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. 1989 Jun 26;17(12):4757–4767. doi: 10.1093/nar/17.12.4757

Contribution of DNA polymerase delta to DNA replication in permeable CHO cells synchronized in S phase.

A Basnakian 1, G Banfalvi 1, N Sarkar 1
PMCID: PMC318030  PMID: 2748336

Abstract

To evaluate the relative contributions of DNA polymerase alpha and DNA polymerase delta in chromosome replication during the S phase of the cell cycle, we have used the permeable cell system for replication as a functional assay. We carried out the analysis of DNA polymerases both in quiescent cells stimulated to proliferate and progress through the cell cycle (monolayers) and in actively growing cells separated into progressive stages of the cell cycle by centrifugal elutriation (suspension cultures). DNA polymerase alpha was measured by using the inhibitor butylphenyl dGTP at low concentrations. Using several inhibitors such as aphidicolin, ddTTP and butylphenyl dGTP, we found that DNA polymerase alpha and delta activity were coordinately increased during S phase and declined at the end. However, DNA polymerase delta was performing about 80% of the total replication and DNA polymerase alpha performed only 20%. This high ratio of DNA polymerase delta to DNA polymerase alpha replication activity was maintained throughout S phase in two entirely different experimental approaches.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ashihara T., Baserga R. Cell synchronization. Methods Enzymol. 1979;58:248–262. doi: 10.1016/s0076-6879(79)58141-5. [DOI] [PubMed] [Google Scholar]
  2. Byrnes J. J. Differential inhibitors of DNA polymerases alpha and delta. Biochem Biophys Res Commun. 1985 Oct 30;132(2):628–634. doi: 10.1016/0006-291x(85)91179-9. [DOI] [PubMed] [Google Scholar]
  3. Byrnes J. J., Downey K. M., Black V. L., So A. G. A new mammalian DNA polymerase with 3' to 5' exonuclease activity: DNA polymerase delta. Biochemistry. 1976 Jun 29;15(13):2817–2823. doi: 10.1021/bi00658a018. [DOI] [PubMed] [Google Scholar]
  4. Chang L. M., Brown M., Bollum F. J. Induction of DNA polymerase in mouse L cells. J Mol Biol. 1973 Feb 15;74(1):1–8. doi: 10.1016/0022-2836(73)90349-5. [DOI] [PubMed] [Google Scholar]
  5. Craig R. K., Costello P. A., Keir H. M. Dexyribonucleic acid polymerases of BHK-21/C13cells. Relationship to the physiological state of the cells, and to synchronous indution of synthesis of deoxyribonuleic acid. Biochem J. 1975 Feb;145(2):233–240. doi: 10.1042/bj1450233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crute J. J., Wahl A. F., Bambara R. A. Purification and characterization of two new high molecular weight forms of DNA polymerase delta. Biochemistry. 1986 Jan 14;25(1):26–36. doi: 10.1021/bi00349a005. [DOI] [PubMed] [Google Scholar]
  7. Decker R. S., Yamaguchi M., Possenti R., Bradley M. K., DePamphilis M. L. In vitro initiation of DNA replication in simian virus 40 chromosomes. J Biol Chem. 1987 Aug 5;262(22):10863–10872. [PubMed] [Google Scholar]
  8. Dresler S. L., Frattini M. G. DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase alpha to inhibition by butylphenyl-deoxyguanosine triphosphate. Nucleic Acids Res. 1986 Sep 11;14(17):7093–7102. doi: 10.1093/nar/14.17.7093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dresler S. L., Kimbro K. S. 2',3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: evidence for involvement of DNA polymerase delta. Biochemistry. 1987 May 19;26(10):2664–2668. doi: 10.1021/bi00384a002. [DOI] [PubMed] [Google Scholar]
  10. Focher F., Ferrari E., Spadari S., Hübscher U. Do DNA polymerases delta and alpha act coordinately as leading and lagging strand replicases? FEBS Lett. 1988 Feb 29;229(1):6–10. doi: 10.1016/0014-5793(88)80786-5. [DOI] [PubMed] [Google Scholar]
  11. Gray J. W., Coffino P. Cell cycle analysis by flow cytometry. Methods Enzymol. 1979;58:233–248. doi: 10.1016/s0076-6879(79)58140-3. [DOI] [PubMed] [Google Scholar]
  12. Hammond R. A., Byrnes J. J., Miller M. R. Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication. Biochemistry. 1987 Oct 20;26(21):6817–6824. doi: 10.1021/bi00395a035. [DOI] [PubMed] [Google Scholar]
  13. Khan N. N., Wright G. E., Dudycz L. W., Brown N. C. Butylphenyl dGTP: a selective and potent inhibitor of mammalian DNA polymerase alpha. Nucleic Acids Res. 1984 Apr 25;12(8):3695–3706. doi: 10.1093/nar/12.8.3695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Khan N. N., Wright G. E., Dudycz L. W., Brown N. C. Elucidation of the mechanism of selective inhibition of mammalian DNA polymerase alpha by 2-butylanilinopurines: development and characterization of 2-(p-n-butylanilino)adenine and its deoxyribonucleotides. Nucleic Acids Res. 1985 Sep 11;13(17):6331–6342. doi: 10.1093/nar/13.17.6331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krauss S. W., Linn S. Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states. J Cell Physiol. 1986 Jan;126(1):99–106. doi: 10.1002/jcp.1041260114. [DOI] [PubMed] [Google Scholar]
  16. Krokan H., Schaffer P., DePamphilis M. L. Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid. Biochemistry. 1979 Oct 2;18(20):4431–4443. doi: 10.1021/bi00587a025. [DOI] [PubMed] [Google Scholar]
  17. Lee M. Y., Tan C. K., Downey K. M., So A. G. Structural and functional properties of calf thymus DNA polymerase delta. Prog Nucleic Acid Res Mol Biol. 1981;26:83–96. doi: 10.1016/s0079-6603(08)60396-7. [DOI] [PubMed] [Google Scholar]
  18. Marraccino R. L., Wahl A. F., Keng P. C., Lord E. M., Bambara R. A. Cell cycle dependent activities of DNA polymerases alpha and delta in Chinese hamster ovary cells. Biochemistry. 1987 Dec 1;26(24):7864–7870. doi: 10.1021/bi00398a049. [DOI] [PubMed] [Google Scholar]
  19. Miller M. R., Seighman C., Ulrich R. G. Inhibition of DNA replication and DNA polymerase alpha activity by monoclonal anti-(DNA polymerase alpha) immunoglobulin G and F(ab) fragments. Biochemistry. 1985 Dec 3;24(25):7440–7445. doi: 10.1021/bi00346a061. [DOI] [PubMed] [Google Scholar]
  20. Nishida C., Reinhard P., Linn S. DNA repair synthesis in human fibroblasts requires DNA polymerase delta. J Biol Chem. 1988 Jan 5;263(1):501–510. [PubMed] [Google Scholar]
  21. Prelich G., Stillman B. Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA. Cell. 1988 Apr 8;53(1):117–126. doi: 10.1016/0092-8674(88)90493-x. [DOI] [PubMed] [Google Scholar]
  22. Sarkar N., List J. F., Banfalvi G. Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells. Eur J Biochem. 1987 Oct 15;168(2):263–268. doi: 10.1111/j.1432-1033.1987.tb13415.x. [DOI] [PubMed] [Google Scholar]
  23. Tanabe K., Karawya E., Fewell J. W., Kuff E. L., Wilson S. H. DNA polymerase and simian virus 40 infection of resting monkey cells: induction of aphidicolin resistant alpha-polymerase. Nucleic Acids Res. 1983 Dec 10;11(23):8253–8268. doi: 10.1093/nar/11.23.8253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thömmes P., Reiter T., Knippers R. Synthesis of DNA polymerase alpha analyzed by immunoprecipitation from synchronously proliferating cells. Biochemistry. 1986 Mar 25;25(6):1308–1314. doi: 10.1021/bi00354a018. [DOI] [PubMed] [Google Scholar]
  25. Wahl A. F., Crute J. J., Sabatino R. D., Bodner J. B., Marraccino R. L., Harwell L. W., Lord E. M., Bambara R. A. Properties of two forms of DNA polymerase delta from calf thymus. Biochemistry. 1986 Dec 2;25(24):7821–7827. doi: 10.1021/bi00372a006. [DOI] [PubMed] [Google Scholar]
  26. Wahl A. F., Geis A. M., Spain B. H., Wong S. W., Korn D., Wang T. S. Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle. Mol Cell Biol. 1988 Nov;8(11):5016–5025. doi: 10.1128/mcb.8.11.5016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zuber M., Tan E. M., Ryoji M. Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells. Mol Cell Biol. 1989 Jan;9(1):57–66. doi: 10.1128/mcb.9.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]

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