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. 1996 Jul 1;15(13):3430–3441.

A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases.

V Truniger 1, J M Lázaro 1, M Salas 1, L Blanco 1
PMCID: PMC451907  PMID: 8670845

Abstract

The functional significance of the conserved motif 'YxGG/A', located between the 3'-5' exonuclease and polymerization domains of eukaryotic-type DNA polymerases, has been studied by site-directed mutagenesis in phi29 DNA polymerase. Single substitutions at this region were obtained, and 11 phi29 DNA polymerase mutant derivatives were overproduced in Escherichia coli and purified to homogeneity. Nine mutants showed an altered polymerase/3'-5' exonuclease balance on a template/primer DNA structure, giving rise to three different mutant phenotypes: (i) favored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low pol/exo ratio); and (iii) favored exonucleolysis and null polymerization. Interestingly, these three different phenotypes could be obtained by mutating a single amino acid at the 'YxGG/A' motif. All different phenotypes could be directly related to defects in DNA binding at a particular active site. Thus, a high pol/exo ratio was related to a poor stability at the 3'-5' exonuclease active site. On the contrary, a low pol/exo ratio or null polymerization capacity was related to a poor stability at the polymerization active site and either a normal or an increased accessibility to the exonuclease active site. These results allow us to propose that this motif, located in the connecting region between the N-terminal and C-terminal domains, has a primary role in DNA binding, playing a critical role in the coordination or cross-talk between synthesis and degradation.

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Selected References

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

  1. Beese L. S., Derbyshire V., Steitz T. A. Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science. 1993 Apr 16;260(5106):352–355. doi: 10.1126/science.8469987. [DOI] [PubMed] [Google Scholar]
  2. Beese L. S., Steitz T. A. Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 1991 Jan;10(1):25–33. doi: 10.1002/j.1460-2075.1991.tb07917.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernad A., Blanco L., Lázaro J. M., Martín G., Salas M. A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell. 1989 Oct 6;59(1):219–228. doi: 10.1016/0092-8674(89)90883-0. [DOI] [PubMed] [Google Scholar]
  4. Bernad A., Lázaro J. M., Salas M., Blanco L. The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4610–4614. doi: 10.1073/pnas.87.12.4610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bernad A., Zaballos A., Salas M., Blanco L. Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases. EMBO J. 1987 Dec 20;6(13):4219–4225. doi: 10.1002/j.1460-2075.1987.tb02770.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blanco L., Bernad A., Blasco M. A., Salas M. A general structure for DNA-dependent DNA polymerases. Gene. 1991 Apr;100:27–38. doi: 10.1016/0378-1119(91)90346-d. [DOI] [PubMed] [Google Scholar]
  7. Blanco L., Bernad A., Esteban J. A., Salas M. DNA-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 DNA polymerase. J Biol Chem. 1992 Jan 15;267(2):1225–1230. [PubMed] [Google Scholar]
  8. Blanco L., Bernad A., Lázaro J. M., Martín G., Garmendia C., Salas M. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J Biol Chem. 1989 May 25;264(15):8935–8940. [PubMed] [Google Scholar]
  9. Blanco L., Salas M. Mutational analysis of bacteriophage phi 29 DNA polymerase. Methods Enzymol. 1995;262:283–294. doi: 10.1016/0076-6879(95)62024-9. [DOI] [PubMed] [Google Scholar]
  10. Blasco M. A., Lázaro J. M., Blanco L., Salas M. Phi 29 DNA polymerase active site. Residue ASP249 of conserved amino acid motif "Dx2SLYP" is critical for synthetic activities. J Biol Chem. 1993 Nov 15;268(32):24106–24113. [PubMed] [Google Scholar]
  11. Blasco M. A., Lázaro J. M., Blanco L., Salas M. Phi 29 DNA polymerase active site. The conserved amino acid motif "Kx3NSxYG" is involved in template-primer binding and dNTP selection. J Biol Chem. 1993 Aug 5;268(22):16763–16770. [PubMed] [Google Scholar]
  12. Blasco M. A., Méndez J., Lázaro J. M., Blanco L., Salas M. Primer terminus stabilization at the phi 29 DNA polymerase active site. Mutational analysis of conserved motif KXY. J Biol Chem. 1995 Feb 10;270(6):2735–2740. doi: 10.1074/jbc.270.6.2735. [DOI] [PubMed] [Google Scholar]
  13. Bordo D., Argos P. Suggestions for "safe" residue substitutions in site-directed mutagenesis. J Mol Biol. 1991 Feb 20;217(4):721–729. doi: 10.1016/0022-2836(91)90528-e. [DOI] [PubMed] [Google Scholar]
  14. Braithwaite D. K., Ito J. Compilation, alignment, and phylogenetic relationships of DNA polymerases. Nucleic Acids Res. 1993 Feb 25;21(4):787–802. doi: 10.1093/nar/21.4.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Carthew R. W., Chodosh L. A., Sharp P. A. An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter. Cell. 1985 Dec;43(2 Pt 1):439–448. doi: 10.1016/0092-8674(85)90174-6. [DOI] [PubMed] [Google Scholar]
  16. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  17. Cullmann G., Hindges R., Berchtold M. W., Hübscher U. Cloning of a mouse cDNA encoding DNA polymerase delta: refinement of the homology boxes. Gene. 1993 Dec 8;134(2):191–200. doi: 10.1016/0378-1119(93)90093-i. [DOI] [PubMed] [Google Scholar]
  18. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  19. Dong Q., Copeland W. C., Wang T. S. Mutational studies of human DNA polymerase alpha. Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis. J Biol Chem. 1993 Nov 15;268(32):24163–24174. [PubMed] [Google Scholar]
  20. Esteban J. A., Soengas M. S., Salas M., Blanco L. 3'-->5' exonuclease active site of phi 29 DNA polymerase. Evidence favoring a metal ion-assisted reaction mechanism. J Biol Chem. 1994 Dec 16;269(50):31946–31954. [PubMed] [Google Scholar]
  21. Freemont P. S., Friedman J. M., Beese L. S., Sanderson M. R., Steitz T. A. Cocrystal structure of an editing complex of Klenow fragment with DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8924–8928. doi: 10.1073/pnas.85.23.8924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Garmendia C., Bernad A., Esteban J. A., Blanco L., Salas M. The bacteriophage phi 29 DNA polymerase, a proofreading enzyme. J Biol Chem. 1992 Feb 5;267(4):2594–2599. [PubMed] [Google Scholar]
  23. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  24. Gibbs J. S., Chiou H. C., Bastow K. F., Cheng Y. C., Coen D. M. Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6672–6676. doi: 10.1073/pnas.85.18.6672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hall J. D., Wang Y. S., Pierpont J., Berlin M. S., Rundlett S. E., Woodward S. Aphidicolin resistance in herpes simplex virus type I reveals features of the DNA polymerase dNTP binding site. Nucleic Acids Res. 1989 Nov 25;17(22):9231–9244. doi: 10.1093/nar/17.22.9231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hermanns J., Osiewacz H. D. The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase. Curr Genet. 1992 Dec;22(6):491–500. doi: 10.1007/BF00326415. [DOI] [PubMed] [Google Scholar]
  27. Inciarte M. R., Viñuela E., Salas M. Transcription in vitro of phi29 DNA and EcoRI fragments by Bacillus subtilis RNA polymerase. Eur J Biochem. 1976 Dec;71(1):77–83. doi: 10.1111/j.1432-1033.1976.tb11091.x. [DOI] [PubMed] [Google Scholar]
  28. Joyce C. M. How DNA travels between the separate polymerase and 3'-5'-exonuclease sites of DNA polymerase I (Klenow fragment). J Biol Chem. 1989 Jun 25;264(18):10858–10866. [PubMed] [Google Scholar]
  29. Joyce C. M., Steitz T. A. Function and structure relationships in DNA polymerases. Annu Rev Biochem. 1994;63:777–822. doi: 10.1146/annurev.bi.63.070194.004021. [DOI] [PubMed] [Google Scholar]
  30. Lázaro J. M., Blanco L., Salas M. Purification of bacteriophage phi 29 DNA polymerase. Methods Enzymol. 1995;262:42–49. doi: 10.1016/0076-6879(95)62007-9. [DOI] [PubMed] [Google Scholar]
  31. Morrison A., Christensen R. B., Alley J., Beck A. K., Bernstine E. G., Lemontt J. F., Lawrence C. W. REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase. J Bacteriol. 1989 Oct;171(10):5659–5667. doi: 10.1128/jb.171.10.5659-5667.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Méndez J., Blanco L., Lázaro J. M., Salas M. Primer-terminus stabilization at the psi 29 DNA polymerase active site. Mutational analysis of conserved motif TX2GR. J Biol Chem. 1994 Nov 25;269(47):30030–30038. [PubMed] [Google Scholar]
  33. Nakamaye K. L., Eckstein F. Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1986 Dec 22;14(24):9679–9698. doi: 10.1093/nar/14.24.9679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ollis D. L., Brick P., Hamlin R., Xuong N. G., Steitz T. A. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. 1985 Feb 28-Mar 6Nature. 313(6005):762–766. doi: 10.1038/313762a0. [DOI] [PubMed] [Google Scholar]
  35. Pelletier H., Sawaya M. R., Kumar A., Wilson S. H., Kraut J. Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science. 1994 Jun 24;264(5167):1891–1903. [PubMed] [Google Scholar]
  36. Rodríguez J. M., Yáez R. J., Rodríguez J. F., Viñuela E., Salas M. L. The DNA polymerase-encoding gene of African swine fever virus: sequence and transcriptional analysis. Gene. 1993 Dec 22;136(1-2):103–110. doi: 10.1016/0378-1119(93)90453-a. [DOI] [PubMed] [Google Scholar]
  37. Rohe M., Schrage K., Meinhardt F. The linear plasmid pMC3-2 from Morchella conica is structurally related to adenoviruses. Curr Genet. 1991 Dec;20(6):527–533. doi: 10.1007/BF00334782. [DOI] [PubMed] [Google Scholar]
  38. Salas M. Protein-priming of DNA replication. Annu Rev Biochem. 1991;60:39–71. doi: 10.1146/annurev.bi.60.070191.000351. [DOI] [PubMed] [Google Scholar]
  39. Sawaya M. R., Pelletier H., Kumar A., Wilson S. H., Kraut J. Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. Science. 1994 Jun 24;264(5167):1930–1935. doi: 10.1126/science.7516581. [DOI] [PubMed] [Google Scholar]
  40. Soengas M. S., Esteban J. A., Lázaro J. M., Bernad A., Blasco M. A., Salas M., Blanco L. Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3'-5' exonuclease and strand-displacement activities. EMBO J. 1992 Nov;11(11):4227–4237. doi: 10.1002/j.1460-2075.1992.tb05517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stocki S. A., Nonay R. L., Reha-Krantz L. J. Dynamics of bacteriophage T4 DNA polymerase function: identification of amino acid residues that affect switching between polymerase and 3' --> 5' exonuclease activities. J Mol Biol. 1995 Nov 17;254(1):15–28. doi: 10.1006/jmbi.1995.0595. [DOI] [PubMed] [Google Scholar]
  42. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  43. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tsurumi T., Maeno K., Nishiyama Y. Nucleotide sequence of the DNA polymerase gene of herpes simplex virus type 2 and comparison with the type 1 counterpart. Gene. 1987;52(2-3):129–137. doi: 10.1016/0378-1119(87)90039-4. [DOI] [PubMed] [Google Scholar]
  45. de Vega M., Lazaro J. M., Salas M., Blanco L. Primer-terminus stabilization at the 3'-5' exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. EMBO J. 1996 Mar 1;15(5):1182–1192. [PMC free article] [PubMed] [Google Scholar]

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