Global genome nucleotide excision repair factors and the ubiquitin-proteasome system regulate the DNA damage response in Saccharomyces cerevisiae

These results indicate a role of the Rad7 E3 ubiquitin ligase in regulation of cellular dNTP levels in the DNA damage response.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Global genome nucleotide excision repair factors and the ubiquitin-proteasome system regulate the DNA damage response in Saccharomyces cerevisiae

The DNA damage response triggers a complex regulatory network to protect genomic integrity and promote cell survival by inducing effects such as cell cycle arrest, upregulation of dNTP synthesis and DNA repair. The Nucleotide Excision Repair (NER) pathway removes a broad array of DNA lesions by excising the damage with surrounding DNA and resynthesising the DNA using the undamaged strand as the template. NER consists of two sub-pathways that remove lesions from transcribed DNA (TC-NER), or from the rest of the genome (GG-NER). It has been proposed more recently that NER factors have additional regulatory roles in the DNA damage response. The initial aim of the study was to identify a possible regulatory interaction between the Abf1 protein and the 19S proteasome. abfl and sug (proteasome) mutant alleles were employed to identify a functional interaction between the factors but a significantly altered mutant phenotype could not be identified. The study proceeded to investigate a regulatory role of the Rad4-Rad23 NER complex in the DNA damage response via Rad7-dependent ubiquitination of Rad4. Previous evidence suggested that post-UV Rad4 ubiquitination has a regulatory role in DNA damage-responsive transcription. Global analysis of gene expression revealed that genes involved in dNTP synthesis, including the Ribonucleotide Reductase (RNR) pathway, were misregulated in a strain unable to ubiquitinate Rad4. The study progressed to investigate the function of the Rad4-Rad23 complex in regulation of the RNR pathway. It was discovered that constitutive activation or expression of the RNR pathway could suppress the UV sensitivity of the E3 ligase-defective strain, thus suggesting that the Rad7-E3 ligase has a role in regulating cellular dNTP levels in response to DNA damage. The Rad4-Rad23 complex binds at the DUN1 promoter, a positive regulator of the RNR pathway. E3 ligase-defective mutant strains were also found to exhibit defective cell cycle progression in the presence and absence of DNA damage. This defective cell cycle progression is suppressed by upregulating the RNR pathway. This further supports a role of the Rad7 E3 ligase in regulation of cellular dNTP levels. These results indicate a role of the Rad7 E3 ubiquitin ligase in regulation of cellular dNTP levels in the DNA damage response

Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast

Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI) whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17 (a critical component of the ATR pathway) lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation

The Ecm11-Gmc2 complex promotes synaptonemal complex formation through assembly of transverse filaments in budding yeast

During meiosis, homologous chromosomes pair at close proximity to form the synaptonemal complex (SC). This association is mediated by transverse filament proteins that hold the axes of homologous chromosomes together along their entire length. Transverse filament proteins are highly aggregative and can form an aberrant aggregate called the polycomplex that is unassociated with chromosomes. Here, we show that the Ecm11-Gmc2 complex is a novel SC component, functioning to facilitate assembly of the yeast transverse filament protein, Zip1. Ecm11 and Gmc2 initially localize to the synapsis initiation sites, then throughout the synapsed regions of paired homologous chromosomes. The absence of either Ecm11 or Gmc2 substantially compromises the chromosomal assembly of Zip1 as well as polycomplex formation, indicating that the complex is required for extensive Zip1 polymerization. We also show that Ecm11 is SUMOylated in a Gmc2-dependent manner. Remarkably, in the unSUMOylatable ecm11 mutant, assembly of chromosomal Zip1 remained compromised while polycomplex formation became frequent. We propose that the Ecm11-Gmc2 complex facilitates the assembly of Zip1 and that SUMOylation of Ecm11 is critical for ensuring chromosomal assembly of Zip1, thus suppressing polycomplex formation

Budding Yeast Pch2, a Widely Conserved Meiotic Protein, Is Involved in the Initiation of Meiotic Recombination

Budding yeast Pch2 protein is a widely conserved meiosis-specific protein whose role is implicated in the control of formation and displacement of meiotic crossover events. In contrast to previous studies where the function of Pch2 was implicated in the steps after meiotic double-strand breaks (DSBs) are formed, we present evidence that Pch2 is involved in meiotic DSB formation, the initiation step of meiotic recombination. The reduction of DSB formation caused by the pch2 mutation is most prominent in the sae2 mutant background, whereas the impact remains mild in the rad51 dmc1 double mutant background. The DSB reduction is further pronounced when pch2 is combined with a hypomorphic allele of SPO11. Interestingly, the level of DSB reduction is highly variable between chromosomes, with minimal impact on small chromosomes VI and III. We propose a model in which Pch2 ensures efficient formation of meiotic DSBs which is necessary for igniting the subsequent meiotic checkpoint responses that lead to proper differentiation of meiotic recombinants

Ecm11 and Gmc2 are required for efficient meiotic crossing over.

<p>(A) Sporulation is delayed in the <i>ecm11</i> and <i>gmc2</i> mutants. Diploid cells were introduced into meiosis and spore formation was examined at indicated time points. (B) Crossing over is reduced in the <i>ecm11</i> and <i>gmc2</i> mutants. Diploid cells carrying one circular and one linear chromosome III were introduced into meiosis and crossing over between these chromosomes was measured by Southern blotting. The amount of linear dimer and trimer chromosomes represents the efficiency of crossing over. (C) Quantification of crossover products shown in (B). The amount of recombinants was expressed as the ratio (percentage) of the combined signal of linear dimer and trimer bands per the sum of linear monomer, dimer and trimer bands. Error bars represent SEM.</p

Ecm11 and Gmc2 are necessary for the efficient assembly of Zip1 on chromosomes and in the polycomplex.

<p>(A) Zip1 localization is discontinuous on meiotic prophase chromosomes in the <i>ecm11</i> and <i>gmc2</i> mutants. The localization of Zip1 along with Red1, a component of meiotic chromosome axes, was examined on spread chromosomes. Bar, 5 µm. (B, C) Quantitative analysis of the Zip1 localization. Zip1 stretch area represents the size of one continuous Zip1 staining. See Results and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003194#s4" target="_blank">Materials and Methods</a> for more details. (D) Polycomplex formation was abolished in <i>ecm11</i> and <i>gmc2</i> mutants. Zip1 localization was examined in the <i>spo11</i> mutant background. The white arrowhead indicates the location of the polycomplex. Small speckle-like Zip1 staining likely represents the centromeric localization of Zip1. Chromosome spreads were prepared using cells at 20 hours after introduction into meiosis when, in wild type, cells at the pachytene stage are enriched. (E) Quantification of spread nuclei exhibiting a polycomplex.</p