probing-structure-dynamics-nucleosomes-using-atomic-force-microscopy

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

University of Nebraska Medical Center

As a guardian of the bacterial genome, the RecG DNA helicase repairs DNA replication and rescues stalled replication. We applied atomic force microscopy (AFM) to directly visualize dynamics of RecG upon the interaction with replication fork substrates in the presence and absence of SSB using high-speed AFM. We directly visualized that RecG moves back and forth over dozens of base pairs in the presence of SSB. There is no RecG translocation in the absence of SSB. Computational modeling was performed to build models of Escherichia coli RecG in a free state and in complex with the fork. The simulations revealed the formation of complexes of RecG with the fork and identified conformational transitions that may be responsible for RecG remodeling that can facilitate RecG translocation along the DNA duplex. Such complexes do not form with the DNA duplex, which is in line with experimental data. Overall, our results provide mechanistic insights into the modes of interaction of RecG with the replication fork, suggesting a novel role of RecG in the repair of stalled DNA replication forks.

Dynamics of the Interaction of RecG Protein with Stalled Replication Forks.

Atomic force microscopy (AFM) is an imaging technique that enables single molecule characterization of biological systems at nanometer resolution. Imaging in ambient conditions can provide details of the conformational states and interactions of a population of molecules which is well complemented by single-molecule imaging of the systems dynamics using time-lapse AFM imaging, in which images are capture at rates of 10-15 frames per second in an aqueous buffer. Here we describe the assembly and preparation of nucleosomes containing centromere protein A (CENP-A) for AFM imaging in both static and time-lapse modes. The AFM imaging and data analysis techniques described enable characterization of the extent of DNA wrapping around the histone core and time-resolved visualization of the systems intrinsic dynamic behaviors.

Assembly of Centromere Chromatin for Characterization by High-Speed Time-Lapse Atomic Force Microscopy.

Impairment of microglial functions, such as phagocytosis and/or dysregulation of immune responses, has been implicated as an underlying factor involved in the pathogenesis of various neurodegenerative disorders. Our previous studies have demonstrated that long intergenic noncoding RNA (lincRNA)-Cox2 expression is influenced by nuclear factor κB (NF-κB) signaling and serves as a coactivator of transcriptional factors to regulate the expression of a vast array of immune-related genes in microglia. Extracellular vesicles (EVs) have been recognized as primary facilitators of cell-to-cell communication and cellular regulation. Herein, we show that EVs derived from astrocytes exposed to morphine can be taken up by microglial endosomes, leading, in turn, to activation of Toll-like receptor 7 (TLR7) with a subsequent upregulation of lincRNA-Cox2 expression, ultimately resulting in impaired microglial phagocytosis. This was further validated in vivo, wherein inhibition of microglial phagocytic activity was also observed in brain slices isolated from morphine-administrated mice compared with control mice. Additionally, we also showed that intranasal delivery of EVs containing lincRNA-Cox2 siRNA (small interfering RNA) was able to restore microglial phagocytic activity in mice administered morphine. These findings have ramifications for the development of EV-loaded RNA-based therapeutics for the treatment of various disorders involving functional impairment of microglia.

Article	Year
Dynamics of the Interaction of RecG Protein with Stalled Replication Forks. Biochemistry\| PubMed ID: 29432678	2018
Assembly of Centromere Chromatin for Characterization by High-Speed Time-Lapse Atomic Force Microscopy. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29956236	2018
Astrocyte EV-Induced lincRNA-Cox2 Regulates Microglial Phagocytosis: Implications for Morphine-Mediated Neurodegeneration. Molecular therapy. Nucleic acids\| PubMed ID: 30388619	2018

Zhiqiang Sun

Biography

JoVE Journal Publications

Other Publications