Interaction of simian virus 40 large T-antigen with cellular DNA polymerase ?: studies with various T-antigen mutants of SV40

1991 ◽  
Vol 118 (1-2) ◽  
pp. 113-125 ◽  
Author(s):  
J. M. Pistillo ◽  
J. K. Vishwanatha
Keyword(s):  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Cellular Dna

scholarly journals Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen.

1993 ◽  
Vol 13 (2) ◽  
pp. 809-820 ◽  
Author(s):  
I Dornreiter ◽  
W C Copeland ◽  
T S Wang
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Dna Polymerase Alpha ◽  
Amino Terminal ◽  
Human Dna ◽  
Sv40 Dna

Initiation of cell-free simian virus 40 (SV40) DNA replication requires the interaction of DNA polymerase alpha/primase with a preinitiation complex containing the viral T antigen and cellular proteins, replication protein A, and topoisomerase I or II. To further understand the molecular mechanisms of the transition from preinitiation to initiation, the intermolecular interaction between human DNA polymerase alpha and T antigen was investigated. We have demonstrated that the human DNA polymerase alpha catalytic polypeptide is able to associate with SV40 large T antigen directly under physiological conditions. A physical association between these two proteins was detected by coimmunoprecipitation with monoclonal antibodies from insect cells coinfected with recombinant baculoviruses. A domain of human polymerase alpha physically interacting with T antigen was identified within the amino-terminal region from residues 195 to 313. This domain of human polymerase alpha was able to form a nonproductive complex with T antigen, causing inhibition of the SV40 DNA replication in vitro. Kinetics of the inhibition indicated that this polymerase domain can inhibit viral replication only during the preinitiation stage. Extra molecules of T antigen could partially overcome the inhibition only prior to initiation complex formation. The data support the conclusion that initiation of SV40 DNA replication requires the physical interaction of T antigen in the preinitiation complex with the amino-terminal domain of human polymerase alpha from amino acid residues 195 to 313.

Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen

1993 ◽  
Vol 13 (2) ◽  
pp. 809-820
Author(s):  
I Dornreiter ◽  
W C Copeland ◽  
T S Wang
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Dna Polymerase Alpha ◽  
Amino Terminal ◽  
Human Dna ◽  
Sv40 Dna

Initiation of cell-free simian virus 40 (SV40) DNA replication requires the interaction of DNA polymerase alpha/primase with a preinitiation complex containing the viral T antigen and cellular proteins, replication protein A, and topoisomerase I or II. To further understand the molecular mechanisms of the transition from preinitiation to initiation, the intermolecular interaction between human DNA polymerase alpha and T antigen was investigated. We have demonstrated that the human DNA polymerase alpha catalytic polypeptide is able to associate with SV40 large T antigen directly under physiological conditions. A physical association between these two proteins was detected by coimmunoprecipitation with monoclonal antibodies from insect cells coinfected with recombinant baculoviruses. A domain of human polymerase alpha physically interacting with T antigen was identified within the amino-terminal region from residues 195 to 313. This domain of human polymerase alpha was able to form a nonproductive complex with T antigen, causing inhibition of the SV40 DNA replication in vitro. Kinetics of the inhibition indicated that this polymerase domain can inhibit viral replication only during the preinitiation stage. Extra molecules of T antigen could partially overcome the inhibition only prior to initiation complex formation. The data support the conclusion that initiation of SV40 DNA replication requires the physical interaction of T antigen in the preinitiation complex with the amino-terminal domain of human polymerase alpha from amino acid residues 195 to 313.

scholarly journals Two Immunologically Distinct Human DNA Polymerase α-Primase Subpopulations Are Involved in Cellular DNA Replication

2001 ◽  
Vol 21 (7) ◽  
pp. 2581-2593 ◽  
Author(s):  
Silke Dehde ◽  
Gabor Rohaly ◽  
Oliver Schub ◽  
Heinz-Peter Nasheuer ◽  
Wolfgang Bohn ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Brdu Incorporation ◽  
Metabolic Labeling ◽  
Dna Polymerase Α ◽  
Origin Binding Protein ◽  
Cellular Dna

ABSTRACT Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase α-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G1, whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G2. Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2′-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork.

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