scholarly journals Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

1991 ◽  
Vol 11 (4) ◽  
pp. 2108-2115 ◽  
Author(s):  
K L Collins ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Dna Templates ◽  
Single Stranded Dna ◽  
Dna Polymerase Alpha ◽  
Primase Complex

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

scholarly journals Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase

1991 ◽  
Vol 11 (4) ◽  
pp. 2108-2115 ◽  
Author(s):  
K L Collins ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Dna Templates ◽  
Single Stranded Dna ◽  
Dna Polymerase Alpha ◽  
Primase Complex

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

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 Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

2004 ◽  
Vol 78 (4) ◽  
pp. 1605-1615 ◽  
Author(s):  
Yueh-Ming Loo ◽  
Thomas Melendy
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Replication Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Physical Interaction ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Papillomavirus Dna

ABSTRACT With the exception of viral proteins E1 and E2, papillomaviruses depend heavily on host replication machinery for replication of their viral genome. E1 and E2 are known to recruit many of the necessary cellular replication factors to the viral origin of replication. Previously, we reported a physical interaction between E1 and the major human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA). E1 was determined to bind to the 70-kDa subunit of RPA, RPA70. In this study, using E1-affinity coprecipitation and enzyme-linked immunosorbent assay-based interaction assays, we show that E1 interacts with the major ssDNA-binding domain of RPA. Consistent with our previous report, no measurable interaction between E1 and the two smaller subunits of RPA was detected. The interaction of E1 with RPA was substantially inhibited by ssDNA. The extent of this inhibition was dependent on the length of the DNA. A 31-nucleotide (nt) oligonucleotide strongly inhibited the E1-RPA interaction, while a 16-nt oligonucleotide showed an intermediate level of inhibition. In contrast, a 10-nt oligonucleotide showed no observable effect on the E1-RPA interaction. This inhibition was not dependent on the sequence of the DNA. Furthermore, ssDNA also inhibited the interaction of RPA with papillomavirus E2, simian virus 40 T antigen, human polymerase alpha-primase, and p53. Taken together, our results suggest a potential role for ssDNA in modulating RPA-protein interactions, in particular, the RPA-E1 interactions during papillomavirus DNA replication. A model for recruitment of RPA by E1 during papillomavirus DNA replication is proposed.

scholarly journals Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen.

1992 ◽  
Vol 11 (2) ◽  
pp. 769-776 ◽  
Author(s):  
I. Dornreiter ◽  
L.F. Erdile ◽  
I.U. Gilbert ◽  
D. von Winkler ◽  
T.J. Kelly ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Replication Protein ◽  
Sv40 T Antigen ◽  
Dna Polymerase Alpha ◽  
Cellular Replication

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.

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).

Binding properties of replication protein A from human and yeast cells

1992 ◽  
Vol 12 (7) ◽  
pp. 3050-3059 ◽  
Author(s):  
C Kim ◽  
R O Snyder ◽  
M S Wold
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Simian Virus ◽  
Yeast Cells ◽  
Origin Of Replication ◽  
Binding Properties ◽  
Single Stranded Dna

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.

scholarly journals The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

1994 ◽  
Vol 14 (2) ◽  
pp. 923-933 ◽  
Author(s):  
M Foiani ◽  
F Marini ◽  
D Gamba ◽  
G Lucchini ◽  
P Plevani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Cell Viability ◽  
Dna Polymerase ◽  
Chromosomal Dna ◽  
Dna Polymerase Alpha ◽  
B Subunit ◽  
Primase Complex

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

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