scholarly journals The Retinoblastoma Protein Alters the Phosphorylation State of Polyomavirus Large T Antigen in Murine Cell Extracts and Inhibits Polyomavirus Origin DNA Replication

1999 ◽  
Vol 73 (4) ◽  
pp. 3004-3013 ◽  
Author(s):  
Inga Reynisdóttir ◽  
Subarna Bhattacharyya ◽  
Dong Zhang ◽  
Carol Prives
Keyword(s):  
Dna Replication ◽  
Potential Effect ◽  
T Antigen ◽  
S Phase ◽  
Large T Antigen ◽  
Murine Cell ◽  
Cell Extracts ◽  
Cdk Phosphorylation ◽  
Retinoblastoma Tumor

ABSTRACT The retinoblastoma tumor suppressor protein (pRb) can associate with the transforming proteins of several DNA tumor viruses, including the large T antigen encoded by polyomavirus (Py T Ag). Although pRb function is critical for regulating progression from G1 to S phase, a role for pRb in S phase has not been demonstrated or excluded. To identify a potential effect of pRb on DNA replication, pRb protein was added to reaction mixtures containing Py T Ag, Py origin-containing DNA (Py ori-DNA), and murine FM3A cell extracts. We found that pRb strongly represses Py ori-DNA replication in vitro. Unexpectedly, however, this inhibition only partially depends on the interaction of pRb with Py T Ag, since a mutant Py T Ag (dl141) lacking the pRb interaction region was also significantly inhibited by pRb. This result suggests that pRb interferes with or alters one or more components of the murine cell replication extract. Furthermore, the ability of Py T Ag to be phosphorylated in such extracts is markedly reduced in the presence of pRb. Since cyclin-dependent kinase (CDK) phosphorylation of Py T Ag is required for its replication function, we hypothesize that pRb interferes with this phosphorylation event. Indeed, the S-phase CDK complex (cyclin A-CDK2), which phosphorylates both pRb and Py T Ag, alleviates inhibition caused by pRb. Moreover, hyperphosphorylated pRb is incapable of inhibiting replication of Py ori-DNA in vitro. We propose a new requirement for maintaining pRb phosphorylation in S phase, namely, to prevent deleterious effects on the cellular replication machinery.

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.

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 Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication.

1985 ◽  
Vol 5 (6) ◽  
pp. 1238-1246 ◽  
Author(s):  
J J Li ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Dna Molecules ◽  
Free System ◽  
Cell Free System ◽  
Cell Extracts ◽  
Sv40 Origin

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.

Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process

2000 ◽  
Vol 113 (11) ◽  
pp. 1929-1938 ◽  
Author(s):  
D. Coverley ◽  
C. Pelizon ◽  
S. Trewick ◽  
R.A. Laskey
Keyword(s):  
Dna Replication ◽  
Protein Kinase ◽  
Mammalian Cell ◽  
Cyclin A ◽  
S Phase ◽  
Human Cells ◽  
Cell Extracts ◽  
In Vitro Replication ◽  
Initiation Of Dna Replication

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G(1) nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G(1)phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6. Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G(2) then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.

Multiple subelements within the polyomavirus enhancer function synergistically to activate DNA replication

1988 ◽  
Vol 8 (11) ◽  
pp. 5000-5015
Author(s):  
W J Muller ◽  
D Dufort ◽  
J A Hassell
Keyword(s):  
Dna Replication ◽  
Binding Sites ◽  
T Antigen ◽  
Common Mechanism ◽  
In Vitro Mutagenesis ◽  
Large T Antigen ◽  
Enhancer Element ◽  
The Core ◽  
Beta Enhancer

The polyomavirus origin for DNA replication comprises at least two essential, but functionally distinct, cis-acting components. One of these, the origin core, is required only for DNA replication. It includes binding sites for large T antigen and the origin of bidirectional DNA replication. The other component is required for both transcription and DNA replication and is represented by two functionally redundant regions, alpha and beta, which are elements of the polyomavirus enhancer. Whereas either enhancer element will activate DNA replication, both enhancer elements are required to constitute a functional enhancer of transcription. To identify the sequences that make up each enhancer element, we have subjected them separately to in vitro mutagenesis and measured their capacity to activate replication in cis of the origin core in MOP-8 cells, which provide all trans-acting replicative functions including large T antigen. The results reveal that the beta enhancer element is composed of three subelements, two auxiliary subelements, and a core subelement. The core subelement independently activated DNA replication, albeit poorly. The auxiliary subelements, which were inactive on their own, acted synergistically with the core subelement to increase its activity. Interestingly, dimers of the beta core subelement functioned as well as the combination of a beta auxiliary subelement and a core subelement, suggesting that the subelements are functionally equivalent. The alpha enhancer element is organized similarly; it too comprises an auxiliary subelement and a core subelement. These results lead us to suggest that the polyomavirus enhancer comprises two levels of organization; two or more enhancer elements form an enhancer, and two or more subelements make up an enhancer element. The subelements share few sequences and serve as binding sites for distinct cellular factors. It appears, therefore, that a number of different cellular proteins function cooperatively to activate polyomavirus DNA replication by a common mechanism.

scholarly journals Simian Virus 40 Large T Antigen Can Specifically Unwind the Central Palindrome at the Origin of DNA Replication

2009 ◽  
Vol 83 (7) ◽  
pp. 3312-3322 ◽  
Author(s):  
Weiping Wang ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Critical Role ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Central Origin ◽  
Sv40 Dna

ABSTRACT The hydrophilic channels between helicase domains of simian virus 40 (SV40) large T antigen play a critical role in DNA replication. Previous mutagenesis of residues in the channels identified one class of mutants (class A: D429A, N449S, and N515S) with normal DNA binding and ATPase and helicase activities but with a severely reduced ability to unwind origin DNA and to support SV40 DNA replication in vitro. Here, we further studied these mutants to gain insights into how T antigen unwinds the origin. We found that the mutants were compromised in melting the imperfect palindrome (EP) but normal in untwisting the AT-rich track. However, the mutants' defect in EP melting was not the major reason they failed to unwind the origin because supplying an EP region as a mismatched bubble, or deleting the EP region altogether, did not rescue their unwinding deficiency. These results suggested that specific separation of the central palindrome of the origin (site II) is an essential step in unwinding origin DNA by T antigen. In support of this, wild-type T antigen was able to specifically unwind a 31-bp DNA containing only site II in an ATPase-dependent reaction, whereas D429A and N515S failed to do so. By performing a systematic mutagenesis of 31-bp site II DNA, we identified discrete regions in each pentanucleotide necessary for normal origin unwinding. These data indicate that T antigen has a mechanism to specifically unwind the central palindrome. Various models are proposed to illustrate how T antigen could separate the central origin.

scholarly journals Interaction of the Transcription Factor TFIID with Simian Virus 40 (SV40) Large T Antigen Interferes with Replication of SV40 DNA In Vitro

1999 ◽  
Vol 73 (2) ◽  
pp. 1099-1107 ◽  
Author(s):  
Utz Herbig ◽  
Klaus Weisshart ◽  
Poonam Taneja ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cell Extracts ◽  
Sv40 Dna ◽  
Replication Activity

ABSTRACT Simian virus 40 (SV40) large tumor (T) antigen is the major regulatory protein that directs the course of viral infection, primarily by interacting with host cell proteins and modulating their functions. Initiation of viral DNA replication requires specific interactions of T antigen bound to the viral origin of DNA replication with cellular replication proteins. Transcription factors are thought to stimulate initiation of viral DNA replication, but the mechanism of stimulation is poorly understood. Since the transcription factor TATA-binding protein (TBP) binds to sequences within the origin of replication and interacts specifically with T antigen, we examined whether TBP complexes stimulate SV40 DNA replication in vitro. On the contrary, we found that depletion of TBP complexes from human cell extracts increased their ability to support viral DNA replication, and readdition of TBP complexes to the depleted extracts diminished their activity. We have mapped the sites of interaction between the proteins to residues 181 to 205 of T antigen and 184 to 220 of TBP. Titration of fusion proteins containing either of these peptides into undepleted cell extracts stimulated their replication activity, suggesting that they prevented the T antigen-TBP interaction that interfered with replication activity. TBP complexes also interfered with origin DNA unwinding by purified T antigen, and addition of either the T antigen or the TBP fusion peptide relieved the inhibition. These results suggest that TBP complexes associate with a T-antigen surface that is also required for origin DNA unwinding and viral DNA replication. We speculate that competition among cellular proteins for T antigen may play a role in regulating the course of viral infection.

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