scholarly journals Recognition site of nuclear factor I, a sequence-specific DNA-binding protein from HeLa cells that stimulates adenovirus DNA replication.

1985 ◽  
Vol 4 (6) ◽  
pp. 1515-1521 ◽  
Author(s):  
P.A. Leegwater ◽  
W. van Driel ◽  
P.C. van der Vliet
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Hela Cells ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Recognition Site ◽  
Nuclear Factor ◽  
Factor I ◽  
Nuclear Factor I

Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication

Nature ◽  
1986 ◽  
Vol 322 (6080) ◽  
pp. 656-659 ◽  
Author(s):  
Ger J. M. Pruijn ◽  
Wim van Driel ◽  
Peter C. van der Vliet
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Hela Cells ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Nuclear Factor

scholarly journals Two regions within the DNA binding domain of nuclear factor I interact with DNA and stimulate adenovirus DNA replication independently.

1996 ◽  
Vol 16 (8) ◽  
pp. 4073-4080 ◽  
Author(s):  
J Dekker ◽  
J A van Oosterhout ◽  
P C van der Vliet
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Polymerase ◽  
Binding Domain ◽  
Nuclear Factor ◽  
Dna Binding Domain ◽  
Factor I ◽  
Terminal Domain ◽  
Nuclear Factor I ◽  
Stimulation Of

The cellular transcription factor nuclear factor I (NFI) stimulates adenovirus DNA replication by up to 50-fold. The NFI DNA binding domain (NFI-BD) is sufficient for stimulation and interacts with the viral DNA polymerase, thereby recruiting the precursor terminal protein-DNA polymerase complex (pTP-pol) to the origin of replication. The mechanism of DNA binding by NFI is unknown. To examine DNA binding and stimulation of adenovirus DNA replication by NFI-BD in more detail, we generated a series of deletion mutants and show that the DNA binding domain of NFI consists of two subdomains: a highly basic N-terminal domain that binds nonspecifically to DNA and a C-terminal domain that binds specifically but with very low affinity to the NFI recognition site. Both of these subdomains stimulate DNA replication, although not to the same extent as the intact DNA binding domain. The N-terminal domain has an alpha-helical structure, as shown by circular dichroism spectroscopy. The C-terminal domain interacts with the pTP-pol complex and is able to recruit the pTP-pol complex to DNA, which leads to pTP-pol-dependent stimulation of replication. The N-terminal domain also stimulates replication in a pTP-pol-dependent manner and enhances binding of pTP-pol to DNA. Since we could not detect a direct protein-protein interaction between pTP-pol and the N-terminal domain, we suggest that this domain stimulates replication by inducing structural changes in the DNA.

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.

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