Wurgler S M, Richardson C C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1993 Sep 25;268(27):20046-54.
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.
大肠杆菌的dgt基因编码一种三磷酸脱氧鸟苷三磷酸水解酶（dGTPase），该酶可将dGTP水解为脱氧鸟苷和三聚磷酸。该酶对dGTP具有高度特异性，其水解的米氏常数为2 - 5微摩尔。硝酸纤维素滤膜结合试验表明，在生理盐条件下，dGTPase以明显的协同性与单链DNA结合，结合常数为7.7×10⁶ M⁻¹。在氯化钠存在的情况下，dGTPase与双链DNA的结合较弱。在没有氯化钠的情况下，dGTPase与单链和双链DNA的结合常数均为1×10⁷ M⁻¹。然而，dGTPase - 双链DNA复合物很容易被氯化钠解离。二价阳离子如Mg²⁺或Mn²⁺可增强DNA结合，但不是DNA结合所必需的。dGTP或GTP的存在不影响dGTPase结合DNA的能力。dGTPase可与长度为17 - 35的寡核苷酸结合，但亲和力较低。均聚物聚（dT）和聚（rU）可作为单链DNA与dGTPase结合的有效竞争者。特异性抑制dGTPase酶活性的噬菌体T7基因1.2蛋白也可阻止dGTPase与单链DNA结合。dGTPase可抑制T7 DNA聚合酶在聚（dA） - 寡聚（dT）模板上的活性。预先将dGTPase与T7基因1.2蛋白孵育可逆转这种抑制作用。
