The dynamics of forming a triplex in an artificial telomere inferred by DNA mechanics

Li, Ning; Wang, Junli; Ma, Kangkang; Liang, Lin; Mi, Lipei; Huang, Wei; Ma, Xiaofeng; Wang, Zeyu; Zheng, Wei; Zhou, Xiao‐Nong; Yu, Zhongbo

doi:10.1093/nar/gkz464

Cited by 19 publications

(18 citation statements)

References 75 publications

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“…AmiGO 2 38 , 39 was used for gene enrichment analysis applying Fisher’s exact test in combination with False Discovery Rate (FDR p < 0.05) correction 40 , 41 .…”

Section: Methodsmentioning

confidence: 99%

Gene expression changes and DNA damage after ex vivo exposure of peripheral blood cells to various CT photon spectra

Kaatsch

Becker

Schüle

et al. 2021

Sci Rep

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Dual-energy CT provides enhanced diagnostic power with similar or even reduced radiation dose as compared to single-energy CT. Its principle is based on the distinct physical properties of low and high energetic photons, which, however, may also affect the biological effectiveness and hence the extent of CT-induced cellular damage. Therefore, a comparative analysis of biological effectiveness of dual- and single-energy CT scans with focus on early gene regulation and frequency of radiation-induced DNA double strand breaks (DSBs) was performed. Blood samples from three healthy individuals were irradiated ex vivo with single-energy (80 kV and 150 kV) and dual-energy tube voltages (80 kV/Sn150kV) employing a modern dual source CT scanner resulting in Volume Computed Tomography Dose Index (CTDIvol) of 15.79–18.26 mGy and dose length product (DLP) of 606.7–613.8 mGy*cm. Non-irradiated samples served as a control. Differential gene expression in peripheral blood mononuclear cells was analyzed 6 h after irradiation using whole transcriptome sequencing. DSB frequency was studied by 53BP1 + γH2AX co-immunostaining and microscopic evaluation of their focal accumulation at DSBs. Neither the analysis of gene expression nor DSB frequency provided any evidence for significantly increased biological effectiveness of dual-energy CT in comparison to samples irradiated with particular single-energy CT spectra. Relative to control, irradiated samples were characterized by a significantly higher rate of DSBs (p < 0.001) and the shared upregulation of five genes, AEN, BAX, DDB2, FDXR and EDA2R, which have already been suggested as radiation-induced biomarkers in previous studies. Despite steadily decreasing doses, CT diagnostics remain a genotoxic stressor with impact on gene regulation and DNA integrity. However, no evidence was found that varying X-ray spectra of CT impact the extent of cellular damage.

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“…AmiGO 2 38 , 39 was used for gene enrichment analysis applying Fisher’s exact test in combination with False Discovery Rate (FDR p < 0.05) correction 40 , 41 .…”

Section: Methodsmentioning

confidence: 99%

Gene expression changes and DNA damage after ex vivo exposure of peripheral blood cells to various CT photon spectra

Kaatsch

Becker

Schüle

et al. 2021

Sci Rep

View full text Add to dashboard Cite

show abstract

“…In lane 4, we saw two bands (top:bottom = 0.85:1) at the expected position for the PPI construct (Arrows in Figure B). Because nicked circular DNA migrates more slowly than linear ones in an agarose gel, , we rationalized that PPI between PHD3-Bromo and H3K4me3 closed the DNA circuit, forming a circular conformation (top arrow). On the other hand, the open circuit was in a linear structure (bottom arrow).…”

Section: Resultsmentioning

confidence: 96%

Interactions between PHD3-Bromo of MLL1 and H3K4me3 Revealed by Single-Molecule Magnetic Tweezers in a Parallel DNA Circuit

Zhu

Liu

et al. 2019

Bioconjugate Chem.

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Single-molecule force spectroscopy is a powerful tool to directly measure protein–protein interactions (PPI). The high specificity and precision of PPI measurements made it possible to reveal detailed mechanisms of intermolecular interactions. However, protein aggregation due to specific or nonspecific interactions is among the most challenging problems in PPI examination. Here, we propose a strategy of a parallel DNA circuit to probe PPI using single-molecule magnetic tweezers. In contrast to PPI examination using atomic force microscopy, microspheres as probes used in magnetic tweezers avoided the single-probe issue of a cantilever. Negatively charged DNA as a linker circumvented the severe aggregation in the PPI construct with a protein linker. The unnatural amino acid encoded in proteins of interest expanded the choices of biorthogonal conjugation. We demonstrated how to apply our strategy to probe the PPI between the PHD3-Bromo and the histone H3 methylated at K4, a critical epigenetic event in leukemia development. We found a rupture force of 12 pN for breaking the PPI, which is much higher than that required to peel DNA off from a nucleosome, 3 pN. We expect that our methods will make PPI measurements of mechanics and kinetics with great precision, facilitating PPI-related research, e.g., PPI-targeted drug discovery.

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“…We use home-made magnetic tweezers ( Fig. S1 ), similar to the setup previously described ( 37 , 38 , 39 , 40 ), which consists of microscopy, motors manipulating magnets, Piezo stages, and a flow cell connecting to a pump. The inverted microscope uses an oil immersion objective (UPLFLN 100 × O2, Numerical aperture (N.A.)…”

Section: Methodsmentioning

confidence: 99%

Single-molecule multiplexed profiling of protein–DNA complexes using magnetic tweezers

Liang

Wang

et al. 2021

Journal of Biological Chemistry

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Epigenetics, such as the dynamic interplay between DNA methylation and demethylation, play diverse roles in critical cellular events. Enzymatic activity at CpG sites, where cytosines are methylated or demethylated, is known to be influenced by the density of CpGs, methylation states, and the flanking sequences of a CpG site. However, how the relevant enzymes are recruited to and recognize their target DNA is less clear. Moreover, although DNA-binding epigenetic enzymes are ideal targets for therapeutic intervention, these targets have been rarely exploited. Single-molecule techniques offer excellent capabilities to probe site-specific protein–DNA interactions and unravel the dynamics. Here, we develop a single-molecule approach that allows multiplexed profiling of protein–DNA complexes using magnetic tweezers. When a DNA hairpin with multiple binding sites is unzipping, strand separation pauses at the positions bound by a protein. We can thus measure site-specific binding probabilities and dissociation time directly. Taking the TET1 CXXC domain as an example, we show that TET1 CXXC binds multiple CpG motifs with various flanking nucleotides or different methylation patterns in an AT-rich DNA. We are able to establish for the first time, at nanometer resolution, that TET1 CXXC prefers G/C flanked CpG motif over C/G, A/T, or T/A flanked ones. CpG methylation strengthens TET1 CXXC recruitment but has little effect on dissociation time. Finally, we demonstrate that TET1 CXXC can distinguish five CpG clusters in a CpG island with crowded binding motifs. We anticipate that the feasibility of single-molecule multiplexed profiling assays will contribute to the understanding of protein–DNA interactions.

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The dynamics of forming a triplex in an artificial telomere inferred by DNA mechanics

Cited by 19 publications

References 75 publications

Gene expression changes and DNA damage after ex vivo exposure of peripheral blood cells to various CT photon spectra

Gene expression changes and DNA damage after ex vivo exposure of peripheral blood cells to various CT photon spectra

Interactions between PHD3-Bromo of MLL1 and H3K4me3 Revealed by Single-Molecule Magnetic Tweezers in a Parallel DNA Circuit

Single-molecule multiplexed profiling of protein–DNA complexes using magnetic tweezers

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