Efficient Replication of Polyomavirus DNA in a Cell-Free System Supplemented with Escherichia coli Single-Stranded DNA Binding Protein, Which Exhibits Species-Specificity in the Requirement for DNA Polymerase α-Primase

• Published in: Journal of biochemistry (Tokyo) 1995-08, Vol.118 (2), p.435-441
• Main Authors: Eki, Toshihiko, Hanaoka, Fumio, Kohda, Takeo, Seki, Masayuki, Ui, Michio, Enomoto, Takemi
• Format: Article
• Language: English
• Subjects: Animals; Cell-Free System; DNA polymerase; DNA Primase; DNA Replication; DNA, Viral - biosynthesis; DNA-Binding Proteins - metabolism; Escherichia coli - metabolism; HeLa Cells; Hot Temperature; Humans; Mice; Polyomavirus - genetics; polyomavirus DNA replication; RNA Nucleotidyltransferases - metabolism; single-stranded DNA binding protein; Species Specificity; temperature–sensitivemutant; Tumor Cells, Cultured; α-primase complex
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a124926

We established a modified cell-free system for polyomavirus (Py V) DNA replication, which was supplemented with Escherichia coli single-stranded DNA binding protein (SSB). DNA synthesis in this system was enhanced by 1.4- to over 15-fold depending upon the amount of cell extracts contained in the reaction mixture. By supplementing with E. coli SSB, we were able to reduce the amount of cell extracts in the reaction mixture, and to lower the concentrations of creatine phosphate and Tris, rendering this system more resistant to salts than the conventional Py V DNA replication system. The modified system was characterized using mutant cell extracts which had heat-inactivated DNA polymerase α. DNA synthesis in the system was dependent on Py V T antigen, the Py V origin of DNA replication, mutant cell extracts, and DNA polymerase α-primase complex purified from wild-type cells. The DNA polymerase α-primase complex was not replaced by DNA polymerase α, indicating that this system requires a functional DNA polymerase α-primase complex. This system exhibited species-specificity in the requirement for DNA polymerase α-primase; only mouse DNA polymerase α-primase but not human DNA polymerase α-primase functioned in this system.