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 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.

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.

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.

scholarly journals Interactions of the Papovavirus DNA Replication Initiator Proteins, Bovine Papillomavirus Type 1 E1 and Simian Virus 40 Large T Antigen, with Human Replication Protein A

1999 ◽  
Vol 73 (6) ◽  
pp. 4899-4907 ◽  
Author(s):  
YuFeng Han ◽  
Yueh-Ming Loo ◽  
Kevin T. Militello ◽  
Thomas Melendy
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Bovine Papillomavirus ◽  
Replication Proteins ◽  
E1 Protein ◽  
Cellular Dna

ABSTRACT Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase α-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to bind to the DNA polymerase α-primase complex. Enzyme-linked immunoassay-based protein interaction assays and protein affinity pull-down assays were used to show that the papillomavirus E1 protein also binds to the cellular RPA complex in vitro. Furthermore, SV40 Tag was able to compete with bovine papillomavirus type 1 E1 for binding to RPA. Each of the three RPA subunits was individually overexpressed in Escherichia colias a soluble fusion protein. These fusion proteins were used to show that the E1-RPA and Tag-RPA interactions are primarily mediated through the 70-kDa subunit of RPA. These results suggest that different viruses have evolved similar mechanisms for taking control of the cellular DNA replication machinery.

scholarly journals Bovine papillomavirus type 1 DNA replication: the transcriptional activator E2 acts in vitro as a specificity factor.

1997 ◽  
Vol 71 (9) ◽  
pp. 6805-6815 ◽  
Author(s):  
C Bonne-Andréa ◽  
F Tillier ◽  
G D McShan ◽  
V G Wilson ◽  
P Clertant
Keyword(s):  
Dna Replication ◽  
Transcriptional Activator ◽  
Bovine Papillomavirus ◽  
Specificity Factor

scholarly journals Structure-Based Mutational Analysis of the Bovine Papillomavirus E1 Helicase Domain Identifies Residues Involved in the Nonspecific DNA Binding Activity Required for Double Trimer Formation

2010 ◽  
Vol 84 (9) ◽  
pp. 4264-4276 ◽  
Author(s):  
Xiaofei Liu ◽  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Mutational Analysis ◽  
Binding Activity ◽  
Helicase Domain ◽  
Bovine Papillomavirus ◽  
Dna Binding Activity ◽  
E1 Protein ◽  
Initiation Of Dna Replication ◽  
Dna Template

ABSTRACT The papillomavirus E1 protein is a multifunctional initiator protein responsible for preparing the viral DNA template for initiation of DNA replication. The E1 protein encodes two DNA binding activities that are required for initiation of DNA replication. A well-characterized sequence-specific DNA binding activity resides in the E1 DBD and is used to tether E1 to the papillomavirus ori. A non-sequence-specific DNA binding activity is also required for formation of the E1 double trimer (DT) complex, which is responsible for the local template melting that precedes loading of the E1 helicase. This DNA binding activity is very poorly understood. We use a structure-based mutagenesis approach to identify residues in the E1 helicase domain that are required for the non-sequence-specific DNA binding and DT formation. We found that three groups of residues are involved in nonspecific DNA binding: the E1 β-hairpin structure containing R505, K506, and H507; a hydrophobic loop containing F464; and a charged loop containing K461 together generate the binding surface involved in nonspecific DNA binding. These residues are well conserved in the T antigens from the polyomaviruses, indicating that the polyomaviruses share this nonspecific DNA binding activity.

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