In vitro synthesis of simian virus 40 DNA: III. - Preliminary characterization of the active components in the system

Summary The repair synthesis of Simian Virus 40 (SV40) DNA in vitro using cytoplasmic extracts from CV 1 cells [ 9 ] was investigated. Extracts were chromatographed on DEAE cellulose at pH 7.5. The majority of the endonuclease activity of the extract was excluded from the DEAE cellulose column (fraction A). The majority of the DNA polymerase activity measured with calf thymus DNA as a template was bound to the column, and eluted with KCl concentrations greater than 150 mM (fractions C and D). In addition, a minor peak of DNA polymerase was recovered in fraction A. When the same fractions were tested with SV40 DNA component I molecules as a template, the majority of the activity incorporating desoxyribonucleoside triphosphates was however recovered in fraction A, and not in fraction C or D. The DNA product labeled in vitro by fraction A sedimented at 4-8S at neutral pH, and at 4S at pH 13. The ability of fraction A to synthesize SV40 DNA component II and relaxed component I species in vitro could be restored by supplementing fraction A with fractions C or D, or with purified DNA ligase from phage T4. It is concluded from these results that the in vitro repair synthesis of SV40 DNA by cytoplasmic extracts is based upon the balanced activities of the endonucleases, and of DNA polymerase β, which are recovered in fraction A ; and of the cell DNA ligase, which is recovered in fractions C and D. Although present in excess in the extract, DNA polymerase α seemed to be mostly inactive in the reaction. No virus specific endonuclease nor DNA polymerase could be detected in the infected cell extracts.