A novel function for the Mre11-Rad50-Xrs2 complex in base excision repair

The Mre11/Rad50/Xrs2 (MRX) complex in Saccharomyces cerevisiae has well-characterized functions in DNA double-strand break processing, checkpoint activation, telomere length maintenance and meiosis. In this study, we demonstrate an involvement of the complex in the base excision repair (BER) pathway. We studied the repair of methyl-methanesulfonate-induced heat-labile sites in chromosomal DNA in vivo and the in vitro BER capacity for the repair of uracil- and 8-oxoG-containing oligonucleotides in MRX-deficient cells. Both approaches show a clear BER deficiency for the xrs2 mutant as compared to wildtype cells. The in vitro analyses revealed that both subpathways, long-patch and short-patch BER, are affected and that all components of the MRX complex are similarly important for the new function in BER. The investigation of the epistatic relationship of XRS2 to other BER genes suggests a role of the MRX complex downstream of the AP-lyases Ntg1 and Ntg2. Analysis of individual steps in BER showed that base recognition and strand incision are not affected by the MRX complex. Reduced gap-filling activity and the missing effect of aphidicoline treatment, an inhibitor for polymerases, on the BER efficiency indicate an involvement of the MRX complex in providing efficient polymerase activity

Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae

The integrase of the Saccharomyces cerevisiae retrotransposon Ty1 integrates Ty1 cDNA into genomic DNA likely via a transesterification reaction. Little is known about the mechanisms ensuring that integrase does not integrate non-Ty DNA fragments. In an effort to elucidate the conditions under which Ty1 integrase accepts non-Ty DNA as substrate, PCR fragments encompassing a selectable marker gene were transformed into yeast strains overexpressing Ty1 integrase. These fragments do not exhibit similarity to Ty1 cDNA except for the presence of the conserved terminal dinucleotide 5′-TG-CA-3′. The frequency of fragment insertion events increased upon integrase overexpression. Characterization of insertion events by genomic sequencing revealed that most insertion events exhibited clear hallmarks of integrase-mediated reactions, such as 5 bp target site duplication and target site preferences. Alteration of the terminal dinucleotide abolished the suitability of the PCR fragments to serve as substrates. We hypothesize that substrate specificity under normal conditions is mainly due to compartmentalization of integrase and Ty cDNA, which meet in virus-like particles. In contrast, recombinant integrase, which is not confined to virus-like particles, is able to accept non-Ty DNA, provided that it terminates in the proper dinucleotide sequence

Promoter-trapping in Saccharomyces cerevisiae by radiation-assisted fragment insertion

Non-homologous insertion (NHI) of DNA fragments into genomic DNA is a method widely used in insertional mutagenesis screens. In the yeast Saccharomyces cerevisiae, the efficiency of NHI is very low. Here we report that its efficiency can be increased by γ-irradiation of recipient cells at the time of transformation. Radiation-assisted NHI depends on YKU70, but its efficiency is not improved by inactivation of RAD5 or RAD52. In a pilot study, we generated 102 transformant clones expressing a lacZ reporter gene under standard conditions (30°C, rich medium). The site of insertion was determined in a subset of eight clones in which lacZ expression was altered by UV-irradiation. A comparison with published data revealed that three of the eight genes identified in our screen have not been targeted by large-scale transposon-based insertion screens. This suggests that radiation-assisted NHI offers a more homogeneous coverage of the genome than methods relying on transposons or retroviral elements

Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae

The integrase of the Saccharomyces cerevisiae retrotransposon Ty1 integrates Ty1 cDNA into genomic DNA likely via a transesterification reaction. Little is known about the mechanisms ensuring that integrase does not integrate non-Ty DNA fragments. In an effort to elucidate the conditions under which Ty1 integrase accepts non-Ty DNA as substrate, PCR fragments encompassing a selectable marker gene were transformed into yeast strains overexpressing Ty1 integrase. These fragments do not exhibit similarity to Ty1 cDNA except for the presence of the conserved terminal dinucleotide 5′-TG-CA-3′. The frequency of fragment insertion events increased upon integrase overexpression. Characterization of insertion events by genomic sequencing revealed that most insertion events exhibited clear hallmarks of integrase-mediated reactions, such as 5 bp target site duplication and target site preferences. Alteration of the terminal dinucleotide abolished the suitability of the PCR fragments to serve as substrates. We hypothesize that substrate specificity under normal conditions is mainly due to compartmentalization of integrase and Ty cDNA, which meet in virus-like particles. In contrast, recombinant integrase, which is not confined to virus-like particles, is able to accept non-Ty DNA, provided that it terminates in the proper dinucleotide sequence

The Yeast TEL1 Gene Partially Substitutes for Human ATM in Suppressing Hyperrecombination, Radiation-Induced Apoptosis and Telomere Shortening in A-T Cells

Homozygous mutations in the human ATM gene lead to a pleiotropic clinical phenotype of ataxia-telangiectasia (A-T) patients and correlating cellular deficiencies in cells derived from A-T donors. Saccharomyces cerevisiae tel1 mutants lacking Tel1p, which is the closest sequence homologue to the ATM protein, share some of the cellular defects with A-T. Through genetic complementation of A-T cells with the yeast TEL1 gene, we provide evidence that Tel1p can partially compensate for ATM in suppressing hyperrecombination, radiation-induced apoptosis, and telomere shortening. Complementation appears to be independent of p53 activation. The data provided suggest that TEL1 is a functional homologue of human ATM in yeast, and they help to elucidate different cellular and biochemical pathways in human cells regulated by the ATM protein

Quantitative Phosphoproteomic Analysis of Early Alterations in Protein Phosphorylation by 2,3,7,8-Tetrachlorodibenzo‑<i>p</i>‑dioxin

A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0.5 to 2 h, 5648 phosphorylated peptides corresponding to 2156 phosphoproteins were identified. Eight peptides exhibited a statistically significantly altered phosphorylation because of TCDD exposure and 22 showed a regulation factor of ≥1.5 in one of the experiments per time point. The vast majority of the TCCD-induced phosphorylation changes had not been reported before. The transcription factor ARNT, the obligate partner for gene activation by the TCDD-bound Ah receptor, exhibited an up-regulation of its Ser77 phosphorylation, a modification known to control the differential binding of ARNT homodimers and heterodimers to different enhancers suggesting that this phosphorylation represents a novel mechanism contributing to the alteration of gene expression by TCDD. Other proteins with altered phosphorylation included, among others, various transcriptional coregulators previously unknown to participate in TCDD-induced gene activation, regulators of small GTPases of the Ras superfamily, UBX domain-containing proteins and the oncogenic protein LYRIC. The results open up new directions for research on the molecular mechanisms of dioxin action and toxicity

Quantitative Phosphoproteomic Analysis of Early Alterations in Protein Phosphorylation by 2,3,7,8-Tetrachlorodibenzo‑<i>p</i>‑dioxin

A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0.5 to 2 h, 5648 phosphorylated peptides corresponding to 2156 phosphoproteins were identified. Eight peptides exhibited a statistically significantly altered phosphorylation because of TCDD exposure and 22 showed a regulation factor of ≥1.5 in one of the experiments per time point. The vast majority of the TCCD-induced phosphorylation changes had not been reported before. The transcription factor ARNT, the obligate partner for gene activation by the TCDD-bound Ah receptor, exhibited an up-regulation of its Ser77 phosphorylation, a modification known to control the differential binding of ARNT homodimers and heterodimers to different enhancers suggesting that this phosphorylation represents a novel mechanism contributing to the alteration of gene expression by TCDD. Other proteins with altered phosphorylation included, among others, various transcriptional coregulators previously unknown to participate in TCDD-induced gene activation, regulators of small GTPases of the Ras superfamily, UBX domain-containing proteins and the oncogenic protein LYRIC. The results open up new directions for research on the molecular mechanisms of dioxin action and toxicity

Quantitative Phosphoproteomic Analysis of Early Alterations in Protein Phosphorylation by 2,3,7,8-Tetrachlorodibenzo‑<i>p</i>‑dioxin

A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0.5 to 2 h, 5648 phosphorylated peptides corresponding to 2156 phosphoproteins were identified. Eight peptides exhibited a statistically significantly altered phosphorylation because of TCDD exposure and 22 showed a regulation factor of ≥1.5 in one of the experiments per time point. The vast majority of the TCCD-induced phosphorylation changes had not been reported before. The transcription factor ARNT, the obligate partner for gene activation by the TCDD-bound Ah receptor, exhibited an up-regulation of its Ser77 phosphorylation, a modification known to control the differential binding of ARNT homodimers and heterodimers to different enhancers suggesting that this phosphorylation represents a novel mechanism contributing to the alteration of gene expression by TCDD. Other proteins with altered phosphorylation included, among others, various transcriptional coregulators previously unknown to participate in TCDD-induced gene activation, regulators of small GTPases of the Ras superfamily, UBX domain-containing proteins and the oncogenic protein LYRIC. The results open up new directions for research on the molecular mechanisms of dioxin action and toxicity