Mutational and pH Studies of the 3′ → 5′ Exonuclease Activity of Bacteriophage T4 DNA Polymerase

The 3′ → 5′ exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3′ → 5′ exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10–100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 105-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 103- to 104-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the p Kaof the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation. The 3′ → 5′ exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3′ → 5′ exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10–100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 105-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 103- to 104-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the p Kaof the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation. E. coli DNA polymerase I poly(dT)16 Many DNA polymerases have the ability to proofread newly replicated DNA by transferring the 3′-end of the primer-strand from the polymerase to the 3′ → 5′ exonuclease active center where the terminal nucleotide is removed. Mutant DNA polymerases that have reduced ability to carry out the transfer process (1Stocki S.A. Nonay R.L. Reha-Krantz L.J. J. Mol. Biol. 1995; 254: 15-28Crossref PubMed Scopus (82) Google Scholar, 2Marquez L.A. Reha-Krantz L.J. J. Biol. Chem. 1996; 271: 28903-28911Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 3Baker R.P. Reha-Krantz L.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3507-3512Crossref PubMed Scopus (49) Google Scholar, 4Reha-Krantz L.J. Marquez L.A. Elisseeva E. Baker R.P. Bloom L.B. Dunford H.B. Goodman M.F. J. Biol. Chem. 1998; 273: 22969-22976Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) or are defective in the hydrolysis reaction (4Reha-Krantz L.J. Marquez L.A. Elisseeva E. Baker R.P. Bloom L.B. Dunford H.B. Goodman M.F. J. Biol. Chem. 1998; 273: 22969-22976Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 6Reha-Krantz L.J. Nonay R.L. J. Biol. Chem. 1993; 268: 27100-27108Abstract Full Text PDF PubMed Google Scholar, 7Frey M.W. Nossal N.G. Capson T.L. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2579-2583Crossref PubMed Scopus (111) Google Scholar) replicate DNA with more errors (1Stocki S.A. Nonay R.L. Reha-Krantz L.J. J. Mol. Biol. 1995; 254: 15-28Crossref PubMed Scopus (82) Google Scholar, 5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 6Reha-Krantz L.J. Nonay R.L. J. Biol. Chem. 1993; 268: 27100-27108Abstract Full Text PDF PubMed Google Scholar, 7Frey M.W. Nossal N.G. Capson T.L. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2579-2583Crossref PubMed Scopus (111) Google Scholar, 8Muzyczka N. Poland R.L. Bessman M.J. J. Biol. Chem. 1972; 247: 7116-7122Abstract Full Text PDF PubMed Google Scholar, 9Reha-Krantz L.J. J. Mol. Biol. 1988; 202: 711-724Crossref PubMed Scopus (64) Google Scholar). A two-metal ion mechanism for the 3′ → 5′ exonuclease activity of Escherichia coli DNA polymerase I (DNA pol I)1 has been proposed from structural and mutational studies (10Freemont P.S. Friedman J.M. Beese L.S. Sanderson M.R. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8924-8928Crossref PubMed Scopus (324) Google Scholar, 11Derbyshire V. Grindley N.D.F. Joyce C.M. EMBO J. 1991; 10: 17-24Crossref PubMed Scopus (232) Google Scholar, 12Beese L. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (914) Google Scholar). The two divalent metal ions, which may be Mg2+, Mn2+, or Zn2+ (10Freemont P.S. Friedman J.M. Beese L.S. Sanderson M.R. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8924-8928Crossref PubMed Scopus (324) Google Scholar), are bound by conserved carboxylate residues in the exonuclease active centers of proofreading DNA polymerases (reviewed in Refs. 13Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (569) Google Scholar and 14Blasco L. Bernad A. Salas M. Gene (Amst.). 1992; 112: 139-144Crossref PubMed Scopus (72) Google Scholar). According to the model (Fig.1), a water molecule, coordinated by metal ion A, forms an attacking hydroxide ion, which is positioned in-line with the target phosphodiester bond. Metal ion B is proposed to stabilize the leaving 3′-hydroxy group and to position the O-P-O bond angles in the transition state (12Beese L. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (914) Google Scholar). The two metal ions are central to the model because amino acid substitutions that prevent binding of one or both metal ions reduce exonuclease activity several thousandfold. The conservation of metal ion binding residues in the exonuclease active centers of all proofreading DNA polymerases, and the severe reduction in exonuclease activity when the metal binding residues are replaced by non-carboxylate residues, suggest a common mechanism for the hydrolysis reaction catalyzed by DNA polymerases (13Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (569) Google Scholar).The two-metal ion mechanism may extend to other enzymes that catalyze phosphoryl transfer, for example, bacterial alkaline phosphatase, RNase H of the human immunodeficiency virus reverse transcriptase, single-stranded P1 nuclease, and phospholipase (reviewed in Ref. 15Steitz T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6498-6502Crossref PubMed Scopus (1019) Google Scholar). As observed for E. coli DNA pol I, a distance of about 3.9 Å separates two essential metal ions in the active centers of these enzymes. These observations led Steitz and Steitz (15Steitz T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6498-6502Crossref PubMed Scopus (1019) Google Scholar) to propose that RNA molecules involved in hydrolysis and splicing reactions may similarly position two divalent metal ions to carry out phosphoryl transfer reactions. A two-metal ion model for the hammerhead ribozyme mechanism is illustrated in Fig. 2, but catalysis does not require a metal hydroxide ion (16Pontius B.W. Lott W.B. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2290-2294Crossref PubMed Scopus (95) Google Scholar, 17Lott W.B. Pontius B.W. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 542-547Crossref PubMed Scopus (84) Google Scholar), although single-metal-hydroxide-ion models have been proposed (reviewed in Ref. 18Scott W.G. Klug A. Trends Biochem. Sci. 1996; 21: 220-224Abstract Full Text PDF PubMed Scopus (89) Google Scholar). Both one- and two-metal ion models for hammerhead ribozyme activity require the ribose 2′-OH group, which is not present in DNA (Fig.2). Since DNA does not have the 2′-OH, different mechanisms of metal-assisted hydrolysis of RNA and DNA are predicted.Figure 2Proposed two-metal ion model for the hydrolysis reaction catalyzed by the hammerhead ribozyme, adapted from Pontius et al. (16Pontius B.W. Lott W.B. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2290-2294Crossref PubMed Scopus (95) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Structural studies of the Klenow fragment of DNA pol I suggest that a water molecule or hydroxide ion is bound to metal ion A (Ref. 12Beese L. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (914) Google Scholar; Fig.1); however, only one study has examined the pH dependence of the exonuclease reaction and correlation to metal ion p Ka values (11Derbyshire V. Grindley N.D.F. Joyce C.M. EMBO J. 1991; 10: 17-24Crossref PubMed Scopus (232) Google Scholar). We present data here from pH and buffer studies of the exonuclease reaction catalyzed by wild-type and mutant bacteriophage T4 DNA polymerases that are consistent with formation of a metal hydroxide ion. Our studies focus on the ExoIII motif, which is conserved in the 3′ → 5′ exonuclease active centers of proofreading DNA polymerases (13Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (569) Google Scholar, 14Blasco L. Bernad A. Salas M. Gene (Amst.). 1992; 112: 139-144Crossref PubMed Scopus (72) Google Scholar). The motif sequence is a tyrosine residue, followed by three amino acids, and then an aspartate residue, which is a ligand to metal ion A (5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 13Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (569) Google Scholar). The conserved ExoIII residues in E. coli DNA pol I are Tyr-497 and Asp-501 (Fig. 1). The corresponding residues in T4 DNA polymerase are Tyr-320 and Asp-324 (5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 19Wang J., Yu, P. Lin T.C. Konigsberg W.H. Steitz T.A. Biochemistry. 1996; 35: 8110-8119Crossref PubMed Scopus (104) Google Scholar, 20Sattar A.K.M.A. Lin T-C. Jones C. Konigsberg W.H. Biochemistry. 1996; 35: 16621-16629Crossref PubMed Scopus (40) Google Scholar) and Tyr-323 and Asp-327 in the T4-like RB69 DNA polymerase (Refs. 21Wang C.-C. Yeh L.-S. Karam J.D. J. Biol. Chem. 1995; 270: 26558-26564Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar and 22Wang J., A.K.M. Sattar Wang C.C. Karam J.D. Konigsberg W.H. Steitz W.H. Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar; Fig.3). the structural in the exonuclease active centers of the bacterial and enzymes (Ref. 22Wang J., A.K.M. Sattar Wang C.C. Karam J.D. Konigsberg W.H. Steitz W.H. Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar; and The other essential residues for the 3′ → 5′ exonuclease activity of E. coli DNA pol I are which may ligand to metal ion A, and residue which a ligand to metal ion B (Fig. 1). The corresponding DNA polymerase residues are Asp-112 and in the T4 DNA polymerase and and in the RB69 DNA polymerase (Fig. for the bacterial and DNA polymerases have been example, alanine substitutions for residue Asp-501 in E. coli DNA pol I and for residue Asp-324 in T4 DNA polymerase reduce 3′ → 5′ exonuclease activity by of (5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 6Reha-Krantz L.J. Nonay R.L. J. Biol. Chem. 1993; 268: 27100-27108Abstract Full Text PDF PubMed Google Scholar, 7Frey M.W. Nossal N.G. Capson T.L. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2579-2583Crossref PubMed Scopus (111) Google Scholar, 11Derbyshire V. Grindley N.D.F. Joyce C.M. EMBO J. 1991; 10: 17-24Crossref PubMed Scopus (232) Google Scholar, 20Sattar A.K.M.A. Lin T-C. Jones C. Konigsberg W.H. Biochemistry. 1996; 35: 16621-16629Crossref PubMed Scopus (40) Google Scholar). The in exonuclease activity indicate that Asp-501 in E. coli DNA pol I and Asp-324 in T4 DNA polymerase are essential for in exonuclease activity are detected when is for Tyr-497 in E. coli DNA pol I (11Derbyshire V. Grindley N.D.F. Joyce C.M. EMBO J. 1991; 10: 17-24Crossref PubMed Scopus (232) Google Scholar) and when or alanine are for Tyr-320 in T4 DNA pol A.K.M.A. Lin T-C. Jones C. Konigsberg W.H. Biochemistry. 1996; 35: 16621-16629Crossref PubMed Scopus (40) Google the conserved tyrosine residue in the ExoIII motif an but in 3′ → 5′ exonuclease active center of the RB69 DNA The by Wang at from the RB69 DNA polymerase J., A.K.M. Sattar Wang C.C. Karam J.D. Konigsberg W.H. Steitz W.H. Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). The exonuclease active center is a with the active site residues and by The bound is a Metal ion have been data from the T4 exonuclease J., Yu, P. Lin T.C. Konigsberg W.H. Steitz T.A. Biochemistry. 1996; 35: 8110-8119Crossref PubMed Scopus (104) Google Scholar). residues are in the exonuclease active centers of the and bacterial DNA Residues and Asp-112 to metal ions A and observed for residue in E. coli DNA pol I (Fig. 1). Residues and a ligand to metal ion observed for residue in E. coli DNA pol Residues Asp-327 and Asp-324 a ligand to metal ion A, observed for residue Asp-501 in E. coli DNA pol Large Image Figure ViewerDownload Hi-res image Download extend studies (5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 6Reha-Krantz L.J. Nonay R.L. J. Biol. Chem. 1993; 268: 27100-27108Abstract Full Text PDF PubMed Google Scholar, 7Frey M.W. Nossal N.G. Capson T.L. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2579-2583Crossref PubMed Scopus (111) Google Scholar, 11Derbyshire V. Grindley N.D.F. Joyce C.M. EMBO J. 1991; 10: 17-24Crossref PubMed Scopus (232) Google Scholar, 20Sattar A.K.M.A. Lin T-C. Jones C. Konigsberg W.H. Biochemistry. 1996; 35: 16621-16629Crossref PubMed Scopus (40) Google Scholar) of mutant DNA polymerases with reduced ability to metal ions in the exonuclease active center by that the exonuclease activity for of the is to the most A exonuclease activity detected for the T4 D324A-DNA polymerase in compared with a activity detected in buffer for the and The in exonuclease activity for the D324A-DNA polymerase in compared with buffer that residue Asp-324 be by We propose that may with the T4 DNA polymerase in the exonuclease active center to a of hydroxide ions to a The of proposal for the wild-type T4 DNA polymerase is that metal ion A, bound by residue Asp-324, by a water molecule to an attacking metal-hydroxide ion, proposed for E. coli DNA pol I (Ref. 12Beese L. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (914) Google Scholar; Fig. 1). T4 DNA residues Glu-114 and Lys-299 may assist formation of the attacking hydroxide ion at T4 DNA polymerase residue Tyr-320 to assist of the DNA in the catalytically active conformation. Many DNA polymerases have the ability to proofread newly replicated DNA by transferring the 3′-end of the primer-strand from the polymerase to the 3′ → 5′ exonuclease active center where the terminal nucleotide is removed. Mutant DNA polymerases that have reduced ability to carry out the transfer process (1Stocki S.A. Nonay R.L. Reha-Krantz L.J. J. Mol. Biol. 1995; 254: 15-28Crossref PubMed Scopus (82) Google Scholar, 2Marquez L.A. Reha-Krantz L.J. J. Biol. Chem. 1996; 271: 28903-28911Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 3Baker R.P. Reha-Krantz L.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3507-3512Crossref PubMed Scopus (49) Google Scholar, 4Reha-Krantz L.J. Marquez L.A. Elisseeva E. Baker R.P. Bloom L.B. Dunford H.B. Goodman M.F. J. Biol. Chem. 1998; 273: 22969-22976Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) or are defective in the hydrolysis reaction (4Reha-Krantz L.J. Marquez L.A. Elisseeva E. Baker R.P. Bloom L.B. Dunford H.B. Goodman M.F. J. Biol. Chem. 1998; 273: 22969-22976Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 6Reha-Krantz L.J. Nonay R.L. J. Biol. Chem. 1993; 268: 27100-27108Abstract Full Text PDF PubMed Google Scholar, 7Frey M.W. Nossal N.G. Capson T.L. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2579-2583Crossref PubMed Scopus (111) Google Scholar) replicate DNA with more errors (1Stocki S.A. Nonay R.L. Reha-Krantz L.J. J. Mol. Biol. 1995; 254: 15-28Crossref PubMed Scopus (82) Google Scholar, 5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 6Reha-Krantz L.J. Nonay R.L. J. Biol. Chem. 1993; 268: 27100-27108Abstract Full Text PDF PubMed Google Scholar, 7Frey M.W. Nossal N.G. Capson T.L. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2579-2583Crossref PubMed Scopus (111) Google Scholar, 8Muzyczka N. Poland R.L. Bessman M.J. J. Biol. Chem. 1972; 247: 7116-7122Abstract Full Text PDF PubMed Google Scholar, 9Reha-Krantz L.J. J. Mol. Biol. 1988; 202: 711-724Crossref PubMed Scopus (64) Google Scholar). A two-metal ion mechanism for the 3′ → 5′ exonuclease activity of Escherichia coli DNA polymerase I (DNA pol I)1 has been proposed from structural and mutational studies (10Freemont P.S. Friedman J.M. Beese L.S. Sanderson M.R. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8924-8928Crossref PubMed Scopus (324) Google Scholar, 11Derbyshire V. Grindley N.D.F. Joyce C.M. EMBO J. 1991; 10: 17-24Crossref PubMed Scopus (232) Google Scholar, 12Beese L. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (914) Google Scholar). The two divalent metal ions, which may be Mg2+, Mn2+, or Zn2+ (10Freemont P.S. Friedman J.M. Beese L.S. Sanderson M.R. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8924-8928Crossref PubMed Scopus (324) Google Scholar), are bound by conserved carboxylate residues in the exonuclease active centers of proofreading DNA polymerases (reviewed in Refs. 13Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (569) Google Scholar and 14Blasco L. Bernad A. Salas M. Gene (Amst.). 1992; 112: 139-144Crossref PubMed Scopus (72) Google Scholar). According to the model (Fig.1), a water molecule, coordinated by metal ion A, forms an attacking hydroxide ion, which is positioned in-line with the target phosphodiester bond. Metal ion B is proposed to stabilize the leaving 3′-hydroxy group and to position the O-P-O bond angles in the transition state (12Beese L. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (914) Google Scholar). The two metal ions are central to the model because amino acid substitutions that prevent binding of one or both metal ions reduce exonuclease activity several thousandfold. The conservation of metal ion binding residues in the exonuclease active centers of all proofreading DNA polymerases, and the severe reduction in exonuclease activity when the metal binding residues are replaced by non-carboxylate residues, suggest a common mechanism for the hydrolysis reaction catalyzed by DNA polymerases (13Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (569) Google Scholar). The two-metal ion mechanism may extend to other enzymes that catalyze phosphoryl transfer, for example, bacterial alkaline phosphatase, RNase H of the human immunodeficiency virus reverse transcriptase, single-stranded P1 nuclease, and phospholipase (reviewed in Ref. 15Steitz T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6498-6502Crossref PubMed Scopus (1019) Google Scholar). As observed for E. coli DNA pol I, a distance of about 3.9 Å separates two essential metal ions in the active centers of these enzymes. These observations led Steitz and Steitz (15Steitz T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6498-6502Crossref PubMed Scopus (1019) Google Scholar) to propose that RNA molecules involved in hydrolysis and splicing reactions may similarly position two divalent metal ions to carry out phosphoryl transfer reactions. A two-metal ion model for the hammerhead ribozyme mechanism is illustrated in Fig. 2, but catalysis does not require a metal hydroxide ion (16Pontius B.W. Lott W.B. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2290-2294Crossref PubMed Scopus (95) Google Scholar, 17Lott W.B. Pontius B.W. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 542-547Crossref PubMed Scopus (84) Google Scholar), although single-metal-hydroxide-ion models have been proposed (reviewed in Ref. 18Scott W.G. Klug A. Trends Biochem. Sci. 1996; 21: 220-224Abstract Full Text PDF PubMed Scopus (89) Google Scholar). Both one- and two-metal ion models for hammerhead ribozyme activity require the ribose 2′-OH group, which is not present in DNA (Fig.2). Since DNA does not have the 2′-OH, different mechanisms of metal-assisted hydrolysis of RNA and DNA are studies of the Klenow fragment of DNA pol I suggest that a water molecule or hydroxide ion is bound to metal ion A (Ref. 12Beese L. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (914) Google Scholar; Fig.1); however, only one study has examined the pH dependence of the exonuclease reaction and correlation to metal ion p Ka values (11Derbyshire V. Grindley N.D.F. Joyce C.M. EMBO J. 1991; 10: 17-24Crossref PubMed Scopus (232) Google Scholar). We present data here from pH and buffer studies of the exonuclease reaction catalyzed by wild-type and mutant bacteriophage T4 DNA polymerases that are consistent with formation of a metal hydroxide ion. Our studies focus on the ExoIII motif, which is conserved in the 3′ → 5′ exonuclease active centers of proofreading DNA polymerases (13Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (569) Google Scholar, 14Blasco L. Bernad A. Salas M. Gene (Amst.). 1992; 112: 139-144Crossref PubMed Scopus (72) Google Scholar). The motif sequence is a tyrosine residue, followed by three amino acids, and then an aspartate residue, which is a ligand to metal ion A (5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 13Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (569) Google Scholar). The conserved ExoIII residues in E. coli DNA pol I are Tyr-497 and Asp-501 (Fig. 1). The corresponding residues in T4 DNA polymerase are Tyr-320 and Asp-324 (5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 19Wang J., Yu, P. Lin T.C. Konigsberg W.H. Steitz T.A. Biochemistry. 1996; 35: 8110-8119Crossref PubMed Scopus (104) Google Scholar, 20Sattar A.K.M.A. Lin T-C. Jones C. Konigsberg W.H. Biochemistry. 1996; 35: 16621-16629Crossref PubMed Scopus (40) Google Scholar) and Tyr-323 and Asp-327 in the T4-like RB69 DNA polymerase (Refs. 21Wang C.-C. Yeh L.-S. Karam J.D. J. Biol. Chem. 1995; 270: 26558-26564Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar and 22Wang J., A.K.M. Sattar Wang C.C. Karam J.D. Konigsberg W.H. Steitz W.H. Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar; Fig.3). the structural in the exonuclease active centers of the bacterial and enzymes (Ref. 22Wang J., A.K.M. Sattar Wang C.C. Karam J.D. Konigsberg W.H. Steitz W.H. Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar; and The other essential residues for the 3′ → 5′ exonuclease activity of E. coli DNA pol I are which may ligand to metal ion A, and residue which a ligand to metal ion B (Fig. 1). The corresponding DNA polymerase residues are Asp-112 and in the T4 DNA polymerase and and in the RB69 DNA polymerase (Fig. for the bacterial and DNA polymerases have been example, alanine substitutions for residue Asp-501 in E. coli DNA pol I and for residue Asp-324 in T4 DNA polymerase reduce 3′ → 5′ exonuclease activity by of (5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 6Reha-Krantz L.J. Nonay R.L. J. Biol. Chem. 1993; 268: 27100-27108Abstract Full Text PDF PubMed Google Scholar, 7Frey M.W. Nossal N.G. Capson T.L. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2579-2583Crossref PubMed Scopus (111) Google Scholar, 11Derbyshire V. Grindley N.D.F. Joyce C.M. EMBO J. 1991; 10: 17-24Crossref PubMed Scopus (232) Google Scholar, 20Sattar A.K.M.A. Lin T-C. Jones C. Konigsberg W.H. Biochemistry. 1996; 35: 16621-16629Crossref PubMed Scopus (40) Google Scholar). The in exonuclease activity indicate that Asp-501 in E. coli DNA pol I and Asp-324 in T4 DNA polymerase are essential for in exonuclease activity are detected when is for Tyr-497 in E. coli DNA pol I (11Derbyshire V. Grindley N.D.F. Joyce C.M. EMBO J. 1991; 10: 17-24Crossref PubMed Scopus (232) Google Scholar) and when or alanine are for Tyr-320 in T4 DNA pol A.K.M.A. Lin T-C. Jones C. Konigsberg W.H. Biochemistry. 1996; 35: 16621-16629Crossref PubMed Scopus (40) Google the conserved tyrosine residue in the ExoIII motif an but in We extend studies (5Reha-Krantz L.J. Stocki S. Nonay R.L. Dimayuga E. Goodrich L.D. Konigsberg W.H. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2417-2421Crossref PubMed Scopus (55) Google Scholar, 6Reha-Krantz L.J. Nonay R.L. J. Biol. Chem. 1993; 268: 27100-27108Abstract Full Text PDF PubMed Google Scholar, 7Frey M.W. Nossal N.G. Capson T.L. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2579-2583Crossref PubMed Scopus (111) Google Scholar, 11Derbyshire V. Grindley N.D.F. Joyce C.M. EMBO J. 1991; 10: 17-24Crossref PubMed Scopus (232) Google Scholar, 20Sattar A.K.M.A. Lin T-C. Jones C. Konigsberg W.H. Biochemistry. 1996; 35: 16621-16629Crossref PubMed Scopus (40) Google Scholar) of mutant DNA polymerases with reduced ability to metal ions in the exonuclease active center by that the exonuclease activity for of the is to the most A exonuclease activity detected for the T4 D324A-DNA polymerase in compared with a activity detected in buffer for the and The in exonuclease activity for the D324A-DNA polymerase in compared with buffer that residue Asp-324 be by We propose that may with the T4 DNA polymerase in the exonuclease active center to a of hydroxide ions to a The of proposal for the wild-type T4 DNA polymerase is that metal ion A, bound by residue Asp-324, by a water molecule to an attacking metal-hydroxide ion, proposed for E. coli DNA pol I (Ref. 12Beese L. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (914) Google Scholar; Fig. 1). T4 DNA residues Glu-114 and Lys-299 may assist formation of the attacking hydroxide ion at T4 DNA polymerase residue Tyr-320 to assist of the DNA in the catalytically active conformation. We M. for the that the 3′ → 5′ exonuclease activity of the D324A-DNA polymerase is reduced in L. Bloom for J. and Steitz for Fig. B. for in Fig. and C. and E. for on the by Nonay and