DNA binding properties of the deoxyguanosine triphosphate triphosphohydrolase of Escherichia coli.
The dgt gene of Escherichia coli encodes a deoxyguanosine triphosphate triphosphohydrolase (dGTPase) that hydrolyzes dGTP to deoxyguanosine and tripolyphosphate. The enzyme is highly specific for dGTP which is hydrolyzed with a Km of 2-5 microM. Nitrocellulose filter binding assays demonstrate that, under physiological salt conditions, dGTPase binds with apparent cooperativity to single-stranded DNA with an association constant of 7.7 x 10(6) M-1. In the presence of NaCl, dGTPase binds weakly to double-stranded DNA. In the absence of NaCl, dGTPase binds both single- and double-stranded DNA with an association constant of 1 x 10(7) M-1. The dGTPase-double-stranded DNA complex, however, is readily dissociated with NaCl. Divalent cations such as Mg2+ or Mn2+ enhance, but are not required for DNA binding. The presence of dGTP or GTP does not effect the ability of dGTPase to bind DNA. dGTPase binds to oligonucleotides of length 17-35, but with lower affinities. The homopolymers poly(dT) and poly(rU) act as effective competitors of single-stranded DNA for binding to dGTPase. The bacteriophage T7 gene 1.2 protein, which specifically inhibits the enzymatic activity of dGTPase, also prevents dGTPase from binding to single-stranded DNA. dGTPase inhibits the activity of T7 DNA polymerase on a poly(dA)-oligo(dT) template. This inhibition is reversed by prior incubation of dGTPase with the T7 gene 1.2 protein.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
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Wurgler
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Richardson
D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D010744:Phosphoric Monoester Hydrolases