RESUMO
During meiosis, homologous chromosomes are physically connected by crossovers and sister chromatid cohesion. Interhomolog crossovers are generated by the highly regulated repair of programmed double strand breaks (DSBs). The meiosis-specific kinase Mek1 is critical for this regulation. Mek1 downregulates the mitotic recombinase Rad51, indirectly promoting interhomolog strand invasion by the meiosis-specific recombinase Dmc1. Mek1 also promotes the formation of crossovers that are distributed throughout the genome by interference and is the effector kinase for a meiosis-specific checkpoint that delays entry into Meiosis I until DSBs have been repaired. The target of this checkpoint is a meiosis-specific transcription factor, Ndt80, which is necessary to express the polo-like kinase CDC5 and the cyclin CLB1 thereby allowing completion of recombination and meiotic progression. This work shows that Mek1 and Ndt80 negatively feedback on each other such that when DSB levels are high, Ndt80 is inactive due to high levels of Mek1 activity. As DSBs are repaired, chromosomes synapse and Mek1 activity is reduced below a threshold that allows activation of Ndt80. Ndt80 transcription of CDC5 results in degradation of Red1, a meiosis-specific protein required for Mek1 activation, thereby abolishing Mek1 activity completely. Elimination of Mek1 kinase activity allows Rad51-mediated repair of any remaining DSBs. In this way, cells do not enter Meiosis I until recombination is complete and all DSBs are repaired.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , MAP Quinase Quinase 1/genética , Meiose/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Cromátides/genética , Pareamento Cromossômico , Segregação de Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Retroalimentação Fisiológica , Fosforilação , Recombinação Genética , Saccharomyces cerevisiae/genéticaRESUMO
During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.
Assuntos
Proteínas de Ciclo Celular/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , MAP Quinase Quinase 1/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Segregação de Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Meiose/genética , Mitose/genética , Proteínas Mutantes/genética , Fosforilação , Saccharomyces cerevisiae/genéticaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1006226.].
RESUMO
The highly conserved Kinase, Endopeptidase and Other Proteins of small Size (KEOPS)/Endopeptidase-like and Kinase associated to transcribed Chromatin (EKC) protein complex has been implicated in transcription, telomere maintenance and chromosome segregation, but its exact function remains unknown. The complex consists of five proteins, Kinase-Associated Endopeptidase (Kae1), a highly conserved protein present in bacteria, archaea and eukaryotes, a kinase (Bud32) and three additional small polypeptides. We showed that the complex is required for a universal tRNA modification, threonyl carbamoyl adenosine (t6A), found in all tRNAs that pair with ANN codons in mRNA. We also showed that the bacterial ortholog of Kae1, YgjD, is required for t6A modification of Escherichia coli tRNAs. The ATPase activity of Kae1 and the kinase activity of Bud32 are required for the modification. The yeast protein Sua5 has been reported previously to be required for t6A synthesis. Using yeast extracts, we established an in vitro system for the synthesis of t6A that requires Sua5, Kae1, threonine, bicarbonate and ATP. It remains to be determined whether all reported defects of KEOPS/EKC mutants can be attributed to the lack of t6A, or whether the complex has multiple functions.
Assuntos
Adenosina/análogos & derivados , Metaloendopeptidases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA de Transferência/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Metaloendopeptidases/genética , Dados de Sequência Molecular , Complexos Multiproteicos , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Sir3, a component of the transcriptional silencing complex in the yeast Saccharomyces cerevisiae, has an N-terminal BAH domain that is crucial for the protein's silencing function. Previous work has shown that the N-terminal alanine residue of Sir3 (Ala2) and its acetylation play an important role in silencing. Here we show that the silencing defects of Sir3 Ala2 mutants can be suppressed by mutations in histones H3 and H4, specifically, by H3 D77N and H4 H75Y mutations. Additionally, a mutational analysis demonstrates that three separate regions of the Sir3 BAH domain are important for its role in silencing. Many of these BAH mutations also can be suppressed by the H3 D77N and H4 H75Y mutations. In agreement with the results of others, in vitro experiments show that the Sir3 BAH domain can interact with partially purified nucleosomes. The silencing-defective BAH mutants are defective for this interaction. These results, together with the previously characterized interaction between the C-terminal region of Sir3 and the histone H3/H4 tails, suggest that Sir3 utilizes multiple domains to interact with nucleosomes.
