Sayers J R, Eckstein F
Max-Planck Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
J Biol Chem. 1990 Oct 25;265(30):18311-7.
The D15 gene of the bacteriophage T5, thought to encode an exonuclease, was cloned into an M13 phage on a 1344-base pair fragment. The deduced amino acid sequence of 291 residues (Kaliman, A. V., Krutilina, A. I., Kryukov, V. M., and Bayev, A. A. (1986) FEBS Lett. 195, 61-64) shows a high degree of homology with the first 320 amino acid residues of Escherichia coli DNA polymerase I, the region containing the enzyme's 5'-3' exonuclease activity. Recombinant M13 phage DNA was manipulated by oligonucleotide-directed mutagenesis to enable subcloning into a high efficiency expression vector, allowing the production of large amounts of enzyme for physical characterization and crystallization trials. The enzyme was purified to homogeneity. The purified enzyme is active on both native and heat-denatured DNA and shows no endonuclease activity on either double-stranded closed-circular or nicked DNA. The enzyme is also able to degrade some oligonucleotides in a manner which depends not only on the nucleotide sequence but also on the state of hybridization of the potential substrate. The mode of action of this enzyme is similar to, although not identical to that of the 5'-3' exonuclease activity of E. coli DNA polymerase I.
噬菌体T5的D15基因被认为编码一种核酸外切酶，它被克隆到一个M13噬菌体上，位于一个1344碱基对的片段上。推导的291个氨基酸残基的氨基酸序列（卡里曼，A. V.，克鲁季利娜，A. I.，克里乌科夫，V. M.，和巴耶夫，A. A.（1986年）《欧洲生物化学学会联合会快报》195，61 - 64）与大肠杆菌DNA聚合酶I的前320个氨基酸残基高度同源，该区域包含该酶的5'-3'核酸外切酶活性。通过寡核苷酸定向诱变对重组M13噬菌体DNA进行操作，以便亚克隆到高效表达载体中，从而能够大量生产该酶用于物理特性鉴定和结晶试验。该酶被纯化至同质。纯化后的酶对天然DNA和热变性DNA均有活性，对双链闭环或切口DNA均无内切酶活性。该酶还能够以一种不仅取决于核苷酸序列而且取决于潜在底物杂交状态的方式降解一些寡核苷酸。这种酶的作用模式与大肠杆菌DNA聚合酶I的5'-3'核酸外切酶活性相似，但不完全相同。
