scholarly journals ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein

2007 ◽  
Vol 81 (7) ◽  
pp. 3293-3302 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Double Helix ◽  
Minor Groove ◽  
Bovine Papillomavirus ◽  
Base Pairs ◽  
Initiator Protein ◽  
Initiation Of Dna Replication ◽  
Multiple Interactions

ABSTRACT Template melting is an essential step in the initiation of DNA replication, but the mechanism of template melting is unknown for any replicon. Here we demonstrate that melting of the bovine papillomavirus type 1 ori is a sequence-dependent process which relies on specific recognition of TA base pairs in the minor groove by the E1 initiator. We show that correct template melting is a prerequisite for the formation of a stable double hexamer with helicase activity and that ori mutants that fail to melt correctly are defective for ori unwinding and DNA replication in vivo. Our results also indicate that melting of the DNA is achieved by destabilization of the double helix along its length through multiple interactions with E1, each of which is responsible for melting of a few base pairs, resulting in the extensive melting that is required for initiation of DNA replication.

scholarly journals Competition for DNA Binding Sites between the Short and Long Forms of E2 Dimers Underlies Repression in Bovine Papillomavirus Type 1 DNA Replication Control

1998 ◽  
Vol 72 (3) ◽  
pp. 1931-1940 ◽  
Author(s):  
Daniel A. Lim ◽  
Manfred Gossen ◽  
Chris W. Lehman ◽  
Michael R. Botchan
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Short Form ◽  
Bovine Papillomavirus ◽  
Binding Studies ◽  
Proliferating Cells ◽  
Dna Binding Sites ◽  
Replication Control

ABSTRACT Papillomaviruses establish a long-term latency in vivo by maintaining their genomes as nuclear plasmids in proliferating cells. Bovine papillomavirus type 1 encodes two proteins required for viral DNA replication: the helicase E1 and the positive regulator E2. The homodimeric E2 is known to cooperatively bind to DNA with E1 to form a preinitiation complex at the origin of DNA replication. The virus also codes for two short forms of E2 that can repress viral functions when overexpressed, and at least one copy of the repressor is required for stable plasmid maintenance in transformed cells. Employing a tetracycline-regulated system to control E1 and E2 production from integrated loci, we show that the short form of E2 negatively regulates DNA replication. We also found that the short form could repress replication in a cell-free replication system and that the repression requires the DNA binding domain of the protein. In contrast, heterodimers of the short and long forms were activators and, by footprint analysis, were shown to be as potent as homodimeric E2 in loading E1 to its cognate site. DNA binding studies show that when E1 levels are low and are dependent upon E2 for occupancy of the origin site, the repressor can block E1-DNA interactions. We conclude that DNA replication modulation results from competition between the different forms of E2 for DNA binding. Given that heterodimers are active and that the repressor form of E2 shows little cooperativity with E1 for DNA binding, this protein is a weak repressor.

Evidence that replication initiates at only some of the potential origins in each oligomeric form of bovine papillomavirus type 1 DNA

1990 ◽  
Vol 10 (6) ◽  
pp. 3078-3086
Author(s):  
J B Schvartzman ◽  
S Adolph ◽  
L Martín-Parras ◽  
C L Schildkraut
Keyword(s):  
Dna Replication ◽  
The Other ◽  
Bovine Papillomavirus ◽  
Two Dimensional Gel Electrophoresis ◽  
Dna Oligomers ◽  
Latently Infected ◽  
Oligomeric Form ◽  
Multiple Copies ◽  
Initiation Of Dna Replication

In a subclone of ID13 mouse fibroblasts latently infected with bovine papillomavirus type 1 (BPV-1) DNA, the viral genome occurred as a mixture of extrachromosomal circular monomers and oligomers. Multiple copies were also associated with the host cell genome, predominantly at a single site in a head-to-tail tandem array. We examined the replicative intermediates of extrachromosomal forms of BPV-1 DNA by using two-dimensional gel electrophoresis. The results obtained indicate that initiation of DNA replication occurred near the center of the EcoRI-BamHI 5.6-kilobase fragment. In some molecules, however, this fragment was replicated from one end to the other by means of a single fork initiated elsewhere. Termination also occurred within this fragment. The EcoRI-BamHI 2.3-kilobase fragment replicated as a DNA molecule containing a termination site for DNA replication and also by means of a single fork traversing the fragment from one end to the other. Thus, replication forks proceeded through these fragments in different manners, apparently depending on whether they were part of a monomer, a dimer, a trimer, or higher oligomers. These observations lead to the conclusion that initiation of DNA replication in BPV-1 DNA takes place at or close to plasmid maintenance sequence 1. From this point, replication proceeds bidirectionally and termination occurs approximately 180 degrees opposite the origin. The results obtained are consistent with one or more replication origins being quiescent in BPV-1 DNA oligomers.

scholarly journals Evidence that replication initiates at only some of the potential origins in each oligomeric form of bovine papillomavirus type 1 DNA.

1990 ◽  
Vol 10 (6) ◽  
pp. 3078-3086 ◽  
Author(s):  
J B Schvartzman ◽  
S Adolph ◽  
L Martín-Parras ◽  
C L Schildkraut
Keyword(s):  
Dna Replication ◽  
The Other ◽  
Bovine Papillomavirus ◽  
Two Dimensional Gel Electrophoresis ◽  
Dna Oligomers ◽  
Latently Infected ◽  
Oligomeric Form ◽  
Multiple Copies ◽  
Initiation Of Dna Replication

In a subclone of ID13 mouse fibroblasts latently infected with bovine papillomavirus type 1 (BPV-1) DNA, the viral genome occurred as a mixture of extrachromosomal circular monomers and oligomers. Multiple copies were also associated with the host cell genome, predominantly at a single site in a head-to-tail tandem array. We examined the replicative intermediates of extrachromosomal forms of BPV-1 DNA by using two-dimensional gel electrophoresis. The results obtained indicate that initiation of DNA replication occurred near the center of the EcoRI-BamHI 5.6-kilobase fragment. In some molecules, however, this fragment was replicated from one end to the other by means of a single fork initiated elsewhere. Termination also occurred within this fragment. The EcoRI-BamHI 2.3-kilobase fragment replicated as a DNA molecule containing a termination site for DNA replication and also by means of a single fork traversing the fragment from one end to the other. Thus, replication forks proceeded through these fragments in different manners, apparently depending on whether they were part of a monomer, a dimer, a trimer, or higher oligomers. These observations lead to the conclusion that initiation of DNA replication in BPV-1 DNA takes place at or close to plasmid maintenance sequence 1. From this point, replication proceeds bidirectionally and termination occurs approximately 180 degrees opposite the origin. The results obtained are consistent with one or more replication origins being quiescent in BPV-1 DNA oligomers.

scholarly journals Bovine papillomavirus type 1 E2 protein heterodimer is functional in papillomavirus DNA replication in vivo

Virology ◽  
2009 ◽  
Vol 386 (2) ◽  
pp. 353-359 ◽  
Author(s):  
Reet Kurg ◽  
Piia Uusen ◽  
Toomas Sepp ◽  
Mari Sepp ◽  
Aare Abroi ◽  
...  
Keyword(s):  
Dna Replication ◽  
Bovine Papillomavirus ◽  
E2 Protein ◽  
Papillomavirus Dna

A Novel Fluorescence-Based Screen for Inhibitors of the Initiation of DNA Replication in Bacteria

2019 ◽  
Vol 16 (3) ◽  
pp. 272-277 ◽  
Author(s):  
Rasmus N. Klitgaard ◽  
Anders Løbner-Olesen
Keyword(s):  
Dna Replication ◽  
High Throughput ◽  
Cyclic Peptide ◽  
Replication Initiation ◽  
False Positive Result ◽  
Over Expression ◽  
Initiator Protein ◽  
Dna Replication Initiation ◽  
Cellular Processes ◽  
Initiation Of Dna Replication

Background:One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited.Materials and Methods:Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria.Results:The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen.Conclusion:We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product

1989 ◽  
Vol 9 (2) ◽  
pp. 406-414
Author(s):  
H Romanczuk ◽  
W M Wormington
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
Gene Product ◽  
Mammalian Cells ◽  
Xenopus Laevis Oocytes ◽  
Bovine Papillomavirus ◽  
In Vitro Synthesis ◽  
E6 Gene ◽  
E6 Protein

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

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