Genotoxic therapy such as radiation serves as a frontline cancer treatment, yet acquired resistance that leads to tumor reoccurrence is frequent. We found that cancer cells maintain viability during irradiation by reversibly increasing genome-wide DNA breaks, thereby limiting premature mitotic progression. We identify caspase-activated DNase (CAD) as the nuclease inflicting these de novo DNA lesions at defined loci, which are in proximity to chromatin-modifying CCCTC-binding factor (CTCF) sites. CAD nuclease activity is governed through phosphorylation by DNA damage response kinases, independent of caspase activity. In turn, loss of CAD activity impairs cell fate decisions, rendering cancer cells vulnerable to radiation-induced DNA double-strand breaks. Our observations highlight a cancer-selective survival adaptation, whereby tumor cells deploy regulated DNA breaks to delimit the detrimental effects of therapy-evoked DNA damage.
R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.
- MeSH
- chromozomální nestabilita účinky léků MeSH
- DNA biosyntéza MeSH
- G1 fáze účinky léků MeSH
- inhibitory enzymů farmakologie MeSH
- komplex rozpoznávající replikační počátek metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- R-smyčka * MeSH
- rekombinasa Rad51 * antagonisté a inhibitory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. RecQ helicases promote recovery of replication forks being stalled due to different replication roadblocks of either exogenous or endogenous source. They prevent generation of aberrant replication fork structures and replication fork collapse, and are involved in proper checkpoint signaling. The essential role of human RecQ helicases in the genome maintenance during DNA replication is underlined by association of defects in their function with cancer predisposition.
- MeSH
- helikasy RecQ fyziologie MeSH
- lidé MeSH
- nádory etiologie MeSH
- nestabilita genomu MeSH
- replikace DNA * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Collisions between replication and transcription machineries represent a significant source of genomic instability. RECQ5 DNA helicase binds to RNA-polymerase (RNAP) II during transcription elongation and suppresses transcription-associated genomic instability. Here, we show that RECQ5 also associates with RNAPI and enforces the stability of ribosomal DNA arrays. We demonstrate that RECQ5 associates with transcription complexes in DNA replication foci and counteracts replication fork stalling in RNAPI- and RNAPII-transcribed genes, suggesting that RECQ5 exerts its genome-stabilizing effect by acting at sites of replication-transcription collisions. Moreover, RECQ5-deficient cells accumulate RAD18 foci and BRCA1-dependent RAD51 foci that are both formed at sites of interference between replication and transcription and likely represent unresolved replication intermediates. Finally, we provide evidence for a novel mechanism of resolution of replication-transcription collisions wherein the interaction between RECQ5 and proliferating cell nuclear antigen (PCNA) promotes RAD18-dependent PCNA ubiquitination and the helicase activity of RECQ5 promotes the processing of replication intermediates.
- MeSH
- biologické modely MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- elongace genetické transkripce MeSH
- fyziologický stres genetika MeSH
- genetická transkripce * MeSH
- HEK293 buňky MeSH
- helikasy RecQ metabolismus MeSH
- interakční proteinové domény a motivy MeSH
- lidé MeSH
- multienzymové komplexy metabolismus MeSH
- otevřené čtecí rámce genetika MeSH
- prekurzory RNA genetika MeSH
- proliferační antigen buněčného jádra metabolismus MeSH
- protein BRCA1 metabolismus MeSH
- rekombinasa Rad51 metabolismus MeSH
- replikace DNA * MeSH
- ribozomální DNA metabolismus MeSH
- ubikvitinace MeSH
- ubikvitinligasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Various helicases and single-stranded DNA (ssDNA) binding proteins are known to destabilize G-quadruplex (GQ) structures, which otherwise result in genomic instability. Bulk biochemical studies have shown that Bloom helicase (BLM) unfolds both intermolecular and intramolecular GQ in the presence of ATP. Using single molecule FRET, we show that binding of RecQ-core of BLM (will be referred to as BLM) to ssDNA in the vicinity of an intramolecular GQ leads to destabilization and unfolding of the GQ in the absence of ATP. We show that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to ssDNA overhang, as modulated by the nucleotide state, ionic conditions, overhang length and overhang directionality. In particular, we observed enhanced GQ unfolding by BLM in the presence of non-hydrolysable ATP analogs, which has implications for the underlying mechanism. We also show that increasing GQ stability, via shorter loops or higher ionic strength, reduces BLM-mediated GQ unfolding. Finally, we show that while WRN has similar activity as BLM, RecQ and RECQ5 helicases do not unfold GQ in the absence of ATP at physiological ionic strength. In summary, our study points to a novel and potentially very common mechanism of GQ destabilization mediated by proteins binding to the vicinity of these structures.
- MeSH
- adenosindifosfát metabolismus MeSH
- adenosintrifosfát analogy a deriváty metabolismus MeSH
- G-kvadruplexy * MeSH
- helikasy RecQ chemie metabolismus MeSH
- jednovláknová DNA metabolismus MeSH
- lidé MeSH
- telomery chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Metallothioneins (MTs) are cysteine-rich peptides involved in heavy metal tolerance of many eukaryotes. Here, we examined their involvement in intracellular binding of silver (Ag) in the ectomycorrhizal fungus Amanita strobiliformis. The Ag complexes and their peptide ligands were characterized using chromatography and mass spectrometry. The full-length coding sequences obtained from a cDNA library were used for complementation assays in yeast mutant strains. Abundance of respective transcripts in A. strobiliformis was measured by quantitative real-time reverse-transcribed polymerase chain reaction (qRT-PCR). Ag-speciation analyses showed that intracellular Ag was in wild-grown fruit bodies and cultured extraradical mycelia of A. strobiliformis sequestered by metallothioneins. The determined sequence of the peptide facilitated isolation of three cDNA clones, AsMT1a, AsMT1b and AsMT1c. These encode isomorphic MTs consisting of 34 amino acid residues and sharing 82% identity. In mycelia the expression of AsMT1s is induced by Ag. All AsMT1s expressed in yeasts complemented hypersensitivity of mutants to cadmium (Cd) and copper (Cu) and formed Ag complexes. Only the Ag-AsMT1a complex was detected in the A. strobiliformis fruit body in which AsMT1a was the prevailing transcript. The present study identified the existence of metallothionein isoforms in ectomycorrhizal fungi. We demonstrated that intracellular sequestration of Ag in fruit bodies and mycelia of hyperaccumulating A. strobiliformis is dominated by metallothioneins.
- MeSH
- Amanita genetika metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- genetické vektory MeSH
- genová knihovna MeSH
- kadmium metabolismus MeSH
- měď metabolismus MeSH
- metalothionein genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- mycelium metabolismus MeSH
- plodnice hub genetika metabolismus MeSH
- protein - isoformy genetika metabolismus MeSH
- Saccharomyces cerevisiae MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- stříbro metabolismus MeSH
- testy genetické komplementace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
