scholarly journals Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding.

1987 ◽  
Vol 61 (11) ◽  
pp. 3373-3380 ◽  
Author(s):  
F A Grässer ◽  
K Mann ◽  
G Walter
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen

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 Stimulation of the Human Heat Shock Protein 70 Promoter in Vitro by Simian Virus 40 Large T Antigen

1989 ◽  
Vol 264 (27) ◽  
pp. 16160-16164
Author(s):  
I C Taylor ◽  
W Solomon ◽  
B M Weiner ◽  
E Paucha ◽  
M Bradley ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Human Heat Shock Protein ◽  
Stimulation Of

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.

scholarly journals Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen.

1987 ◽  
Vol 61 (10) ◽  
pp. 3326-3330 ◽  
Author(s):  
M Strauss ◽  
P Argani ◽  
I J Mohr ◽  
Y Gluzman
Keyword(s):  
Dna Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Large T Antigen

Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein

1984 ◽  
Vol 4 (2) ◽  
pp. 232-239
Author(s):  
F Van Roy ◽  
L Fransen ◽  
W Fiers
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Complex ◽  
Immune Complexes ◽  
Kinase Activity ◽  
P53 Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen

Immune complex kinase assays in the simian virus 40 system were performed by incubation of immunoprecipitates containing tumor antigens with [gamma-32P]ATP, followed by analysis of any phosphoacceptor proteins. These assays yielded mainly the viral large T-antigen and, in particular, the associated cellular p53 as endogenous substrates. The nature of these substrates was confirmed by proteolysis techniques. Under specific conditions, casein could be used as an exogenous substrate as well. The kinase reactions showed preference for ATP and MgCl2 instead of GTP or MnCl2. Both phosphoserine and phosphothreonine, but in no case phosphotyrosine, were detected after an immune complex kinase reaction. Apparently, several in vivo phosphorylation sites were recognized in vitro in both large T-antigen and p53, but the presence of some artifactual sites could not be completely excluded. Although contaminating kinases were detectable in the immune complexes, at least the p53 molecules were phosphorylated in vitro in a more specific way. This followed from several characteristics of the immune complex kinase reactions and especially from the strong inhibition of p53 phosphorylation by two anti-large-T monoclonal antibodies. It was shown that large T-antigen showed associated kinase activity, although none of our results could unambiguously demonstrate an intrinsic kinase activity of this protein. Finally, anti-p53 monoclonal antibodies only slightly affected in vitro phosphorylation reactions, whereas a p53 molecule from a simian virus 40-free, chemically transformed human cell line was not phosphorylated in vitro under any condition tested. Thus, it is highly unlikely that the p53 molecule per se carries intrinsic or even associated kinase activities.

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 Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein.

1984 ◽  
Vol 4 (2) ◽  
pp. 232-239 ◽  
Author(s):  
F Van Roy ◽  
L Fransen ◽  
W Fiers
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Complex ◽  
Immune Complexes ◽  
Kinase Activity ◽  
P53 Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen

Immune complex kinase assays in the simian virus 40 system were performed by incubation of immunoprecipitates containing tumor antigens with [gamma-32P]ATP, followed by analysis of any phosphoacceptor proteins. These assays yielded mainly the viral large T-antigen and, in particular, the associated cellular p53 as endogenous substrates. The nature of these substrates was confirmed by proteolysis techniques. Under specific conditions, casein could be used as an exogenous substrate as well. The kinase reactions showed preference for ATP and MgCl2 instead of GTP or MnCl2. Both phosphoserine and phosphothreonine, but in no case phosphotyrosine, were detected after an immune complex kinase reaction. Apparently, several in vivo phosphorylation sites were recognized in vitro in both large T-antigen and p53, but the presence of some artifactual sites could not be completely excluded. Although contaminating kinases were detectable in the immune complexes, at least the p53 molecules were phosphorylated in vitro in a more specific way. This followed from several characteristics of the immune complex kinase reactions and especially from the strong inhibition of p53 phosphorylation by two anti-large-T monoclonal antibodies. It was shown that large T-antigen showed associated kinase activity, although none of our results could unambiguously demonstrate an intrinsic kinase activity of this protein. Finally, anti-p53 monoclonal antibodies only slightly affected in vitro phosphorylation reactions, whereas a p53 molecule from a simian virus 40-free, chemically transformed human cell line was not phosphorylated in vitro under any condition tested. Thus, it is highly unlikely that the p53 molecule per se carries intrinsic or even associated kinase activities.

Sign in / Sign up

Export Citation Format

Share Document