Human Adenovirus (Ad) is a non-enveloped, icosahedral virus with a linear, double-stranded DNA genome. The Ad IVa2 protein is involved in multiple viral processes including viral late gene transcription and virus assembly. Previous studies have shown that IVa2 loads additional viral proteins onto conserved DNA elements within the Ad genome to regulate these viral processes. IVa2 also possesses strong non-specific DNA binding activity, and it is likely it uses this activity to recruit proteins to the conserved DNA elements. Here we have investigated the non-specific DNA binding activity of IVa2 using nitrocellulose/DEAE filter binding and sedimentation equilibrium techniques. We have analyzed our data using the McGhee and Von Hippel approach [1], and find that IVa2 binds with strong, positive nearest-neighbor cooperativity. In addition, we describe how to apply the McGhee and von Hippel approach to directly analyze sedimentation equilibrium data using non-linear least-squares methods. We discuss the implications of these results with respect to current virus assembly mechanisms.
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http://dx.doi.org/10.1016/j.bpc.2014.06.005 | DOI Listing |
CRISPR-Cas12a is widely used for genome editing and biomarker detection since it can create targeted double-stranded DNA breaks and promote non-specific DNA cleavage after identifying specific DNA. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Cas12a variant (FnoCas12a ) by introducing double proline substitutions (K969P/D970P) in a conserved helix called the bridge helix (BH). In this work, we used cryogenic electron microscopy (cryoEM) to understand the molecular mechanisms of BH- mediated activation of Cas12a.
View Article and Find Full Text PDFZhonghua Zhong Liu Za Zhi
January 2025
Department of Pathology, China-Japan Friendship Hospital, Beijing100029, China.
To analyze the clinical significance of molecular classification and hereditary phenotype in endometrial carcinoma (EC) based on high throughput sequencing (NGS). 97 EC samples were collected retrospectively from December 2019 to October 2022 in China-Japan Friendship Hospital. NGS technique was used to analyze the molecular classification, POLE hypermutation, microsatellite high Instability/mismatch repair dysfunction (MSI-H/MMRd), P53 protein abnormality (P53 abn), and non-specific molecular profile (NSMP).
View Article and Find Full Text PDFBiophys J
January 2025
Department of Physics and Astronomy, Department of Chemistry, NSF-Simons Center for Multiscale Cell Fate Research, University of California, Irvine, California, USA. Electronic address:
In this work we present a minimal structure-based model of protein diffusional search along local DNA amid protein binding and unbinding events on the DNA, taking into account protein-DNA electrostatic interactions and hydrogen-bonding (HB) interactions or contacts at the interface. We accordingly constructed the protein diffusion-association/dissociation free energy surface and mapped it to 1D as the protein slides along DNA, maintaining the protein-DNA interfacial HB contacts that presumably dictate the DNA sequence information detection. Upon DNA helical path correction, the protein 1D diffusion rates along local DNA can be physically derived to be consistent with experimental measurements.
View Article and Find Full Text PDFJ Vis Exp
December 2024
Institute for Biological and Medical Engineering, Pontificia Universidad Católica de Chile;
Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy.
View Article and Find Full Text PDFNucleic Acids Res
January 2025
Department of Physics, 845 W Taylor St, University of Illinois Chicago, Chicago, IL 60607, USA.
Altered DNA dynamics at lesion sites are implicated in how DNA repair proteins sense damage within genomic DNA. Using laser temperature-jump (T-jump) spectroscopy combined with cytosine-analog Förster Resonance Energy Transfer (FRET) probes that sense local DNA conformations, we measured the intrinsic dynamics of DNA containing 3 base-pair mismatches recognized in vitro by Rad4 (yeast ortholog of XPC). Rad4/XPC recognizes diverse lesions from environmental mutagens and initiates nucleotide excision repair.
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