A novel approach for organelle-specific DNA damage targeting reveals different susceptibility of mitochondrial DNA to the anticancer drugs camptothecin and topotecan

DNA is susceptible of being damaged by chemicals, UV light or gamma irradiation. Nuclear DNA damage invokes both a checkpoint and a repair response. By contrast, little is known about the cellular response to mitochondrial DNA damage. We designed an experimental system that allows organelle-specific DNA damage targeting in Saccharomyces cerevisiae. DNA damage is mediated by a toxic topoisomerase I allele which leads to the formation of persistent DNA single-strand breaks. We show that organelle-specific targeting of a toxic topoisomerase I to either the nucleus or mitochondria leads to nuclear DNA damage and cell death or to loss of mitochondrial DNA and formation of respiration-deficient ‘petite’ cells, respectively. In wild-type cells, toxic topoisomerase I–DNA intermediates are formed as a consequence of topoisomerase I interaction with camptothecin-based anticancer drugs. We reasoned that targeting of topoisomerase I to the mitochondria of top1Δ cells should lead to petite formation in the presence of camptothecin. Interestingly, camptothecin failed to generate petite; however, its derivative topotecan accumulates in mitochondria and induces petite formation. Our findings demonstrate that drug modifications can lead to organelle-specific DNA damage and thus opens new perspectives on the role of mitochondrial DNA-damage in cancer treatment

Silenced yeast chromatin is maintained by Sir2 in preference to permitting histone acetylations for efficient NER

Very little is currently known about how nucleotide excision repair (NER) functions at the ends of chromosomes. To examine this, we introduced the URA3 gene into either transcriptionally active or repressed subtelomeric regions of the yeast genome. This enabled us to examine the repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in identical sequences under both circumstances. We found that NER is significantly more efficient in the non-repressed subtelomere than the repressed one. At the non-repressed subtelomere, UV radiation stimulates both histones H3 and H4 acetylation in a similar fashion to that seen at other regions of the yeast genome. These modifications occur regardless of the presence of the Sir2 histone deacetylase. On the other hand, at the repressed subtelomere, where repair is much less efficient, UV radiation is unable to stimulate histone H4 or H3 acetylation in the presence of Sir2. In the absence of Sir2 both of these UV-induced modifications are detected, resulting in a significant increase in NER efficiency in the region. Our experiments reveal that there are instances in the yeast genome where the maintenance of the existing chromatin structures dominates over the action of chromatin modifications associated with efficient NER

Novel Phosphorylation Sites in the S. cerevisiae Cdc13 Protein Reveal New Targets for Telomere Length Regulation

The S. cerevisiae Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). We also determined phosphorylation abundance on both wild type (WT) and a telomerase deficient form of Cdc13, encoded by the cdc13-2 allele, in both G1 phase cells, when telomerase is not active, and G2/M phase cells, when it is. We identified 21 sites of in vivo phosphorylation, of which only five had been reported previously. In contrast, phosphorylation of two in vitro targets of the ATM-like Tel1 kinase, S249 and S255, was not detected. This result helps resolve conflicting data on the importance of phosphorylation of these residues in telomerase recruitment. multiple residues showed differences in their cell cycle pattern of modification. For example, phosphorylation of S314 was significantly higher in the G2/M compared to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation negatively affected telomere length. Our findings provide new targets in a key telomerase regulatory protein for modulation of telomere dynamics. [Image: see text

Methylated H3K4, a Transcription-Associated Histone Modification, Is Involved in the DNA Damage Response Pathway

Eukaryotic genomes are associated with a number of proteins such as histones that constitute chromatin. Post-translational histone modifications are associated with regulatory aspects executed by chromatin and all transactions on genomic DNA are dependent on them. Thus, it will be relevant to understand how histone modifications affect genome functions. Here we show that the mono ubiquitylation of histone H2B and the tri-methylation of histone H3 on lysine 4 (H3K4me3), both known for their involvement in transcription, are also important for a proper response of budding yeast cells to DNA damaging agents and the passage through S-phase. Cells that cannot methylate H3K4 display a defect in double-strand break (DSB) repair by non-homologous end joining. Furthermore, if such cells incur DNA damage or encounter a stress during replication, they very rapidly lose viability, underscoring the functional importance of the modification. Remarkably, the Set1p methyltransferase as well as the H3K4me3 mark become detectable on a newly created DSB. This recruitment of Set1p to the DSB is dependent on the presence of the RSC complex, arguing for a contribution in the ensuing DNA damage repair process. Taken together, our results demonstrate that Set1p and its substrate H3K4me3, which has been reported to be important for the transcription of active genes, also plays an important role in genome stability of yeast cells. Given the high degree of conservation for the methyltransferase and the histone mark in a broad variety of organisms, these results could have similar implications for genome stability mechanisms in vertebrate and mammalian cells

Genome Instability and Transcription Elongation Impairment in Human Cells Depleted of THO/TREX

THO/TREX connects transcription with genome integrity in yeast, but a role of mammalian THO in these processes is uncertain, which suggests a differential implication of mRNP biogenesis factors in genome integrity in yeast and humans. We show that human THO depletion impairs transcription elongation and mRNA export and increases instability associated with DNA breaks, leading to hyper-recombination and γH2AX and 53BP1 foci accumulation. This is accompanied by replication alteration as determined by DNA combing. Genome instability is R-loop–dependent, as deduced from the ability of the AID enzyme to increase DNA damage and of RNaseH to reduce it, or from the enhancement of R-loop–dependent class-switching caused by THOC1-depletion in CH12 murine cells. Therefore, mammalian THO prevents R-loop formation and has a role in genome dynamics and function consistent with an evolutionary conservation of the functional connection between these mRNP biogenesis factors and genome integrity that had not been anticipated

Electron microscopic analysis reveals that replication factor C is sequestered by single-stranded DNA.

Replication factor C (RF-C) is a eukaryotic heteropentameric protein required for DNA replication and repair processes by loading proliferating cell nuclear antigen (PCNA) onto DNA in an ATP-dependent manner. Prior to loading PCNA, RF-C binds to DNA. This binding is thought to be restricted to a specific DNA structure, namely to a primer/template junction. Using the electron microscope we have examined the affinity of human heteropentameric RF-C and the DNA-binding region within the large subunit of RF-C from Drosophila melanogaster (dRF-Cp140) to heteroduplex DNA. The electron microscopic data indicate that both human heteropentameric RF-C and the DNA-binding region within dRF-Cp140 are sequestered by single-stranded DNA. No preferential affinity for the 3' or 5' transition points from single- to double-stranded DNA was evident