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 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-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 pK(a) of 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.
Elisseeva
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Department of Biological Science, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Mandal
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SS
Reha-Krantz
L J
LJ
D001665:Binding Sites; D004256:DNA Polymerase I / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000201:enzymology; D005092:Exonucleases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011071:Poly T / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D016337:RNA, Catalytic / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*; D014867:Water / Q000737:chemistry
D008670:Metals; D016337:RNA, Catalytic; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D014867:Water; D011071:Poly T; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases