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. 1989 Jun;8(6):1833–1839. doi: 10.1002/j.1460-2075.1989.tb03578.x

Exonucleolytic proofreading increases the accuracy of DNA synthesis by human lymphocyte DNA polymerase alpha-DNA primase.

G Bialek 1, H P Nasheuer 1, H Goetz 1, F Grosse 1
PMCID: PMC401030  PMID: 2527747

Abstract

DNA polymerase-primase complex, isolated with an apparently undegraded alpha-subunit, was immunoaffinity-purified to near homogeneity from the human lymphoblast line HSC93. The undegraded state of the alpha-subunit was monitored by Western-blot analysis of crude cellular extracts and all active fractions obtained during purification. The human polymerase-primase consists of four subunits with molecular weights of 195, 68, 55 and 48 kd. The fidelity of the polymerase-primase in copying bacteriophage phi X174am16 DNA in vitro was determined by measuring the frequency of production of different revertent phages. The overall accuracy was between 4 x 10(-6) and 10 x 10(-6). This value reflects the spontaneous mutation frequency of phi X174am16 phages in Escherichia coli, and is 10- to 20-fold higher than the accuracy of a conventionally purified enzyme from calf thymus. The frequencies of base pairing mismatches, estimated from pool bias measurements, were 3.5 x 10(-7) (1/2 880,000) for dGMP:Ttemplate mispairs, between 10(-7) and 10(-8) for dCMP:Ttemplate (1/35,000,000), dCMP:Atemplate (1/18,200,000) and dAMP:Gtemplate mispairs (1/16,500,000), and below 10(-8) (1/100,000,000) for dTMP:Ttemplate, dGMP:Atemplate and dGMP:Gtemplate mispairs. In contrast to previous preparations, the intact polymerase-primase possesses a 3'----5' exonuclease activity. This exonuclease removes both matched and mismatched 3'-OH ends, with a preference for mismatched bases. Fidelity was reduced 8-fold by increasing the concentration of the next nucleotide following the incorporated mismatch nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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  1. Banks G. R., Boezi J. A., Lehman I. R. A high molecular weight DNA polymerase from Drosophila melanogaster embryos. Purification, structure, and partial characterization. J Biol Chem. 1979 Oct 10;254(19):9886–9892. [PubMed] [Google Scholar]
  2. Brosius S., Grosse F., Krauss G. Subspecies of DNA polymerase alpha from calf thymus with different fidelity in copying synthetic template-primers. Nucleic Acids Res. 1983 Jan 11;11(1):193–202. doi: 10.1093/nar/11.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brutlag D., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. 36. A proofreading function for the 3' leads to 5' exonuclease activity in deoxyribonucleic acid polymerases. J Biol Chem. 1972 Jan 10;247(1):241–248. [PubMed] [Google Scholar]
  4. Chang L. M., Bollum F. J. A comparison of associated enzyme activities in various deoxyribonucleic acid polymerases. J Biol Chem. 1973 May 25;248(10):3398–3404. [PubMed] [Google Scholar]
  5. Chen Y. C., Bohn E. W., Planck S. R., Wilson S. H. Mouse DNA polymerase alpha. Subunit structure and identification of a species with associated exonuclease. J Biol Chem. 1979 Nov 25;254(22):11678–11687. [PubMed] [Google Scholar]
  6. Cotterill S. M., Reyland M. E., Loeb L. A., Lehman I. R. A cryptic proofreading 3'----5' exonuclease associated with the polymerase subunit of the DNA polymerase-primase from Drosophila melanogaster. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5635–5639. doi: 10.1073/pnas.84.16.5635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deng G., Wu R. Terminal transferase: use of the tailing of DNA and for in vitro mutagenesis. Methods Enzymol. 1983;100:96–116. doi: 10.1016/0076-6879(83)00047-6. [DOI] [PubMed] [Google Scholar]
  8. Fersht A. R. Fidelity of replication of phage phi X174 DNA by DNA polymerase III holoenzyme: spontaneous mutation by misincorporation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4946–4950. doi: 10.1073/pnas.76.10.4946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fersht A. R., Knill-Jones J. W. DNA polymerase accuracy and spontaneous mutation rates: frequencies of purine.purine, purine.pyrimidine, and pyrimidine.pyrimidine mismatches during DNA replication. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4251–4255. doi: 10.1073/pnas.78.7.4251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fersht A. R., Knill-Jones J. W. Fidelity of replication of bacteriophage phi X174 DNA in vitro and in vivo. J Mol Biol. 1983 Apr 25;165(4):633–654. doi: 10.1016/s0022-2836(83)80271-x. [DOI] [PubMed] [Google Scholar]
  11. Fersht A. R., Knill-Jones J. W., Tsui W. C. Kinetic basis of spontaneous mutation. Misinsertion frequencies, proofreading specificities and cost of proofreading by DNA polymerases of Escherichia coli. J Mol Biol. 1982 Mar 25;156(1):37–51. doi: 10.1016/0022-2836(82)90457-0. [DOI] [PubMed] [Google Scholar]
  12. Fisher P. A., Korn D. DNA polymerase-alpha. Purification and structural characterization of the near homogeneous enzyme from human KB cells. J Biol Chem. 1977 Sep 25;252(18):6528–6535. [PubMed] [Google Scholar]
  13. Glickman B. W., Radman M. Escherichia coli mutator mutants deficient in methylation-instructed DNA mismatch correction. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1063–1067. doi: 10.1073/pnas.77.2.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grosse F., Krauss G., Knill-Jones J. W., Fersht A. R. Accuracy of DNA polymerase-alpha in copying natural DNA. EMBO J. 1983;2(9):1515–1519. doi: 10.1002/j.1460-2075.1983.tb01616.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grosse F., Krauss G. Purification and partial characterization of a DNA polymerase alpha species from calf thymus. Nucleic Acids Res. 1980 Dec 11;8(23):5703–5714. doi: 10.1093/nar/8.23.5703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grosse F., Krauss G. Purification of a 9S DNA polymerase alpha species from calf thymus. Biochemistry. 1981 Sep 15;20(19):5470–5475. doi: 10.1021/bi00522a019. [DOI] [PubMed] [Google Scholar]
  17. Grosse F., Krauss G. Replication of M13mp7 single-stranded DNA in vitro by the 9-S DNA polymerase alpha from calf thymus. Eur J Biochem. 1984 May 15;141(1):109–114. doi: 10.1111/j.1432-1033.1984.tb08164.x. [DOI] [PubMed] [Google Scholar]
  18. Grosse F., Krauss G. The primase activity of DNA polymerase alpha from calf thymus. J Biol Chem. 1985 Feb 10;260(3):1881–1888. [PubMed] [Google Scholar]
  19. Hussy P., Maass G., Tümmler B., Grosse F., Schomburg U. Effect of 4-quinolones and novobiocin on calf thymus DNA polymerase alpha primase complex, topoisomerases I and II, and growth of mammalian lymphoblasts. Antimicrob Agents Chemother. 1986 Jun;29(6):1073–1078. doi: 10.1128/aac.29.6.1073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ishida R., Buchwald M. Susceptibility of Fanconi's anemia lymphoblasts to DNA-cross-linking and alkylating agents. Cancer Res. 1982 Oct;42(10):4000–4006. [PubMed] [Google Scholar]
  21. Kaguni L. S., DiFrancesco R. A., Lehman I. R. The DNA polymerase-primase from drosophila melanogaster embryos. Rate and fidelity of polymerization on single-stranded DNA templates. J Biol Chem. 1984 Jul 25;259(14):9314–9319. [PubMed] [Google Scholar]
  22. Karawya E., Swack J., Albert W., Fedorko J., Minna J. D., Wilson S. H. Identification of a higher molecular weight DNA polymerase alpha catalytic polypeptide in monkey cells by monoclonal antibody. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7777–7781. doi: 10.1073/pnas.81.24.7777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kunkel T. A., Eckstein F., Mildvan A. S., Koplitz R. M., Loeb L. A. Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6734–6738. doi: 10.1073/pnas.78.11.6734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kunkel T. A. Exonucleolytic proofreading. Cell. 1988 Jun 17;53(6):837–840. doi: 10.1016/s0092-8674(88)90189-4. [DOI] [PubMed] [Google Scholar]
  25. Kunkel T. A., Loeb L. A. Fidelity of mammalian DNA polymerases. Science. 1981 Aug 14;213(4509):765–767. doi: 10.1126/science.6454965. [DOI] [PubMed] [Google Scholar]
  26. Kunkel T. A., Sabatino R. D., Bambara R. A. Exonucleolytic proofreading by calf thymus DNA polymerase delta. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4865–4869. doi: 10.1073/pnas.84.14.4865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kunkel T. A., Soni A. Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma. J Biol Chem. 1988 Mar 25;263(9):4450–4459. [PubMed] [Google Scholar]
  28. 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]
  29. Loeb L. A., Kunkel T. A. Fidelity of DNA synthesis. Annu Rev Biochem. 1982;51:429–457. doi: 10.1146/annurev.bi.51.070182.002241. [DOI] [PubMed] [Google Scholar]
  30. Masaki S., Tanabe K., Yoshida S. Large polypeptides of 10S DNA polymerase alpha from calf thymus: rapid isolation using monoclonal antibody and tryptic peptide mapping analysis. Nucleic Acids Res. 1984 Jun 11;12(11):4455–4467. doi: 10.1093/nar/12.11.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Modrich P. DNA mismatch correction. Annu Rev Biochem. 1987;56:435–466. doi: 10.1146/annurev.bi.56.070187.002251. [DOI] [PubMed] [Google Scholar]
  32. Nasheuer H. P., Grosse F. DNA polymerase alpha-primase from calf thymus. Determination of the polypeptide responsible for primase activity. J Biol Chem. 1988 Jun 25;263(18):8981–8988. [PubMed] [Google Scholar]
  33. Nasheuer H. P., Grosse F. Immunoaffinity-purified DNA polymerase alpha displays novel properties. Biochemistry. 1987 Dec 15;26(25):8458–8466. doi: 10.1021/bi00399a064. [DOI] [PubMed] [Google Scholar]
  34. Ottiger H. P., Hübscher U. Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3993–3997. doi: 10.1073/pnas.81.13.3993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ottiger H., Frei P., Hässig M., Hübscher U. Mammalian DNA polymerase alpha: a replication competent holoenzyme form from calf thymus. Nucleic Acids Res. 1987 Jun 25;15(12):4789–4807. doi: 10.1093/nar/15.12.4789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Phear G., Nalbantoglu J., Meuth M. Next-nucleotide effects in mutations driven by DNA precursor pool imbalances at the aprt locus of Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4450–4454. doi: 10.1073/pnas.84.13.4450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Reckmann B., Grosse F., Krauss G. The elongation of mismatched primers by DNA polymerase alpha from calf thymus. Nucleic Acids Res. 1983 Oct 25;11(20):7251–7260. doi: 10.1093/nar/11.20.7251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reyland M. E., Lehman I. R., Loeb L. A. Specificity of proofreading by the 3'----5' exonuclease of the DNA polymerase-primase of Drosophila melanogaster. J Biol Chem. 1988 May 15;263(14):6518–6524. [PubMed] [Google Scholar]
  39. Reyland M. E., Loeb L. A. On the fidelity of DNA replication. Isolation of high fidelity DNA polymerase-primase complexes by immunoaffinity chromatography. J Biol Chem. 1987 Aug 5;262(22):10824–10830. [PubMed] [Google Scholar]
  40. Skarnes W., Bonin P., Baril E. Exonuclease activity associated with a multiprotein form of HeLa cell DNA polymerase alpha. Purification and properties of the exonuclease. J Biol Chem. 1986 May 15;261(14):6629–6636. [PubMed] [Google Scholar]
  41. So A. G., Downey K. M. Mammalian DNA polymerases alpha and delta: current status in DNA replication. Biochemistry. 1988 Jun 28;27(13):4591–4595. doi: 10.1021/bi00413a001. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Topal M. D., Fresco J. R. Complementary base pairing and the origin of substitution mutations. Nature. 1976 Sep 23;263(5575):285–289. doi: 10.1038/263285a0. [DOI] [PubMed] [Google Scholar]
  44. WATSON J. D., CRICK F. H. The structure of DNA. Cold Spring Harb Symp Quant Biol. 1953;18:123–131. doi: 10.1101/sqb.1953.018.01.020. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. 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]

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