Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

Peng, Yucai; Acheson, Nicholas H.

doi:10.1128/jvi.72.9.7330-7340.1998

Cited by 8 publications

(5 citation statements)

References 60 publications

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“…It has previously been shown that the affinity of a DNA-binding protein for DNA increases at low pH. [39][40][41][42] This is also the case with the Tus-TerB and Tus-Ter-lockB complexes and can be explained by an increase in net positive charge of Tus (at low pH), strengthening the electrostatic interactions with the negatively charged phosphate backbone of the DNA. Thus, the difference in stabilization trends observed for Tus-GFP-TerB and Tus-GFP-Ter-lockB at low pH as well as at lower salt conditions is mainly due to the difference in number of nucleotides between these two species.…”

Section: Salt and Ph-dependence Of Free And Dna-bound Tus-gfpmentioning

confidence: 84%

Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins

et al. 2012

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The development of differential scanning fluorimetry and the high-throughput capability of Thermofluor have vastly facilitated the screening of crystallization conditions of proteins and large mutant libraries in structural genomics programs, as well as ligands in drug discovery and functional genomics programs. These techniques are limited by their requirement for both highly purified proteins and solvatochromic dyes, fueling the need for more robust technologies that can be used with crude protein samples. Here, we present the development of a new high-throughput technology for the quantitative determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins. This technology is based on the principle that a change in the proximal environment of GFP, such as unfolding and aggregation of the protein of interest, is measurable through its effect on the fluorescence of the fluorophore. Protein stability data was generated for twelve GFP-tagged proteins including monomeric and multimeric, DNA-binding, RNA-binding, proteolytic, heat-shock and metabolic proteins of Escherichia coli, Burkholderia pseudomallei, Staphylococcus aureus, dengue and influenza (H5N1) viruses. The technology is simple, fast and insensitive to variations in sample volumes, and the useful temperature and pH range is 30-80 uC and 5-11 respectively. The system does not require solvatochromic dyes, reducing the risk of interference. The protein samples are simply mixed with the test conditions in a 96-well plate and subjected to a melt-curve protocol using a real-time thermal cycler. The data are obtained within 1-2 h and include unique quality control measures.

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Section: Salt and Ph-dependence Of Free And Dna-bound Tus-gfpmentioning

confidence: 84%

Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins

et al. 2012

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“…Then, splicing sites were evaluated in the context of protein sequences that they produce, and exons were identified based on homology to known PyV LTag proteins ( S2 Table ). ORI region was analyzed based on identified and defined SV40 and MCPyV ORI sequences [ 37 – 39 ].…”

Section: Methodsmentioning

confidence: 99%

Identification of Two Novel Members of the Tentative Genus Wukipolyomavirus in Wild Rodents

et al. 2015

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Two novel polyomaviruses (PyVs) were identified in kidney and chest-cavity fluid samples of wild bank voles (Myodes glareolus) and common voles (Microtus arvalis) collected in Germany. All cloned and sequenced genomes had the typical PyV genome organization, including putative open reading frames for early regulatory proteins large T antigen and small T antigen on one strand and for structural late proteins (VP1, VP2 and VP3) on the other strand. Virus-like particles (VLPs) were generated by yeast expression of the VP1 protein of both PyVs. VLP-based ELISA and large T-antigen sequence-targeted polymerase-chain reaction investigations demonstrated signs of infection of these novel PyVs in about 42% of bank voles and 18% of common voles. In most cases only viral DNA, but not VP1-specific antibodies were detected. In additional animals exclusively VP1-specific antibodies, but no viral DNA was detected, indicative for virus clearance. Phylogenetic and clustering analysis including all known PyV genomes placed novel bank vole and common vole PyVs amongst members of the tentative Wukipolymavirus genus. The other known four rodent PyVs, Murine PyV and Hamster PyV, and Murine pneumotropic virus and Mastomys PyV belong to different phylogenetic clades, tentatively named Orthopolyomavirus I and Orthopolyomavirus II, respectively. In conclusion, the finding of novel vole-borne PyVs may suggest an evolutionary origin of ancient wukipolyomaviruses in rodents and may offer the possibility to develop a vole-based animal model for human wukipolyomaviruses.

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“… • T-antigen (a specific DNA-binding protein from simian virus) binds with high affinity to unspecific DNA at pH = 6, with the affinity decreasing substantially at pH = 7.5 ( Wessel et al, 1992 ). • DNA-binding activity of polyomavirus large T-antigen decreases steadily from pH = 6.0 to 8.0 ( Peng and Acheson, 1998 ). …”

Section: Resultsmentioning

confidence: 99%

“…• DNA-binding activity of polyomavirus large T-antigen decreases steadily from pH = 6.0 to 8.0 ( Peng and Acheson, 1998 ).…”

Section: Resultsmentioning

confidence: 99%

“…For protein -nucleic acid complexes in general, an earlier theoretical work (Record et al, 1978) concluded that free energy of protein-DNA interactions should decrease with pH. We are aware of only a few relevant experimental data points (Senear and Ackers, 1990;Wessel et al, 1992;Peng and Acheson, 1998;Torigoe et al, 2003;Hamimes et al, 2006;Deegan et al, 2010;Moreau et al, 2012)-these show consistently that complex stability decreases with pH, however, most of the conclusions are qualitative, the strength of the reported effect varies substantially from study to study. Reported computational predictions also vary with respect to the significance of pH dependence or, equivalently, net proton uptake, in proteinnucleic acid binding.…”

Section: Introductionmentioning

confidence: 89%

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Biologically relevant small variations of intra-cellular pH can have significant effect on stability of protein–DNA complexes, including the nucleosome

Onufriev

2023

Front. Mol. Biosci.

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Stability of a protein-ligand complex may be sensitive to pH of its environment. Here we explore, computationally, stability of a set of protein-nucleic acid complexes using fundamental thermodynamic linkage relationship. The nucleosome, as well as an essentially random selection of 20 protein complexes with DNA or RNA, are included in the analysis. An increase in intra-cellular/intra-nuclear pH destabilizes most complexes, including the nucleosome. We propose to quantify the effect by ΔΔG0.3—the change in the binding free energy due to pH increase of 0.3 units, corresponding to doubling of the H+ activity; variations of pH of this amplitude can occur in living cells, including in the course of the cell cycle, and in cancer cells relative to normal ones. We suggest, based on relevant experimental findings, a threshold of biological significance of 12kBT(∼0.3kcal/mol) for changes of stability of chromatin-related protein-DNA complexes: a change in the binding affinity above the threshold may have biological consequences. We find that for 70% of the examined complexes, ΔΔG0.3>12kBT (for 10%, ΔΔG0.3 is between 3 and 4 kBT). Thus, small but relevant variations of intra-nuclear pH of 0.3 may have biological consequences for many protein-nucleic acid complexes. The binding affinity between the histone octamer and its DNA, which directly affects the DNA accessibility in the nucleosome, is predicted to be highly sensitive to intra-nuclear pH. A variation of 0.3 units results in ΔΔG0.3 ∼ 10kBT(∼6kcal/mol); for spontaneous unwrapping of 20 bp long entry/exit fragments of the nucleosomal DNA, ΔΔG0.3 = 2.2kBT; partial disassembly of the nucleosome into the tetrasome is characterized by ΔΔG0.3 = 5.2kBT. The predicted pH -induced modulations of the nucleosome stability are significant enough to suggest that they may have consequences relevant to the biological function of the nucleosome. Accessibility of the nucleosomal DNA is predicted to positively correlate with pH variations during the cell cycle; an increase in intra-cellular pH seen in cancer cells is predicted to lead to a more accessible nucleosomal DNA; a drop in pH associated with apoptosis is predicted to make nucleosomal DNA less accessible. We speculate that processes that depend on accessibility to the DNA in the nucleosomes, such as transcription or DNA replication, might become upregulated due to relatively small, but nevertheless realistic increases of intra-nuclear pH.

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Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

Cited by 8 publications

References 60 publications

Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins

Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins

Identification of Two Novel Members of the Tentative Genus Wukipolyomavirus in Wild Rodents

Biologically relevant small variations of intra-cellular pH can have significant effect on stability of protein–DNA complexes, including the nucleosome

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