved in several cell cycle transitions and checkpoint responses and whose function and own regulation depend on complex phosphorylation/dephosphorylation-mediated processes. Importantly, we also show that the upregulation of Cdc14A phosphatase affects Cdc25A protein levels in human cells. Our results suggest that Cdc14A may be involved in the cell cycle regulation of Cdc25A stability.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfoserina/metabolismo , Fosfatasas cdc25/química , Fosfatasas cdc25/metabolismo , Secuencia de Aminoácidos , Ciclina B/metabolismo , Ciclina B1 , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Fosforilación , Proteínas Tirosina FosfatasasRESUMEN
Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.
Asunto(s)
Fosfoproteínas Fosfatasas/química , Monoéster Fosfórico Hidrolasas/química , Proteínas de Saccharomyces cerevisiae/química , Schizosaccharomyces/enzimología , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Citocinesis , ADN/química , Regulación hacia Abajo , Citometría de Flujo , Prueba de Complementación Genética , Glutatión Transferasa/metabolismo , Humanos , Inmunoprecipitación , Microscopía Fluorescente , Mitosis , Fenotipo , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático , TemperaturaRESUMEN
The Schizosaccaromyces pombe protein Flp1p belongs to a conserved family of serine-threonine-phosphatases. The founding member of this family, Saccharomyces cerevisiae Cdc14p, is required for inactivation of mitotic CDKs and reversal of CDK mediated phosphorylation at the end of mitosis, thereby bringing about the M-G1 transition. Initial studies of Flp1p suggest that it may play a different role to Cdc14p. Here we show that Flp1p is required for rapid degradation of the mitotic inducer Cdc25p at the end of mitosis, and that Cdc25p is a substrate of Flp1p in vitro. Down-regulation of Cdc25p activity by Flp1p may ensure a prompt inactivation of mitotic CDK complexes to trigger cell division. Our results suggest a regulatory mechanism, and a universal role, for Cdc14p like proteins in coordination of cytokinesis with other cell cycle events.