scholarly journals A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication

Virology ◽  
2017 ◽  
Vol 500 ◽  
pp. 190-197 ◽  
Author(s):  
Eriko Ohsaki ◽  
Keiji Ueda
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Chimeric Protein ◽  
Binding Domain ◽  
Dna Binding Domain

scholarly journals Herpes Simplex Virus Type 1 C-Terminal Variants of the Origin Binding Protein (OBP), OBPC-1 and OBPC-2, Cooperatively Regulate Viral DNA Levels In Vitro, and OBPC-2 Affects Mortality in Mice

2007 ◽  
Vol 81 (19) ◽  
pp. 10699-10711 ◽  
Author(s):  
Malen A. Link ◽  
Priscilla A. Schaffer
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Origin Binding Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Two in-frame, C-terminal isoforms of the herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), OBPC-1 and OBPC-2, and a unique C-terminal transcript, UL8.5, are specified by HSV-1 DNA. As the first isoform identified, OBPC-1 was initially assumed to be the product of the UL8.5 transcript. Recent evidence has demonstrated, however, that OBPC-1 is a cathepsin B-mediated cleavage product of OBP, suggesting that OBPC-2 is the product of the UL8.5 transcript. Because both OBPC-1 and -2 contain the majority of the OBP DNA binding domain, we hypothesized that both may be involved in regulating origin-dependent, OBP-mediated viral DNA replication. In this paper, we demonstrate that OBPC-2 is, indeed, the product of the UL8.5 transcript. The translational start site of OBPC-2 was mapped, and a virus (M571A) that does not express this protein efficiently was constructed. Using M571A, we have shown that OBPC-2 is able to bind origin DNA, even though it lacks seven N-terminal amino acid residues of the previously mapped OBP DNA binding domain, resulting in a revision of the limits of the OBP DNA binding domain. Consistent with their proposed roles in regulating viral DNA replication, OBPC-1 and -2 act together to down-regulate viral DNA replication in vitro. During functional studies in vivo, OBPC-2 was identified as a factor that increases mortality in the mouse ocular model of HSV-1 infection.

scholarly journals trans-dominant inhibition of poly(ADP-ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N'-nitro-N-nitrosoguanidine but does not limit DNA replication of a polyomavirus replicon.

1995 ◽  
Vol 15 (6) ◽  
pp. 3154-3163 ◽  
Author(s):  
J H Küpper ◽  
M Müller ◽  
M K Jacobson ◽  
J Tatsumi-Miyajima ◽  
D L Coyle ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Gamma Irradiation ◽  
Cellular Proliferation ◽  
Molecular Genetic ◽  
Genetic System ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dna Binding Domain

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD+ serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible, high-level expression of the DNA-binding domain. Induction of this domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.

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 Surface Mutagenesis of the Bovine Papillomavirus E1 DNA Binding Domain Reveals Residues Required for Multiple Functions Related to DNA Replication

2006 ◽  
Vol 80 (15) ◽  
pp. 7491-7499 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Bovine Papillomavirus ◽  
Dna Binding Domain ◽  
Multifunctional Protein ◽  
Dna Binding Activity ◽  
E1 Protein ◽  
Complex Functions

ABSTRACT The E1 protein from papillomaviruses is a multifunctional protein with complex functions required for the initiation of viral DNA replication. We have performed a surface mutagenesis of the well-characterized E1 DNA binding domain (DBD). We demonstrate that substitutions of multiple residues on the surface of the E1 DBD are defective for DNA replication without affecting the DNA binding activity of the protein. The defects of individual substitutions include failure to form the double trimer that melts the ori and failure to form the double hexamer that unwinds the ori. These results demonstrate that the DBD plays an essential role in multiple DNA replication-related processes apart from DNA binding.

scholarly journals Two Regions of Simian Virus 40 T Antigen Determine Cooperativity of Double-Hexamer Assembly on the Viral Origin of DNA Replication and Promote Hexamer Interactions during Bidirectional Origin DNA Unwinding

1999 ◽  
Vol 73 (3) ◽  
pp. 2201-2211 ◽  
Author(s):  
Klaus Weisshart ◽  
Poonam Taneja ◽  
Andreas Jenne ◽  
Utz Herbig ◽  
Daniel T. Simmons ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Binding Domain ◽  
Dna Unwinding ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Viral Origin

ABSTRACT Phosphorylation of simian virus 40 large tumor (T) antigen on threonine 124 is essential for viral DNA replication. A mutant T antigen (T124A), in which this threonine was replaced by alanine, has helicase activity, assembles double hexamers on viral-origin DNA, and locally distorts the origin DNA structure, but it cannot catalyze origin DNA unwinding. A class of T-antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has remarkably similar properties, although these proteins are phosphorylated on threonine 124, as we show here. By comparing the DNA binding properties of the T124A and class 4 mutant proteins with those of the wild type, we demonstrate that mutant double hexamers bind to viral origin DNA with reduced cooperativity. We report that T124A T-antigen subunits impair the ability of double hexamers containing the wild-type protein to unwind viral origin DNA, suggesting that interactions between hexamers are also required for unwinding. Moreover, the T124A and class 4 mutant T antigens display dominant-negative inhibition of the viral DNA replication activity of the wild-type protein. We propose that interactions between hexamers, mediated through the DNA binding domain and the N-terminal phosphorylated region of T antigen, play a role in double-hexamer assembly and origin DNA unwinding. We speculate that one surface of the DNA binding domain in each subunit of one hexamer may form a docking site that can interact with each subunit in the other hexamer, either directly with the N-terminal phosphorylated region or with another region that is regulated by phosphorylation.

scholarly journals Characterization of the DNA-Binding Domain of the Bovine Papillomavirus Replication Initiator E1

1998 ◽  
Vol 72 (4) ◽  
pp. 2567-2576 ◽  
Author(s):  
Grace Chen ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Sites ◽  
T Antigen ◽  
Binding Domain ◽  
Bovine Papillomavirus ◽  
Dna Binding Domain ◽  
Binding Studies ◽  
Acid Fragment ◽  
Replication Initiator

ABSTRACT The bovine papillomavirus replication initiator protein E1 is an origin of replication (ori)-binding protein absolutely required for viral DNA replication. In the presence of the viral transcription factor E2, E1 binds to the ori and initiates DNA replication. To understand how the E1 initiator recognizes theori and how E2 assists in this process, we have expressed and purified a 166-amino-acid fragment which corresponds to the minimal E1 DNA-binding domain (DBD). DNA binding studies using this protein demonstrate that the E1 DBD can bind to the palindromic E1 binding site in several forms but that binding of two monomers, each recognizing one half-site of the E1 palindrome, is the predominant form. This is reminiscent of the binding of the T-antigen DBD to the SV40ori, and interestingly, the arrangement of E1 binding sites shows striking similarities to the arrangement of T-antigen binding sites in the SV40 ori even though the recognition sequences are unrelated. The E1 DBD is capable of interacting cooperatively with E2; however, the E2 DBD and not the E2 activation domain mediates this interaction. Furthermore, the E2 DBD stimulates binding of two monomers of the E1 DBD to the ori by binding cooperatively with one E1 monomer. Finally, we show that our results concerning the DNA-binding properties of the E1 DBD can be extended to full-length E1.

scholarly journals Association of p107 with Sp1: genetically separable regions of p107 are involved in regulation of E2F- and Sp1-dependent transcription.

1995 ◽  
Vol 15 (10) ◽  
pp. 5444-5452 ◽  
Author(s):  
P K Datta ◽  
P Raychaudhuri ◽  
S Bagchi
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Cell Lines ◽  
Chimeric Protein ◽  
Transient Transfection ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Related Protein ◽  
Activation Domains ◽  
Transcription Factor E2f

The retinoblastoma-related protein p107 has been shown to be a regulator of the transcription factor E2F. p107 associates with E2F via its pocket region and represses E2F-dependent transcription. In this study, we provide evidence for a novel interaction between p107 and the transcription factor Sp1. We show that p107 can be found endogenously associated with Sp1 in the extracts of several different cell lines. Moreover, in transient transfection assays, expression of p107 represses Sp1-dependent transcription. This repression of Sp1-dependent transcription does not require the DNA-binding domain of Sp1. Transcription driven by a chimeric protein containing the Ga14 DNA-binding domain and the Sp1 activation domains is inhibited by p107. Interestingly, unlike the repression of E2F-dependent transcription, the repression of Sp1-dependent transcription does not depend on an intact pocket region. We show that distinct regions of p107 are involved in the control of Sp1 and E2F.

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