Abstract
DNA polymerases alpha, delta, and epsilon have been purified and characterized from the same HeLa cell extract in order to determine their relationship by comparing them from the same cell type. The catalytic properties and the primary structures of the large subunits of the DNA polymerases as compared by partial peptide mapping with N-chlorosuccinimide are different. Likewise, the small subunit of DNA polymerase epsilon appears to be distinct from the large subunit of the same polymerase and from the smaller subunits of DNA polymerase alpha. HeLa DNA polymerase delta is processive only when HeLa proliferating cell nuclear antigen is present, whereas DNA polymerase epsilon is quite processive in its absence. Inhibitor and activator spectra of DNA polymerases alpha, delta, and epsilon also distinguish the three enzymes. These results and immunologic comparisons published elsewhere support the premise that HeLa DNA polymerases alpha, delta, and epsilon are distinct enzymes that have common properties with yeast DNA polymerases I, III, and II, respectively.
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- Bauer G. A., Burgers P. M. The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7506–7510. doi: 10.1073/pnas.85.20.7506. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bauer G. A., Heller H. M., Burgers P. M. DNA polymerase III from Saccharomyces cerevisiae. I. Purification and characterization. J Biol Chem. 1988 Jan 15;263(2):917–924. [PubMed] [Google Scholar]
- Boulet A., Simon M., Faye G., Bauer G. A., Burgers P. M. Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III. EMBO J. 1989 Jun;8(6):1849–1854. doi: 10.1002/j.1460-2075.1989.tb03580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Budd M. E., Sitney K. C., Campbell J. L. Purification of DNA polymerase II, a distinct DNA polymerase, from Saccharomyces cerevisiae. J Biol Chem. 1989 Apr 15;264(11):6557–6565. [PubMed] [Google Scholar]
- Burgers P. M. Mammalian cyclin/PCNA (DNA polymerase delta auxiliary protein) stimulates processive DNA synthesis by yeast DNA polymerase III. Nucleic Acids Res. 1988 Jul 25;16(14A):6297–6307. doi: 10.1093/nar/16.14.6297. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Byrnes J. J., Downey K. M., Que B. G., Lee M. Y., Black V. L., So A. G. Selective inhibition of the 3' to 5' exonuclease activity associated with DNA polymerases: a mechanism of mutagenesis. Biochemistry. 1977 Aug 23;16(17):3740–3746. doi: 10.1021/bi00636a002. [DOI] [PubMed] [Google Scholar]
- 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]
- Davison B. L., Leighton T., Rabinowitz J. C. Purification of Bacillus subtilis RNA polymerase with heparin-agarose. In vitro transcription of phi 29 DNA. J Biol Chem. 1979 Sep 25;254(18):9220–9226. [PubMed] [Google Scholar]
- Focher F., Gassmann M., Hafkemeyer P., Ferrari E., Spadari S., Hübscher U. Calf thymus DNA polymerase delta independent of proliferating cell nuclear antigen (PCNA). Nucleic Acids Res. 1989 Mar 11;17(5):1805–1821. doi: 10.1093/nar/17.5.1805. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Focher F., Spadari S., Ginelli B., Hottiger M., Gassmann M., Hübscher U. Calf thymus DNA polymerase delta: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase alpha, a DNA dependent ATPase and proliferating cell nuclear antigen. Nucleic Acids Res. 1988 Jul 25;16(14A):6279–6295. doi: 10.1093/nar/16.14.6279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hamatake R. K., Hasegawa H., Clark A. B., Bebenek K., Kunkel T. A., Sugino A. Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae. Identification of the catalytic core and a possible holoenzyme form of the enzyme. J Biol Chem. 1990 Mar 5;265(7):4072–4083. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lee M. Y., Tan C. K., Downey K. M., So A. G. Further studies on calf thymus DNA polymerase delta purified to homogeneity by a new procedure. Biochemistry. 1984 Apr 24;23(9):1906–1913. doi: 10.1021/bi00304a003. [DOI] [PubMed] [Google Scholar]
- Lee M. Y., Tan C. K., So A. G., Downey K. M. Purification of deoxyribonucleic acid polymerase delta from calf thymus: partial characterization of physical properties. Biochemistry. 1980 May 13;19(10):2096–2101. doi: 10.1021/bi00551a015. [DOI] [PubMed] [Google Scholar]
- Lee M. Y., Toomey N. L. Differential effects of dimethylsulfoxide on the activities of human DNA polymerases alpha and delta. Nucleic Acids Res. 1986 Feb 25;14(4):1719–1726. doi: 10.1093/nar/14.4.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee M. Y., Toomey N. L. Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting. Biochemistry. 1987 Feb 24;26(4):1076–1085. doi: 10.1021/bi00378a014. [DOI] [PubMed] [Google Scholar]
- Lischwe M. A., Ochs D. A new method for partial peptide mapping using N-chlorosuccinimide/urea and peptide silver staining in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1982 Dec;127(2):453–457. doi: 10.1016/0003-2697(82)90203-2. [DOI] [PubMed] [Google Scholar]
- 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]
- Plevani P., Francesconi S., Lucchini G. The nucleotide sequence of the PRI1 gene related to DNA primase in Saccharomyces cerevisiae. Nucleic Acids Res. 1987 Oct 12;15(19):7975–7989. doi: 10.1093/nar/15.19.7975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schlabach A., Fridlender B., Bolden A., Weissbach A. DNA-dependent DNA polymerases from HeLa cell nuclei. II. Template and substrate utilization. Biochem Biophys Res Commun. 1971 Aug 20;44(4):879–885. doi: 10.1016/0006-291x(71)90793-5. [DOI] [PubMed] [Google Scholar]
- Sitney K. C., Budd M. E., Campbell J. L. DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene. Cell. 1989 Feb 24;56(4):599–605. doi: 10.1016/0092-8674(89)90582-5. [DOI] [PubMed] [Google Scholar]
- Syvaoja J., Linn S. Characterization of a large form of DNA polymerase delta from HeLa cells that is insensitive to proliferating cell nuclear antigen. J Biol Chem. 1989 Feb 15;264(5):2489–2497. [PubMed] [Google Scholar]
- Talanian R. V., Brown N. C., McKenna C. E., Ye T. G., Levy J. N., Wright G. E. Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta. Biochemistry. 1989 Oct 17;28(21):8270–8274. doi: 10.1021/bi00447a002. [DOI] [PubMed] [Google Scholar]
- Tan C. K., Castillo C., So A. G., Downey K. M. An auxiliary protein for DNA polymerase-delta from fetal calf thymus. J Biol Chem. 1986 Sep 15;261(26):12310–12316. [PubMed] [Google Scholar]
- Tanaka S., Hu S. Z., Wang T. S., Korn D. Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase alpha. J Biol Chem. 1982 Jul 25;257(14):8386–8390. [PubMed] [Google Scholar]
- 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]
- Wong S. W., Paborsky L. R., Fisher P. A., Wang T. S., Korn D. Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells. J Biol Chem. 1986 Jun 15;261(17):7958–7968. [PubMed] [Google Scholar]
- Wong S. W., Syvaoja J., Tan C. K., Downey K. M., So A. G., Linn S., Wang T. S. DNA polymerases alpha and delta are immunologically and structurally distinct. J Biol Chem. 1989 Apr 5;264(10):5924–5928. [PubMed] [Google Scholar]
- Wong S. W., Wahl A. F., Yuan P. M., Arai N., Pearson B. E., Arai K., Korn D., Hunkapiller M. W., Wang T. S. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 1988 Jan;7(1):37–47. doi: 10.1002/j.1460-2075.1988.tb02781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]