scholarly journals ALKOXYALKYL ESTERS OF NUCLEOTIDE ANALOGS INHIBIT POLYOMAVIRUS DNA REPLICATION AND LARGE T ANTIGEN ACTIVITIES

2020 ◽  
Author(s):  
Nichodemus O. Onwubiko ◽  
Suraya Diaz ◽  
Marcela Krecmerova ◽  
Heinz Peter Nasheuer
Keyword(s):  
Dna Replication ◽  
Tumor Antigens ◽  
Antiviral Agent ◽  
Dna Helicase ◽  
T Antigen ◽  
Nucleotide Analogs ◽  
Large Tumor ◽  
Nucleotide Analog ◽  
Derivatives Of

Polyomavirus-related infections are ubiqutious in immunocompromised individuals and in some cases are intractable and fatal. Due to lack of approved drugs to treat polyomavirus infections, cidofovir, a phosphonate nucleotide analog approved to treat cytomegalovirus infections has been repurposed as anti-polyomavirus agent. Cidofovir has been modified in various ways to improve its efficacies as broad-spectrum antiviral agent. However, the actual mechanisms and targets of cidofovir and its modified derivatives as anti-polyomavirus agents are still under research. Here, polyomavirus large tumor antigens (Tag) activities were identified as the viral target of cidofovir derivatives. The alkoxyalkyl-ester derivatives of cidofovir efficiently inhibit polyomavirus DNA replication in cell-free human extracts and a viral in vitro replication system only utilizing purified proteins. We present evidence that DNA helicase, and DNA binding activities of polyomavirus Tags are diminished in the presence of low concentrations of alkoxyalkyl-ester derivatives of cidofovir suggesting that the inhibition of viral DNA replication is at least in part mediated by inhibiting ssDNA and dsDNA binding activities of Tags. These findings show that the alkoxyalkyl-ester derivatives of cidofovir are effective in vitro without undergoing further conversions and conclude that the inhibitory mechanisms of nucleotide analog-based drugs are more complex than previously believed.

scholarly journals Mutational Analysis of Simian Virus 40 T-Antigen Primosome Activities in Viral DNA Replication

2002 ◽  
Vol 76 (10) ◽  
pp. 5121-5130 ◽  
Author(s):  
Robert D. Ott ◽  
Yingda Wang ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Mutational Analysis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Helicase ◽  
T Antigen ◽  
Helicase Domain ◽  
In Vitro Synthesis

ABSTRACT The recruitment of DNA polymerase α-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.

scholarly journals Termination complex in Escherichia coli inhibits SV40 DNA replication in vitro by impeding the action of T antigen helicase.

1992 ◽  
Vol 267 (8) ◽  
pp. 5361-5365
Author(s):  
M Hidaka ◽  
T Kobayashi ◽  
Y Ishimi ◽  
M Seki ◽  
T Enomoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
T Antigen ◽  
Sv40 Dna

scholarly journals DNA Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

2004 ◽  
Vol 279 (44) ◽  
pp. 45586-45593 ◽  
Author(s):  
David Shechter ◽  
Carol Y. Ying ◽  
Jean Gautier
Keyword(s):  
Dna Replication ◽  
Neutralizing Antibodies ◽  
Atp Hydrolysis ◽  
Initial Period ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Origin Firing ◽  
Replicative Helicase ◽  
Mcm Proteins

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activitiesin vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) fromXenopus laeviseggs and six purified polyclonal antibodies generated against each of theXenopusMcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2–7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21cip1specifically blocks origin firing but does not prevent helicase action. When p21cip1is added, followed by the non-hydrolyzable analog ATPγS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis

1984 ◽  
Vol 4 (8) ◽  
pp. 1476-1482
Author(s):  
H Ariga
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Cell Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Sv40 Dna

The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixture with BLUR8 as a template was semiconservative and not primed by a putative RNA polymerase III transcript synthesized on the Alu family sequence in vitro. Pulse-chase experiments showed that the small-sized DNA produced in a short-term incubation was converted to full-length closed circular and open circular DNAs in alkaline sucrose gradients. DNA synthesis in extracts began in a region of the Alu family sequence and was inhibited 80% by the addition of anti-T serum. Furthermore, partially purified T antigen bound the Alu family sequence in BLUR8 by the DNA-binding immunoassay. These results suggest that SV40 T antigen recognizes the Alu family sequence, similar to the origin sequence of SV40 DNA, and initiates semiconservative DNA replication in vitro.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

In vitro replication of DNA containing either the SV40 or the polyoma origin

1987 ◽  
Vol 317 (1187) ◽  
pp. 439-453 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Hela Cells ◽  
Dna Binding Protein ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Sv40 Origin ◽  
Sv40 Dna ◽  
Primase Complex

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro . Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo . The host-cell source of DNA polymerase α - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase α - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).

scholarly journals Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

1986 ◽  
Vol 6 (11) ◽  
pp. 3815-3825 ◽  
Author(s):  
R S Decker ◽  
M Yamaguchi ◽  
R Possenti ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Early Gene ◽  
Initial Direction ◽  
Dna Polymerase Alpha

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

scholarly journals Stimulation of DNA Replication from the Polyomavirus Origin by PCAF and GCN5 Acetyltransferases: Acetylation of Large T Antigen

2002 ◽  
Vol 22 (22) ◽  
pp. 7907-7918 ◽  
Author(s):  
An-Yong Xie ◽  
Vladimir P. Bermudez ◽  
William R. Folk
Keyword(s):  
Dna Replication ◽  
T Antigen ◽  
Large T Antigen ◽  
Acetyltransferase Domain ◽  
Stimulation Of

ABSTRACT The PCAF and GCN5 acetyltransferases, but not p300 or CBP, stimulate DNA replication when tethered near the polyomavirus origin. Replication stimulation by PCAF and GCN5 is blocked by mutational inactivation of their acetyltransferase domains but not by deletion of sequences that bind p300 or CBP. Acetylation of histones near the polyomavirus origin assembled into chromatin in vivo is not detectably altered by expression of these acetyltransferases. PCAF and GCN5 interact with polyomavirus large T antigen in vivo, PCAF acetylates large T antigen in vitro, and large T-antigen acetylation in vivo is dependent upon the integrity of the PCAF acetyltransferase domain. These data suggest replication stimulation occurs through recruitment of large T antigen to the origin and acetylation by PCAF or GCN5.

scholarly journals Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro.

1987 ◽  
Vol 7 (1) ◽  
pp. 1-6 ◽  
Author(s):  
H Ariga ◽  
T Itani ◽  
S M Iguchi-Ariga
Keyword(s):  
Dna Replication ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Copy Numbers ◽  
Mouse Cells ◽  
Simplex Virus

We have already reported that the cloned mouse DNA fragment (pMU65) could replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro (H. Ariga, Z. Tsuchihashi, M. Naruto, and M. Yamada, Mol. Cell. Biol. 5:563-568, 1985). The plasmid p65-tk, containing the thymidine kinase (tk) gene of herpes simplex virus and the BglII-EcoRI region of pMU65 homologous to the simian virus 40 origin of DNA replication, was constructed. The p65-tk persisted episomally in tk+ transformants after the transfection of p65-tk into mouse FM3Atk- cells. The copy numbers of p65-tk in FM3Atk+ cells were 100 to 200 copies per cell. Furthermore, the p65-tk replicated semiconservatively, and the initiation of DNA replication started from the mouse DNA sequences when the replicating activity of p65-tk was tested in the in vitro DNA replication system developed from the FM3A cells. These results show that a 2.5-kilobase fragment of mouse DNA contains the autonomously replicating sequences.

scholarly journals Synthetic DNA Replication Bubbles Bound and Unwound with Twofold Symmetry by a Simian Virus 40 T-Antigen Double Hexamer

1998 ◽  
Vol 72 (11) ◽  
pp. 8676-8681 ◽  
Author(s):  
Natalia V. Smelkova ◽  
James A. Borowiec
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Helicase ◽  
T Antigen ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Helicase Activity ◽  
Synthetic Dna ◽  
Stimulation Of

ABSTRACT Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDH with DNA during unwinding, we examined the binding of TAgDH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDH requires ≥40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5′ junction on each strand, with 5 bp of duplex DNA and ∼17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5′ direction into the duplex region, while resulting in no significant changes to the 3′ edge of strongest protection. Our data indicate that each TAgH encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgH interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.

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