scholarly journals Primer-DNA formation during simian virus 40 DNA replication in vitro.

1993 ◽  
Vol 13 (5) ◽  
pp. 2882-2890 ◽  
Author(s):  
D Denis ◽  
P A Bullock
Keyword(s):  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Antigen ◽  
Origin Region ◽  
Sv40 Dna ◽  
Proliferating Cell

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.

Primer-DNA formation during simian virus 40 DNA replication in vitro

1993 ◽  
Vol 13 (5) ◽  
pp. 2882-2890
Author(s):  
D Denis ◽  
P A Bullock
Keyword(s):  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Antigen ◽  
Origin Region ◽  
Sv40 Dna ◽  
Proliferating Cell

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.

scholarly journals Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro.

1989 ◽  
Vol 9 (2) ◽  
pp. 609-619 ◽  
Author(s):  
T Tsurimoto ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Nuclear Antigen ◽  
Nuclear Fraction ◽  
Replication Factor ◽  
Sv40 Dna ◽  
Proliferating Cell

Cell extracts (S100) derived from human 293 cells were separated into five fractions by phosphocellulose chromatography and monitored for their ability to support simian virus 40 (SV40) DNA replication in vitro in the presence of purified SV40 T antigen. Three fractions, designated I, IIA, and IIC, were essential. Fraction IIC contained the known replication factors topoisomerases I and II, but in addition contained a novel replication factor called RF-C. The RF-C activity, assayed in the presence of I, IIA, and excess amounts of purified topoisomerases, was detected in both cytosol and nuclear fractions, but was more abundant in the latter fraction. RF-C was purified from the 293 cell nuclear fraction to near homogeneity by conventional column chromatography. The reconstituted reaction mix containing purified RF-C could replicate SV40 origin-containing plasmid DNA more efficiently than could the S100 extract, and the products were predominantly completely replicated, monomer molecules. Interestingly, in the absence of RF-C, early replicative intermediates accumulated and subsequent elongation was aberrant. Hybridization studies with strand-specific, single-stranded M13-SV40 DNAs showed that in the absence of RF-C, abnormal DNA synthesis occurred preferentially on the lagging strand, and leading-strand replication was inefficient. These products closely resembled those previously observed for SV40 DNA replication in vitro in the absence of proliferating-cell nuclear antigen. These results suggest that an elongation complex containing RF-C and proliferating-cell nuclear antigen is assembled after formation of the first nascent strands at the replication origin. Subsequent synthesis of leading and lagging strands at a eucaryotic DNA replication fork can be distinguished by different requirements for multiple replication components, but we suggest that even though the two polymerases function asymmetrically, they normally progress coordinately.

scholarly journals Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro

1989 ◽  
Vol 9 (2) ◽  
pp. 609-619
Author(s):  
T Tsurimoto ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Nuclear Antigen ◽  
Nuclear Fraction ◽  
Replication Factor ◽  
Sv40 Dna ◽  
Proliferating Cell

Cell extracts (S100) derived from human 293 cells were separated into five fractions by phosphocellulose chromatography and monitored for their ability to support simian virus 40 (SV40) DNA replication in vitro in the presence of purified SV40 T antigen. Three fractions, designated I, IIA, and IIC, were essential. Fraction IIC contained the known replication factors topoisomerases I and II, but in addition contained a novel replication factor called RF-C. The RF-C activity, assayed in the presence of I, IIA, and excess amounts of purified topoisomerases, was detected in both cytosol and nuclear fractions, but was more abundant in the latter fraction. RF-C was purified from the 293 cell nuclear fraction to near homogeneity by conventional column chromatography. The reconstituted reaction mix containing purified RF-C could replicate SV40 origin-containing plasmid DNA more efficiently than could the S100 extract, and the products were predominantly completely replicated, monomer molecules. Interestingly, in the absence of RF-C, early replicative intermediates accumulated and subsequent elongation was aberrant. Hybridization studies with strand-specific, single-stranded M13-SV40 DNAs showed that in the absence of RF-C, abnormal DNA synthesis occurred preferentially on the lagging strand, and leading-strand replication was inefficient. These products closely resembled those previously observed for SV40 DNA replication in vitro in the absence of proliferating-cell nuclear antigen. These results suggest that an elongation complex containing RF-C and proliferating-cell nuclear antigen is assembled after formation of the first nascent strands at the replication origin. Subsequent synthesis of leading and lagging strands at a eucaryotic DNA replication fork can be distinguished by different requirements for multiple replication components, but we suggest that even though the two polymerases function asymmetrically, they normally progress coordinately.

Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form

1990 ◽  
Vol 96 (1) ◽  
pp. 121-129
Author(s):  
N.H. Waseem ◽  
D.P. Lane
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Expression Vectors ◽  
Nuclear Antigen ◽  
Antigenic Structure ◽  
Cellular Dna ◽  
Sv40 Dna ◽  
Proliferating Cell

The proliferating cell nuclear antigen, PCNA, has recently been identified as the polymerase delta accessory protein. PCNA is essential for cellular DNA synthesis and is also required for the in vitro replication of simian virus 40 (SV40) DNA where it acts to coordinate leading and lagging strand synthesis at the replication fork. The cDNA for rat PCNA was cloned into a series of bacterial expression vectors and the resulting protein used to immunize mice. Eleven new monoclonal antibodies to PCNA have been isolated and characterized. Some of the antibodies recognize epitopes conserved from man to fission yeast. Immunocytochemical analysis of primate epithelial cell lines showed that the antibodies recognized antigenically distinct forms of PCNA and that these forms were localized to different compartments of the nucleus. One antibody reacted exclusively with PCNA in the nucleolus. These results suggest that the PCNA protein may fulfil several separate roles in the cell nucleus associated with changes in its antigenic structure.

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 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.

The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro

Nature ◽  
1987 ◽  
Vol 326 (6112) ◽  
pp. 471-475 ◽  
Author(s):  
Gregory Prelich ◽  
Matthew Kostura ◽  
Daniel R. Marshak ◽  
Michael B. Mathews ◽  
Bruce Stillman
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Sv40 Dna ◽  
Proliferating Cell

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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