Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.

Abstract

In vitro replication of DNA containing the polyoma (Py) virus orign of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase a in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. Cell-free extracts of monkey and human cells that replicate SV40 DNA have been described (4-7). The in vitro replication of SV40 DNA requires, in addition to suitably prepared cell extracts, circular DNA containing the viral replication origin (ori) and purified SV40 large tumor (T) antigen. Since SV40 T antigen and DNA containing the SV40 replication origin are the only viral components required, this system should be useful for identifying and characterizing the eukaryotic proteins involved in DNA replication. In previous reports (7-9), it was shown that DNA polymerase a (pol a) and DNA primase are essential for the replication of SV40 DNA in vitro and that the source of these enzymes is important in determining whether SV40 DNA replication will occur. In contrast to extracts of human cells, mouse cell extracts supplemented with SV40 T antigen did not support replication of DNA containing the SV40 origin unless supplemented with the pol a-primase complex from HeLa cells.In this report we describe the establishment of a system that replicates Py DNA in vitro; it requires mouse cell extract, the Py replication origin, and Py T antigen. In addition, we have confirmed and further defined the important role played by pol a-primase in determining the species specificity of papovavirus DNA replication. MATERIALS AND METHODSPurification of Py T Antigen. Py T antigen was purified from CV-1 cells infected with the helper-dependent recombinant adenovirus vector Ad-SVR587 (10) as follows. Cells were infected with wild-type adenovirus [2-5 plaque-forming units (pfu) per cell] and Ad-SVR587 recombinant virus (a gift from S. Mansour, T. Grodzicker, and R. Tjian) (5-10 pfu per cell). After incubation for 36 hr at 370C, cells were scraped from thirty 150-mm plates into -3 ml of cold Dulbecco's phosphate-buffered saline (PBS: Ca2+-and Mg2+-free), washed twice with cold PBS, suspended in 10 volumes of pH 9.0 buffer [20 mM Tris HCl, pH 9.0/0.2 M NaCl/1 mM EDTA/1 mM dithiothreitol/10% (vol/vol) glycerol/1% (vol/vol) Nonidet P-40 (NP-40)/1 mM phenylmethylsulfonyl fluoride (PMSF)], and lysed on ice for 10: min. The lysate was centrifuged for 10 min at 2000 rpm in a Sorvall H-6000A rotor, and the supernatant was then centrifuged for 10 min at 20,000 rpm in a Sorvall SS34 rotor. The supernatant was mixed with 0.5 volume of pH 6.8 buffer (0.1 M Tris'HCl, pH 6.8/1 mM EDTA/1 mM dithiothre...

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

“…Therefore, mouse RPA is not a species-specific factor and is not the source of the inhibitory activities. Similar to RPA, Pol-prim is essential for eukaryotic DNA replication and is the major species-specific factor of mPyV and SV40 (8,32,33,50). Here, we verified the ability of mouse Pol-prim to functionally and physically interact with BKV TAg, similar to its interaction with SV40 TAg in the SV40 system (10,53,57).…”

“…The DNA synthesis on X174 ssDNA was carried out according to Smith et al (46). Briefly, the reaction mixture contained the following: 1 g of X174 ssDNA (NEB); 20 mM Tris-acetate (pH 7.3); 5 mM magnesium acetate; 20 mM potassium acetate; 1 mM dithiothreitol (DTT); 0.1 mg/ml BSA; 1 mM ATP; 0.1 mM each CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP; and 0.1 mM [␣ 32 P]dCTP (3,000 Ci/mmol; Perkin-Elmer). Comparisons between human and mouse Pol-prim were made by titration, and the amount of enzyme complexes was varied from 0.5 to 2.0 units (3.04 U/g for human Pol-prim, 2.88 U/g for mouse Pol-prim, and 1 U to 100 pmol of deoxynucleoside monophosphate [dNMP] incorporation in a 1-h reaction) of each complex per assay.…”

A Specific Docking Site for DNA Polymerase α-Primase on the SV40 Helicase Is Required for Viral Primosome Activity, but Helicase Activity Is Dispensable

“…Clearly, the p180-and primase-docking sites on Tag remain to be determined. Competition of p53 with pol-prim for binding to Tag (47,63,64) suggests that one of these docking sites may overlap with the p53-binding surface at the outer edge of each AAAϩ/D3 domain (44). Because other aspects of the model may also be tested experimentally, we anticipate that it may prove useful in developing a better understanding of eukaryotic primosomes.…”

“…This would be consistent with the extreme species specificity of pol-prim in cell-free SV40 DNA replication. For example, although mouse and human pol-prim are highly conserved overall and Tag interacts physically with both enzymes, the spatial orientation of their Tag docking domains may differ, so that only primate pol-prim is active in the SV40 primosome (47)(48)(49)(50). Moreover, although Lys 425 is conserved among primate polyomavirus Tag proteins (45), it is not conserved among related viral helicases (e.g.…”

Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins.

“…Simian virus 40 (SV40) and polyomavirus have long been used as models for investigations of mammalian DNA replication. Initiation of SV40 or polyomavirus replication requires the homologous large tumor antigen, is species specific, and occurs only in a permissive cell environment (1)(2)(3)(4). However, the uncontrolled replication of these lytic viruses does not reflect the precise regulation of cellular DNA replication, which takes place once per cell cycle in the S phase.…”