Replicating simian virus 40 (SV40) chromosomes that were capable of continuing DNA synthesis in vitro were partially separated from mature SV40 chromosomes on sucrose gradients and analyzed for DNA polymerase activities. The DNA polymerase activities were extracted with salt, fractionated by DEAE-cellulose chromatography, and assayed under conditions selective for each polymerase. Both DNA polymerase α and DNA polymerase γ were found, although DNA polymerase α accounted four about 95% of the total DNA polymerase activity. The amount of both enzyme activities per unit of DNA was greatest in the pool of replicating chromosomes; the activity found in the pool of mature chromosomes was proportional to its contamination with replicating chromosomes. DNA polymerase β was not found associated with SV40 chromosomes. These experiments were complemented by a study of the inhibition of DNA synthesis by 2':3'-dideoxythymidine 5'-triphosphate (d2TTP). SV40 DNA synthesis either in nuclei isolated from infected CV-1 cells, or in a soluble nuclear extract, or in replicating SV40 chromosomes was resistant to inhibition by d2TTP. DNA polymerase α was nearly as resistant to inhibition by d2TTP as were the in vitro DNA replication systems. DNA polymerases β and γ, however, were extremely sensitive to inhibition by d2TTP. 'Okazaki fragments' continued to be synthesized and joined to long chains of nascent DNA, and mature forms of viral DNA were produced, in the presence of d2TTP. Thus it appears that virtually all of the DNA synthesis observed in SV40 chromosomes replicating in vitro is performed by DNA polymerase α.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1978|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology