Properties of overexpressed phage T5 D15 exonuclease. Similarities with Escherichia coli DNA polymerase I 5'-3' exonuclease.
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.
Sayers
J R
JR
Max-Planck Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
Eckstein
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004790:Enzyme Induction; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases