A critical role for the C-terminus of Nej1 protein in Lif1p association, DNA binding and non-homologous end-joining

A predominant pathway implicated in repair of DNA double-strand breaks (DSBs) is the evolutionarily conserved non-homologous end-joining (NHEJ) pathway. Among the major constituents of this pathway in Saccharomyces cerevisiae is Nej1p, for which a biochemical function has yet to be determined. In this work we demonstrate that Nej1p exhibits a DNA binding activity (KD approximately 1.8 microM) comparable to Lif1p. Although binding is enhanced with larger substrates (>300 bp), short approximately 20 bp substrates can suffice. This DNA binding activity is the first biochemical evidence supporting the idea that Nej1p plays a direct role in the repair of double-strand breaks. The C-terminus of Nej1p is required for interaction with Lif1p and is sufficient for DNA binding. Structural characterization reveals that Nej1p exists as a dimer, and that residues 1-244 are sufficient for dimer formation. Nej1p (aa 1-244) is shown to be defective in end-joining in vivo. Preliminary functional and structural studies on the Nej1p-Lif1p complex suggest that the proteins stably co-purify and the complex binds DNA with a higher affinity than each independent component. The significance of these results is discussed with reference to current literature on Nej1p and other end-joining factors (mammalian and yeast), specifically the recently identified putative mammalian homologue of Nej1p, XLF/Cernunnos.     

Rad52 has a role in the repair of sodium selenite-induced DNA damage in Saccharomyces cerevisiae

Selenium (Se) is a chemo-preventive agent that has been shown to have a protective role against cancer. The inorganic form of Se, sodium selenite (Na₂SeO₃), has frequently been included in various chemo-prevention studies, and this commercially available form of Se is used as dietary supplement by the public. Because high doses of this Se compound can be toxic, the underlying molecular mechanisms of sodium selenite toxicity need to be elucidated. Recently, we have reported that sodium selenite is acting as an oxidizing agent in the budding yeast Saccharomyces cerevisiae, producing oxidative damage to DNA. This pro-oxidative activity of sodium selenite likely accounted for the observed DNA double-strand breaks (DSB) and yeast cell death. In this study we determine the genetic factors that are responsible for repair of sodium selenite-induced DSB. We report that the Rad52 protein is indispensable for repairing sodium selenite-induced DSB, suggesting a fundamental role of homologous recombination (HR) in this repair process. These results provide the first evidence that HR may have a fundamental role in the repair of sodium selenite-induced toxic DNA lesions.     

Double-strand breaks trigger MRX- and Mec1-dependent, but Tel1-independent, checkpoint activation

Together with the Tel1 PI3 kinase, the Mre11/Rad50/Xrs2 (MRX) complex is involved in checkpoint activation in response to double-strand breaks (DSBs), a function also conserved in human cells by Mre11/Rad50/Nbs1 acting with ATM. It has been proposed that the yeast Tel1/MRX pathway is activated in the presence of DSBs that cannot be resected. The Mec1 PI3 kinase, by contrast, would be involved in detecting breaks that can be processed. The significance of a Mec1/MRX DSB-activated DNA damage checkpoint has yet to be reported. To understand whether the MRX complex works specifically with Tel1 or Mec1, we investigated MRX function in checkpoint activation in response to endonuclease-induced DSBs in synchronized cells. We found that the expression of EcoRI activated the G1 and intra-S phase checkpoints in a MRX- and Mec1-dependent, but Tel1-independent manner. The pathways identified here are therefore different from the Tel1/MRX pathway that was previously reported. Thus, our results demonstrate that MRX can function in concert with both Mec1 and Tel1 PI3K-like kinases to trigger checkpoint activation in response to DSBs. Importantly, we also describe a novel MRX-independent checkpoint that is activated in late S-phase when cells replicate their DNA in the presence of DSBs. The existence of this novel mode of checkpoint activation explains why several previous studies had reported that mutations in the MRX complex did not abrogate DSB-induced checkpoint activation in asynchronous cells.     

The absence of Ku but not defects in classical non-homologous end-joining is required to trigger PARP1-dependent end-joining

Classical-non-homologous end-joining (C-NHEJ) is considered the main pathway for repairing DNA double strand breaks (DSB) in mammalian cells. When C-NHEJ is defective, cells may switch DSB repair to an alternative-end-joining, which depends on PARP1 and is more erroneous. This PARP1-EJ is suggested to be active especially in tumor cells contributing to their genomic instability. Here, we define conditions under which cells would switch the repair to PARP1-EJ. Using the end jining repair substrate pEJ, we revealed that PARP1-EJ is solely used when Ku is deficient but not when either DNA-PKcs or Xrcc4 is lacking. In the latter case, DSB repair, however, could be shuttled to PARP1-EJ after additional Ku80 down-regulation, which partly rescued the DSB repair in these mutants. We demonstrate here that PARP-EJ may work on DSB ends at high fidelity manner, as evident from the unchanged efficiency upon blocking end resection by either roscovitin or mirin. Furthermore, we demonstrate for that PARP-EJ is likewise involved in the repair of multiple DSBs (I-PpoI- and IR-induced). Importantly, we identified a chromatin signature associated with the switch to PARP1-EJ which is characterized by a strong enrichment of both PARP1 and LigIII at damaged chromatin. Together, these data indicate that Ku is the main regulator for the hierarchal organization between C-NHEJ and PARP1-EJ.     

The role of RecQ helicases in non-homologous end-joining

Abstract

DNA double-strand breaks are highly toxic DNA lesions that cause genomic instability, if not efficiently repaired. RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in several DNA repair pathways, including DNA double-strand break repair. Double-strand breaks can be repaired by homologous recombination (HR) using sister chromatids as templates to facilitate precise DNA repair, or by an HR-independent mechanism known as non-homologous end-joining (NHEJ) (error-prone). NHEJ is a non-templated DNA repair process, in which DNA termini are directly ligated. Canonical NHEJ requires DNA-PKcs and Ku70/80, while alternative NHEJ pathways are DNA-PKcs and Ku70/80 independent. This review discusses the role of RecQ helicases in NHEJ, alternative (or back-up) NHEJ (B-NHEJ) and microhomology-mediated end-joining (MMEJ) in V(D)J recombination, class switch recombination and telomere maintenance.     

Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks

Single‐stranded DNA constitutes an important early intermediate for homologous recombination and damage‐induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double‐strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in the onset of 5′‐strand resection, whereas Sgs1/Top3/Rmi1 with Dna2 and Exo1 are involved in extensive resection. However, the molecular events leading to a switch from the MRX/Sae2‐dependent initiation to the Exo1‐ and Dna2‐dependent resection remain unclear. Here, we show that MRX recruits Dna2 nuclease to DSB ends. MRX also stimulates recruitment of Exo1 and antagonizes excess binding of the Ku complex to DSB ends. Using resection assay with purified enzymes in vitro, we found that Ku and MRX regulate the nuclease activity of Exo1 in an opposite way. Efficient loading of Dna2 and Exo1 requires neither Sae2 nor Mre11 nuclease activities. However, Mre11 nuclease activity is essential for resection in the absence of extensive resection enzymes. The results provide new insights into how MRX catalyses end resection and recombination initiation.     

Characteristics of Flo11-dependent flocculation in Saccharomyces cerevisiae

The FLO11-encoded flocculin is required for a variety of important phenotypes in Saccharomyces cerevisiae, including flocculation, adhesion to agar and plastic, invasive growth, pseudohyphae formation and biofilm development. We present evidence that Flo11p belongs to the Flo1-type class of flocculins rather than to the NewFlo class. Both Flo1-type and NewFlo yeast flocculation are inhibited by mannose. NewFlo flocculation, however, is also inhibited by several other carbohydrates including glucose, maltose and sucrose. These differences have in at least one case been shown to reflect differences in the structure of the carbohydrate-binding site of the flocculins. We report that Flo11p-dependent flocculation is inhibited by mannose, but not by glucose, maltose or sucrose. Furthermore, Flo11p contains a peptide sequence highly similar to one that has been shown to characterise Flo1-type flocculins. Further characterisation of the properties of Flo11p-dependent flocculation revealed that it is dependent on calcium, occurs only at cell densities greater than 1 x 10(8) ml(-1), and only occurs at acidic pH.     

FLO11 is essential for flor formation caused by the C-terminal deletion of NRG1 in Saccharomyces cerevisiae

The flor strains of Saccharomyces cerevisiae form a flor on the surface of wine after alcoholic fermentation. High hydrophobicity of the cell surface is suggested to be important for flor formation by the flor wine yeasts. However, the molecular mechanism of flor formation is not clear. We found that expression of C-terminal deleted NRG1 lacking its two C2H2 zinc finger motifs (NRG1(1-470)) on the multicopy plasmid conferred the ability to form a flor to a non-flor laboratory strain. The cell surface hydrophobicity of NRG1(1-470) was higher than of the non-flor strain. Disruption of the Nrg1p-repressed gene FLO11, which encodes a cell surface glycoprotein that functions as a flocculin or an adhesin, abolished flor formation. Moreover, expression of FLO11 on a multicopy plasmid could also cause flor formation. These results indicate that FLO11 is essential for flor formation by NRG1(1-470). In addition, the results suggest that the C-terminal truncated form of Nrg1p exerts a dominant negative effect on FLO11 repression, resulting in FLO11 expression and, thus, flor formation.     

酿酒酵母SNF4基因敲除缺失菌株的构建

构建一株酿酒酵母SNF4基因缺失菌株并研究其对乙醇产量的影响。扩增带有SNF4基因上下游同源序列和Kanr筛选标记的SNF4基因敲除组件,转化到酿酒酵母YS2获得阳性克隆子,然后将质粒pSH65转到阳性克隆子中,半乳糖诱导pSH65表达Cre酶切除Kanr筛选标记,获得SNF4等位基因完全缺失菌株YS2-△SNF4。发酵实验结果表明,缺失菌株YS2-△SNF4乙醇产量较出发菌株提高了7.57％。利用Cre-LoxP系统,成功构建了SNF4等位基因完全缺失菌株并提高乙醇产生量。     

啤酒酵母乙醇脱氢酶Ⅰ(ADHI)的遗传变异

在啤酒酵母(Saccharomyces cerevisiae)中观察到存在一种新的ADHI:ADHIF,电泳迁移率明显快于文献报道的ADHI:ADHIS。这种ADHIF在10％葡萄糖的培养条件下,以及在呼吸缺陷型菌株进行无氧呼吸时都能够出现。ADHIF性状的遗传分析表明,它受1个与结构基因ADC-1S(编码ADHIS)等位的基因ADC-1F控制。     

Production of Extracellular lipases by Saccharomyces cerevisiae

Seven strains of Saccharomyces cerevisiae all produced lipase when grown in shake flask culture. The best strain, DSM 1848, produced 4.0U of lipase in the medium containing olive oil and yeast extract. Production of the lipase was growth-associated.     

PCR differentiation of Saccharomyces cerevisiae from Saccharomyces bayanus/Saccharomyces pastorianus using specific primers

The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5'-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3'-SSU end) and the 3'-specific primers were located in the LSU gene (positions 560-590 from the 5'-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes.     

Defective signal joint recombination in fanconi anemia fibroblasts reveals a role for Rad50 in V(D)J recombination

V(D)J recombination of immunoglobulin loci is dependent on the immune cell-specific Rag1 and Rag2 proteins as well as a number of ubiquitously expressed cellular DNA repair proteins that catalyze non-homologous end-joining of DNA double-strand breaks. The evolutionarily conserved Rad50/Mre11/Nibrin protein complex has a role in DNA double-strand break-repair, suggesting that these proteins, too, may participate in V(D)J recombination. Recent findings demonstrating that Rad50 function is defective in cells from patients afflicted with Fanconi anemia provide a possible mechanistic explanation for previous findings that lymphoblasts derived from these patients exhibit subtle defects in V(D)J recombination of extrachromosomal plasmid molecules. Here, we describe a series of findings that provide convincing evidence for a role of the Rad50 protein complex in V(D)J recombination. We found that the fidelity of V(D)J signal joint recombination in fibroblasts from patients afflicted with Fanconi anemia was reduced by nearly tenfold, compared to that observed in fibroblasts from normal donors. Second, we observed that antibody-mediated inhibition of the Rad50, Mre11, or Nibrin proteins reduced the fidelity of signal joint recombination significantly in wild-type cells. The latter finding was somewhat unexpected, because signal joint rejoining in cells from patients with Nijmegen breakage syndrome, which results from mutations in the Nibrin gene, occurs with normal fidelity. However, introduction of anti-Nibrin antibodies into these cells reduced the fidelity of signal joint recombination dramatically. These data reveal for the first time a role for the Rad50 complex in V(D)J recombination, and demonstrate that the protein product of the disease-causing allele responsible for Nijmegen breakage syndrome encodes a protein with residual DNA double-strand break repair activity.     

Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex

DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast.     

The characterization of Saccharomyces cerevisiae Mre11/Rad50/Xrs2 complex reveals that Rad50 negatively regulates Mre11 endonucleolytic but not the exonucleolytic activity

The evolutionarily conserved heterotrimeric Mre11/Rad50/Xrs2 (Nbs1) (MRX/N) complex plays a central role in an array of cellular responses involving DNA damage, telomere length homeostasis, cell-cycle checkpoint control and meiotic recombination. The underlying biochemical functions of MRX/N complex, or each of its individual subunits, at telomeres and the importance of complex formation are poorly understood. Here, we show that the Saccharomyces cerevisiae MRX complex, or its subunits, display an overwhelming preference for G-quadruplex DNA than for telomeric single-stranded or double-stranded DNA implicating the possible existence of this DNA structure in vivo. Although these alternative DNA substrates failed to affect Rad50 ATPase activity, kinetic analyses revealed that interaction of Rad50 with Xrs2 and/or Mre11 led to a twofold increase in the rates of ATP hydrolysis. Significantly, we show that Mre11 displays sequence-specific double-stranded DNA endonuclease activity, and Rad50, but not Xrs2, abrogated endonucleolytic but not the exonucleolytic activity. This repression was alleviated upon ATP hydrolysis by Rad50, suggesting that complex formation between Rad50 and Mre11 might be important for blocking the inappropriate cleavage of genomic DNA. Mre11 alone, or in the presence of ATP, MRX, MR or MX sub-complexes cleaved at the 5' end of an array of G residues in single-stranded DNA, at G quartets in G4 DNA, and at the center of TGTG repeats in duplex DNA. We propose that negative regulation of Mre11 endonuclease activity by Rad50 might be important for native as well as de novo telomere length homeostasis.     

Involvement of Nha1 antiporter in regulation of intracellular pH in Saccharomyces cerevisiae

The Nha1 antiporter is involved in regulation of intracellular pH in Saccharomyces cerevisiae. We report that deletion of the NHA1 gene resulted in an increase of cytoplasmic pH in cells suspended in water or acidic buffers. Addition of KCl or NaCl to exponentially growing cells lowered the internal pH but the difference between cells with or without NHA1 was maintained. Addition of KCl to starved cells resulted in much higher alkalinization of cytoplasmic pH in a strain lacking Nha1p compared to the wild-type or Nha1p-overexpressing strains. The H+/K+(Na+) exchange mechanism of Nha1p was confirmed in reconstituted plasma membrane vesicles.