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.

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.

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.

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

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 DNA replication in vitro: identification of multiple stages of initiation.

1989 ◽  
Vol 9 (9) ◽  
pp. 3839-3849 ◽  
Author(s):  
T Tsurimoto ◽  
M P Fairman ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Time Lag ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Antigen ◽  
Replication Factor ◽  
Cellular Fraction ◽  
Template Dna

A cell-free DNA replication system dependent upon five purified cellular proteins, one crude cellular fraction, and the simian virus 40 (SV40)-encoded large tumor antigen (T antigen) initiated and completed replication of plasmids containing the SV40 origin sequence. DNA synthesis initiated at or near the origin sequence after a time lag of approximately 10 min and then proceeded bidirectionally from the origin to yield covalently closed, monomer daughter molecules. The time lag could be completely eliminated by a preincubation of SV40 ori DNA in the presence of T antigen, a eucaryotic single-stranded DNA-binding protein (replication factor A [RF-A]), and topoisomerases I and II. In contrast, if T antigen and the template DNA were incubated alone, the time lag was only partially decreased. Kinetic analyses of origin recognition by T antigen, origin unwinding, and DNA synthesis suggest that the time lag in replication was due to the formation of a complex between T antigen and DNA called the T complex, followed by formation of a second complex called the unwound complex. Formation of the unwound complex required RF-A. When origin unwinding was coupled to DNA replication by the addition of a partially purified cellular fraction (IIA), DNA synthesis initiated at the ori sequence, but the template DNA was not completely replicated. Complete DNA replication in this system required the proliferating-cell nuclear antigen and another cellular replication factor, RF-C, during the elongation stage. In a less fractionated system, another cellular fraction, SSI, was previously shown to be necessary for reconstitution of DNA replication. The SSI fraction was required in the less purified system to antagonize the inhibitory action of another cellular protein(s). This inhibitor specifically blocked the earliest stage of DNA replication, but not the later stages. The implications of these results for the mechanisms of initiation and elongation of DNA replication are discussed.

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