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Dual targeting of higher-order DNA structures by azacryptands induces DNA junction-mediated DNA damage in cancer cells.

ABSTRACT: DNA is intrinsically dynamic and folds transiently into alternative higher-order structures such as G-quadruplexes (G4s) and three-way DNA junctions (TWJs). G4s and TWJs can be stabilised by small molecules (ligands) that have high chemotherapeutic potential, either as standalone DNA damaging agents or combined in synthetic lethality strategies. While previous approaches have claimed to use ligands that specifically target either G4s or TWJs, we report here on a new approach in which ligands targeting both TWJs and G4s in vitro demonstrate cellular effects distinct from that of G4 ligands, and attributable to TWJ targeting. The DNA binding modes of these new, dual TWJ-/G4-ligands were studied by a panel of in vitro methods and theoretical simulations, and their cellular properties by extensive cell-based assays. We show here that cytotoxic activity of TWJ-/G4-ligands is mitigated by the DNA damage response (DDR) and DNA topoisomerase 2 (TOP2), making them different from typical G4-ligands, and implying a pivotal role of TWJs in cells. We designed and used a clickable ligand, TrisNP-α, to provide unique insights into the TWJ landscape in cells and its modulation upon co-treatments. This wealth of data was exploited to design an efficient synthetic lethality strategy combining dual ligands with clinically relevant DDR inhibitors.

SUBMITTER: Zell J

PROVIDER: S-EPMC8501980 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Disruption of Higher Order DNA Structures in Friedreich's Ataxia (GAA)n Repeats by PNA or LNA Targeting.

Project description:Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich's ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression.

| S-EPMC5112992 | biostudies-literature

A model of photon cell killing based on the spatio-temporal clustering of DNA damage in higher order chromatin structures.

Project description:We present a new approach to model dose rate effects on cell killing after photon radiation based on the spatio-temporal clustering of DNA double strand breaks (DSBs) within higher order chromatin structures of approximately 1-2 Mbp size, so called giant loops. The main concept of this approach consists of a distinction of two classes of lesions, isolated and clustered DSBs, characterized by the number of double strand breaks induced in a giant loop. We assume a low lethality and fast component of repair for isolated DSBs and a high lethality and slow component of repair for clustered DSBs. With appropriate rates, the temporal transition between the different lesion classes is expressed in terms of five differential equations. These allow formulating the dynamics involved in the competition of damage induction and repair for arbitrary dose rates and fractionation schemes. Final cell survival probabilities are computable with a cell line specific set of three parameters: The lethality for isolated DSBs, the lethality for clustered DSBs and the half-life time of isolated DSBs. By comparison with larger sets of published experimental data it is demonstrated that the model describes the cell line dependent response to treatments using either continuous irradiation at a constant dose rate or to split dose irradiation well. Furthermore, an analytic investigation of the formulation concerning single fraction treatments with constant dose rates in the limiting cases of extremely high or low dose rates is presented. The approach is consistent with the Linear-Quadratic model extended by the Lea-Catcheside factor up to the second moment in dose. Finally, it is shown that the model correctly predicts empirical findings about the dose rate dependence of incidence probabilities for deterministic radiation effects like pneumonitis and the bone marrow syndrome. These findings further support the general concepts on which the approach is based.

| S-EPMC3879277 | biostudies-literature

Co-opted transposons help perpetuate conserved higher-order chromosomal structures

Project description:Co-option of transposable elements maintains conserved 3D genome structures via CTCF binding site turnover in human and mouse.

2019-12-08 | GSE141550 | GEO

Interrogating the higher order structures of snake venom proteins

Project description:Snake venoms contain complex mixtures of proteins that play vital roles in the survival of venomous snakes. In line with their diverse pharmacological activities, the protein compositions of snake venoms can be highly variable, and efforts to characterise the primary structures of such proteins are extensive and ongoing. In addition, a significant knowledge gap exists in terms of higher-order interactionsbetweenproteinsproposedto modulate venom potency, which poses a challenge for treatment of envenomation and development of successful therapeutic applications.Here we use a multifaceted mass spectrometrybased approach to characteriseproteinsfrom the medically significant venomsof Collett’s snake Pseudechis collettiand the puff adder Bitis arietans.Following chromatographic fractionation and bottom-up proteomics identification, native mass spectrometry identified, among other components, a 117 kDa non-covalent L-amino acid oxidase dimer in the P. collettivenom and a 60 kDa C-type lectin tetramer in the B. arietansvenom. Furthermore, a 27.7 kDa covalently-linked phospholipase A2(PLA2) dimer was identified in P. collettivenom, and thePLA2species were shownto adopt a highly compact geometry based on ion mobility measurements. Exploration of the catalytic efficiencies of the monomeric and dimeric forms of PLA2revealed that the dimeric PLA2 possessed greater bioactivity than the monomeric PLA2s.This work contributes to ongoing efforts to catalogue the protein components of snake venoms, and notably,itemphasises the importance of understanding higher-order protein interactions in venomsand the utility of a combined mass spectrometric approachfor this task.

2020-02-17 | PXD016503 | Pride

PcG-mediated higher-order chromatin structures modulate replication programs at the Drosophila BX-C.

Project description:Polycomb group proteins (PcG) exert conserved epigenetic functions that convey maintenance of repressed transcriptional states, via post-translational histone modifications and high order structure formation. During S-phase, in order to preserve cell identity, in addition to DNA information, PcG-chromatin-mediated epigenetic signatures need to be duplicated requiring a tight coordination between PcG proteins and replication programs. However, the interconnection between replication timing control and PcG functions remains unknown. Using Drosophila embryonic cell lines, we find that, while presence of specific PcG complexes and underlying transcription state are not the sole determinants of cellular replication timing, PcG-mediated higher-order structures appear to dictate the timing of replication and maintenance of the silenced state. Using published datasets we show that PRC1, PRC2, and PhoRC complexes differently correlate with replication timing of their targets. In the fully repressed BX-C, loss of function experiments revealed a synergistic role for PcG proteins in the maintenance of replication programs through the mediation of higher-order structures. Accordingly, replication timing analysis performed on two Drosophila cell lines differing for BX-C gene expression states, PcG distribution, and chromatin domain conformation revealed a cell-type-specific replication program that mirrors lineage-specific BX-C higher-order structures. Our work suggests that PcG complexes, by regulating higher-order chromatin structure at their target sites, contribute to the definition and the maintenance of genomic structural domains where genes showing the same epigenetic state replicate at the same time.

| S-EPMC3578750 | biostudies-literature

Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin

Project description:Packaging of DNA into chromatin regulates DNA accessibility and consequently all DNA-dependent processes. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This organization has been recently questioned using microscopy techniques, proposing an irregular structure. To address the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. We assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions, performed statistical analyses and computer modelling. We found no difference in MNase accessibility, suggesting no difference in fiber folding between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially organized domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization.

2018-12-04 | GSE100401 | GEO

Higher-order chromatin structures of chromosomally-integrated HHV-6A predict Integration Sites

Project description:We used 4C sequencing technology for high-throughput profiling of HHV6A integration in both HEK293T cells and SMC cells. We identified genome-wide host-virus and virus-virus interactions profiles.

2020-12-09 | GSE162821 | GEO

Holliday junction-containing DNA structures persist in cells lacking Sgs1 or Top3 following exposure to DNA damage.

Project description:The Sgs1-Rmi1-Top3 "dissolvasome" is required for the maintenance of genome stability and has been implicated in the processing of various types of DNA structures arising during DNA replication. Previous investigations have revealed that unprocessed (X-shaped) homologous recombination repair (HRR) intermediates persist when S-phase is perturbed by using methyl methanesulfonate (MMS) in Saccharomyces cerevisiae cells with impaired Sgs1 or Top3. However, the precise nature of these persistent DNA structures remains poorly characterized. Here, we report that ectopic expression of either of two heterologous and structurally unrelated Holliday junction (HJ) resolvases, Escherichia coli RusA or human GEN1(1-527), promotes the removal of these X-structures in vivo. Moreover, other types of DNA replication intermediates, including stalled replication forks and non-HRR-dependent X-structures, are refractory to RusA or GEN1(1-527), demonstrating specificity of these HJ resolvases for MMS-induced X-structures in vivo. These data suggest that the X-structures persisting in cells with impaired Sgs1 or Top3 contain HJs. Furthermore, we demonstrate that Sgs1 directly promotes X-structure removal, because the persistent structures arising in Sgs1-deficient strains are eliminated when Sgs1 is reactivated in vivo. We propose that HJ resolvases and Sgs1-Top3-Rmi1 comprise two independent processes to deal with HJ-containing DNA intermediates arising during HRR in S-phase.

| S-EPMC3064375 | biostudies-literature

Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin.

| S-EPMC6379673 | biostudies-literature

Dual Targeting of DNA Damage Response Proteins Implicated in Cancer Radioresistance.

Project description:Ionizing radiation can induce different types of DNA lesions, leading to genomic instability and ultimately cell death. Radiation therapy or radiotherapy, a major modality in cancer treatment, harnesses the genotoxic potential of radiation to target and destroy cancer cells. Nevertheless, cancer cells have the capacity to develop resistance to radiation treatment (radioresistance), which poses a major obstacle in the effective management of cancer. It has been shown that administration of platinum-based drugs to cancer patients can increase tumor radiosensitivity, but despite this, it is associated with severe adverse effects. Several lines of evidence support that activation of the DNA damage response and repair machinery in the irradiated cancer cells enhances radioresistance and cellular survival through the efficient repair of DNA lesions. Therefore, targeting of key DNA damage repair factors would render cancer cells vulnerable to the irradiation effects, increase cancer cell killing, and reduce the risk of side effects on healthy tissue. Herein, we have employed a computer-aided drug design approach for generating ab initio a chemical compound with drug-like properties potentially targeting two proteins implicated in multiple DNA repair pathways. The findings of this study could be taken into consideration in clinical decision-making in terms of co-administering radiation with DNA damage repair factor-based drugs.

| S-EPMC10742610 | biostudies-literature

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