Open AccessSpecies-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.
Author(s) -
Yasufumi Murakami,
Toshihiko Eki,
Mami Yamada,
Carol Prives,
Jerard Hurwitz
Publication year - 1986
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.83.17.6347
Subject(s) - polyoma virus , in vitro , dna , dna replication , replication (statistics) , biology , dna synthesis , virology , microbiology and biotechnology , chemistry , genetics
In vitro replication of DNA containing the polyoma (Py) virus origin 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 alpha 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. The addition of purified DNA polymerase alpha-primase complex isolated from FM3A cells enabled HeLa cell extracts to replicate Py DNA with the same efficiency as FM3A cell extracts. Complementary experiments have shown that FM3A cell extracts do not support SV40 DNA replication unless supplemented with DNA polymerase alpha-primase complex from HeLa cells [Murakami, Y., Wobbe, C.R., Weissbach, L., Dean, F.B. & Hurwitz, J. (1986) Proc. Natl. Acad. Sci. USA 83, 2869-2873]. These results indicate that the host-cell source of the DNA polymerase alpha-primase complex plays an important role in discriminating between SV40 T antigen- and Py T antigen-dependent replication of their homologous DNA in vitro. This may explain the host-range specificity of these viruses in vivo.