Abstract
The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.
Similar content being viewed by others
References
Araki, H., Ropp, P.A., Johnson, A.L., Johnston, L.H., Morrison, A. and Sugino, A. (1992) DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeastSaccharomyces cerevisiae. EMBO J. 11, 733–740.
Bambara, R.A. and Jessee, C.B. (1991) Properties of DNA polymerases δ and ɛ, and their roles in eukaryotic DNA replication. Biochim. Biophys. Acta 1088, 11–24.
Cotteril, S.M., Reyland, M.E., Loeb, L.A. and Lehman, I.R. (1987) A cryptic proofreading 3′–5′ exonuclease associated with the polymerase subunit of the DNA polymerase-primase fromDrosophila melanogaster. Proc. Natl. Acad. Sci. USA 84, 5635–5639.
Crute, J.J., Wahl, A.F. and Bambara, R.A. (1986) Purification and characterization of two new high molecular weight forms of DNA polymerase delta. Biochemistry 25, 26–36.
Echols, H. & Goodman, M. (1991) Fidelity mechanism in DNA replication. Annu. Rev. Biochem. 14, 477–511.
Focher, F., Spadari, S., Ginelli, B., Hottiger, M., Grassmann, M. and Hübscher, U. (1988) Calf thymus DNA polymerase δ: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase α, a DNA dependent ATPase and proliferating cell nuclear antigen. Nucl. Acids Res. 14, 6279–6295.
Hübscher, U. and Thömmes P. (1992) DNA polymerase epsilon: in search of a function. TIBS 17, 55–58.
Kesti, T. and Syväoja, J.E. (1991) Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase ɛ. J. Biol Chem. 266, 6336–6341.
Kornberg, A. and Baker, T.A., DNA replication. Freeman 1991, New York, USA.
Lee, M.Y.W.T., Tan, C.-K., Downey, K.M. and So, A.G. (1984) Further studies on calf thymus DNA polymerase δ purified to homogenity by a new procedure. Biochemistry 23, 1906–1913.
Linn, S. (1991) How many pols does it take to replicate nuclear DNA? Cell 66, 185–187.
Nishida, C., Reinhard, P. and Linn, S. (1988) DNA repair synthesis in human fibroblasts requires DNA polymerase δ. J. Biol. Chem. 263, 501–510.
Nickel, W., Austermann, S., Bialek, G., and Grosse, F. (1992) Interactions of azidothymidine triphosphate with the cellular DNA polymerases α, δ and ɛ and with DNA primase. J. Biol. Chem. 267, 848–854.
Sabatino, R.D. and Bambara, R.A. (1988) Properties of the 3′ to 5′ exonuclease associated with calf DNA polymerase delta, Biochem. 27, 2266–2271.
Sabatino, R.D., Myers, T.W., Bambara, R.A. (1990) Substrate specificity of the exonuclease associated with calf DNA polymerase epsilon, Cancer Res. 50, 5340–5344.
Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
Siegal, G., Turchi, J.J., Jessee, C.B., Mallaber, L.M., Bambara, R.A. and Myers, T.W. (1992) Structural relationships between two forms of DNA polymerase epsilon from calf thymus. J. Biol. Chem. 267, 3991–3999.
Smith, H.O. (1980) Recovery of DNA from gels. Methods Enzymol. 65, 371–391.
Syväoja, J.E. (1990) DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases. BioEssays 12, 533–536.
Syväoja, J.E. and Linn, S. (1989) Characterization of a large form of DNA polymerase δ from HeLa cells that is insensitive to proliferating cell nuclear antigen. J. Biol. Chem. 264, 2489–2497.
Thömmes, P. and Hübscher, U. (1990) Eukaryotic DNA replication. Eur. J. Biochem. 194, 699–712.
Wang, T.S.-F. (1991) Eukaryotic DNA polymerases. Annu. Rev. Biochem. 60, 513–552.
Weiser, T., Gassmann, M., Thömmes, P., Ferrari, E., Hafkemeyer, P., and Hübscher, U. (1991) Biochemical and functional comparison of DNA polymerases α, δ and ɛ from calf thymus. J. Biol. Chem. 266, 10420–10428.
Yang, C.-L., Chang, L.-S., Zhang, P., Hao, H., Zhu, L., Toomey, N.L. and Lee, M.Y.W.T. (1992) Molecular cloning of the catalytic subunit of human DNA polymerase δ. Nucl. Acids Res. 20, 735–745.
Zhang, J., Chung, D.W., Tan, C.-K., Downey, K.M., Davie, E.W. and So, A.G. (1991) Primary structure of the catalytic subunit of calf thymus DNA polymerase δ: sequence similarities with other DNA polymerases. Biochem. 30, 11742–11750.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Uitto, L., Halleeen, J., Remes, P. et al. The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon. Chromosoma 102 (Suppl 1), S142–S146 (1992). https://doi.org/10.1007/BF02451798
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02451798