scholarly journals Thioltransferase (Glutaredoxin) Reactivates the DNA-binding Activity of Oxidation-inactivated Nuclear Factor I

1998 ◽  
Vol 273 (1) ◽  
pp. 392-397 ◽  
Author(s):  
Smarajit Bandyopadhyay ◽  
David W. Starke ◽  
John J. Mieyal ◽  
Richard M. Gronostajski
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Factor I ◽  
Dna Binding Activity ◽  
Nuclear Factor I

scholarly journals Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay.

1986 ◽  
Vol 6 (5) ◽  
pp. 1363-1373 ◽  
Author(s):  
J F Diffley ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Assay ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Factor I ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Nuclear Factor I ◽  
Dna Fragment

A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.

Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay

1986 ◽  
Vol 6 (5) ◽  
pp. 1363-1373
Author(s):  
J F Diffley ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Assay ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Factor I ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Nuclear Factor I ◽  
Dna Fragment

A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.

scholarly journals Identification of DNA binding-site preferences for nuclear factor I-A

FEBS Letters ◽  
1996 ◽  
Vol 390 (1) ◽  
pp. 44-46 ◽  
Author(s):  
Shigehiro Osada ◽  
Shoko Daimon ◽  
Tsutomu Nishihara ◽  
Masayoshi Imagawa
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Nuclear Factor ◽  
Factor I ◽  
Site Preferences ◽  
Dna Binding Site ◽  
Nuclear Factor I

UV stimulation induces nuclear factor κB (NF-κB) DNA-binding activity but not transcriptional activation

2001 ◽  
Vol 29 (6) ◽  
pp. 688-691 ◽  
Author(s):  
K. J. Campbell ◽  
N. R. Chapman ◽  
N. D. Perkins
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Cellular Response ◽  
Uv Light ◽  
Binding Protein ◽  
Nuclear Factor Κb ◽  
Camp Response Element Binding ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Dna Binding Activity

The cellular response to DNA-damaging agents is partly mediated by DNA-binding transcription factors such as p53 and nuclear factor κB (NF-κB). Typically NF-κB activation is associated with resistance to apoptosis. Following stimulation with UV light however, NF-κB activation has been shown to be required for programmed cell death. To study this effect further and to determine the relationship between NF-κB and p53 function, we have examined the effect of UV light on U2OS cells. UV stimulation resulted in the activation of NF-κB DNA-binding and the induction of p53. Surprisingly, and in contrast with tumour necrosis factor α stimulation, this UV-induced NF-κB was transcriptionally inert. These observations suggest a model in which the NF-κB switch from an anti-apoptotic to a pro-apoptotic role within the cell results from modulation of its ability to stimulate gene expression, possibly as a result of the ability of p53 to sequester transcriptional co-activator proteins such as p300/CREB (cAMP-response-element-binding protein)-binding protein.

scholarly journals DNA binding activity of nuclear factor of activated T cells in mononuclear cells from renal transplant patients with and without BK virus viruria

2013 ◽  
Vol 25 (2) ◽  
pp. 112-116 ◽  
Author(s):  
Wen-Yao Yin ◽  
Ning-Sheng Lai ◽  
Ming-Che Lee ◽  
Chia-Li Yu ◽  
Shiang-Long Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Renal Transplant ◽  
Dna Binding ◽  
Mononuclear Cells ◽  
Binding Activity ◽  
Bk Virus ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Transplant Patients ◽  
Dna Binding Activity
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