THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 253, No. 9, Issue of May 10, pp. 3273-3280, 1978 Printed III U.S.A Involvement of DNA Polymerase a in Simian Virus 40 DNA Replication* (Received for publication, October 31, 1977) HOWARD J. EDENBERG,* STEPHEN ANDERSON,§ AND MELVIN L. DEPAMPHILISB From the Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115 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 activi- ties. The DNA polymerase activities were extracted with salt, fractionated by DEAE-cellulose chromatography, and assayed under conditions selective for each polymerase. Both DNA polymerase (Y and DNA polymerase y were found, although DNA polymerase (Yaccounted for 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 /3 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 (d,TTP). 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 d,TTP. DNA polymerase (Y was nearly as resistant to inhibition by d,TTP as were the in vitro DNA replication systems. DNA polymerases /3 and y, however, were extremely sensitive to inhibition by d,TTP. “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 d,TTP. Thus it appears that virtually all of the DNA synthesis observed in SV40 chromosomes replicating in vitro is performed by DNA polymerase (Y. Of the many enzymes and proteins that might play a role in eukaryotic DNA replication (for example, DNA polymerase, DNA ligase, superhelix-relaxing enzyme, and single-stranded DNA binding proteins) none have been unambiguously shown to be required (1). A promising recent approach to this * This work was supported by National Institutes of Health Grant CA 15579. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebv marked “‘uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by a National Institutes of Health postdoctoral fellow- ship. Present address, Department of Biochemistry, Indiana Univer- sity School of Medicine, Indianapolis, Ind. 46202. 5 Supported by a National Service Award from the Public Health Service. ll Established Investigator of the American Heart Association. problem has been the development of soluble systems (2-4) in which the replication of simian virus 40 DNA can be studied. SV401 DNA replication is an excellent model for cellular chromosome replication, since for all processes other than the initiation of new replicons (5) host cell enzymes are used exclusively, and events at the viral DNA replication forks appear identical with those at mammalian replication forks (1). There is extensive evidence for the presence of at least three classes of DNA polymerases in mammalian cells called (Y, p, and y (6-9). A fourth DNA polymerase, associated with mitochondria, may be a form of y-polymerase (10). DNA polymerase (Y is a high molecular weight enzyme sensitive to N-ethylmaleimide. DNA polymerase /3 is a smaller enzyme, insensitive to N-ethylmaleimide, which can use a poly(rA).oligo(dT) template-primer combination nearly as well as it uses activated DNA. DNA polymerase y activity, present in relatively small amounts, is characterized by its ability to use poly(rA).oligo(dT) as a template-primer more effectively than it uses activated DNA. It is also of high molecular weight and sensitive to N-ethylmaleimide. These three enzymes can be fractionated by ion exchange chromatog- raphy. We have analyzed the DNA polymerase composition of replicating SV40 chromosomes that are capable of continuing DNA synthesis in vitro. Both DNA polymerase (Y and DNA polymerase y are associated with these replicating chromo- somes; DNA polymerase p is not. We have also found that the nucleotide analog 2’:3’-dideoxythymidine 5’-triphosphate does not inhibit in vitro SV40 DNA replication but does inhibit DNA polymerases p and y. However, DNA polymerase e is nearly as resistant to d,TTP as is in vitro viral DNA synthesis. These results suggest that DNA polymerase CY is primarily responsible for SV40 DNA synthesis. MATERIALS AND METHODS Materials - 2’:3’-Dideoxythymidine and 2’:3’-dideoxythymidine 5’-triphosphate were purchased from P-L Biochemicals. Poly(rA).oligo(dT),,_,, was made by annealing poly(rA) (Miles Lab- oratories) with olieo(dTl,,_,, (P-L Biochemicalsl in a molar nucleo- tide ratio of 251. ~~-32P]dN”TPs and [&2Pld,TTP were synthesized according to Symons (11). la-3’P]d,TTP was identified by chromatog- raphy in a solvent composed of isobutyric acid, 1 M NH,OH, and 0.1 M EDTA (100:60:1.6). [3H]dThd and [‘*CldThd were obtained from New England Nuclear. Activated DNA was salmon DNA (Sigma) 1 The abbreviations used are: SV40, simian virus 40; d,TTP, 2’:3’- dideoxythymidine 5’-triphosphate; Hepes, 4-(2-hydroxyethyll-l-pi- perazineethanesulfonic acid. 3273 by guest, on July 9, 2011 www.jbc.org Downloaded from