Stoichiometry and Mechanism of Assembly of SV40 T Antigen Complexes with the Viral Origin of DNA Replication and DNA Polymerase α-Primase†

Biochemistry ◽  
1998 ◽  
Vol 37 (44) ◽  
pp. 15345-15352 ◽  
Author(s):  
Shu-Gui Huang ◽  
Klaus Weisshart ◽  
Ilka Gilbert ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Viral Origin ◽  
Dna Polymerase Α ◽  
Origin Of Dna Replication

ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication

Nature ◽  
1989 ◽  
Vol 338 (6217) ◽  
pp. 658-662 ◽  
Author(s):  
Iris A. Mastrangelo ◽  
Paul V. C. Hough ◽  
Joseph S. Wall ◽  
Mark Dodson ◽  
Frank B. Dean ◽  
...  
Keyword(s):  
Dna Replication ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Viral Origin ◽  
Origin Of Dna Replication

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).

p53 and DNA polymerase α compete for binding to SV40 T antigen

Nature ◽  
1987 ◽  
Vol 329 (6138) ◽  
pp. 456-458 ◽  
Author(s):  
J. V. Gannon ◽  
D. P. Lane
Keyword(s):  
Dna Polymerase ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Dna Polymerase Α

scholarly journals Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

2001 ◽  
Vol 75 (18) ◽  
pp. 8569-8578 ◽  
Author(s):  
Armin R. Kautz ◽  
Klaus Weisshart ◽  
Annerose Schneider ◽  
Frank Grosse ◽  
Heinz-Peter Nasheuer
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Complexes ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Specific Factor ◽  
Dna Polymerase Α

ABSTRACT Although p48 is the most conserved subunit of mammalian DNA polymerase α-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.

scholarly journals The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box

Open Biology ◽  
2017 ◽  
Vol 7 (11) ◽  
pp. 170217 ◽  
Author(s):  
Mairi L. Kilkenny ◽  
Aline C. Simon ◽  
Jack Mainwaring ◽  
David Wirthensohn ◽  
Sandro Holzer ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Specific Interaction ◽  
Dna Helicase ◽  
T Antigen ◽  
Molecular Architecture ◽  
Biochemical Basis ◽  
Hmg Box ◽  
Dna Polymerase Α

A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.

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.

scholarly journals Role of the p68 Subunit of Human DNA Polymerase α-Primase in Simian Virus 40 DNA Replication

2002 ◽  
Vol 22 (16) ◽  
pp. 5669-5678 ◽  
Author(s):  
Robert D. Ott ◽  
Christoph Rehfuess ◽  
Vladimir N. Podust ◽  
Jill E. Clark ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Essential Role ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
S Phase ◽  
Dna Polymerase Α

ABSTRACT DNA polymerase α-primase (pol-prim) is a heterotetramer with DNA polymerase and primase activities. The polymerase (p180) and primase (p48 and p58) subunits synthesize primers and extend them, but the function of the remaining subunit (p68) is poorly understood. Genetic studies in yeast suggested an essential role for the p68 ortholog in early S phase prior to the hydroxyurea-sensitive step, possibly a regulatory role in initiation of DNA replication, but found no evidence for an essential function of p68 later in S phase. To investigate whether the human p68 subunit has an essential role in DNA replication, we examined the ability of a purified trimeric human pol-prim lacking p68 to initiate simian virus 40 DNA replication in vitro and to synthesize and elongate primers on single-stranded DNA in the presence of T antigen and replication protein A (RPA). Both activities of trimeric pol-prim were defective, but activity was recovered upon addition of separately purified p68. Phosphorylation of p68 by cyclin A-dependent protein kinase also inhibited both activities of pol-prim. The data strongly suggest that the p68 subunit is required for priming activity of pol-prim in the presence of RPA and T antigen, both during initiation at the origin and during lagging strand replication.

scholarly journals Phosphorylation-dependent interaction of adenovirus preterminal protein with the viral origin of DNA replication.

1994 ◽  
Vol 269 (3) ◽  
pp. 2189-2196
Author(s):  
J. Kusukawa ◽  
M. Ramachandra ◽  
R. Nakano ◽  
R. Padmanabhan
Keyword(s):  
Dna Replication ◽  
Viral Origin ◽  
Origin Of Dna Replication ◽  
Dependent Interaction

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.

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