ISOLATION, CHARACTERIZATION, AND KINETIC-PROPERTIES OF TRUNCATED FORMS OF T4 DNA-POLYMERASE THAT EXHIBIT 3'-5' EXONUCLEASE ACTIVITY

Limited proteolysis of T4 DNA polymerase generated a 45-kDa and 35-kDa protein complex, which had 3'-5' exonucleolytic activity but lacked p olymerase activity. After partial chymotryptic digestion of the cloned and expressed 45-kDa protein derived from T4 DNA polymerase, we isola ted a 27-kDa fragment (residues 96-331) that still had 3'-5' exonuclea se activity, thus demonstrating that the amino acid residues required for catalysis are included within this fragment. We also show that the apparent K-m values for the 3'-5' exonuclease activity exhibited by t he 27-kDa fragment are considerably greater than the apparent K-m valu es determined for the intact DNA polymerase on deoxyoligonucleotide su bstrates having more than 3 bases. In contrast, the k(cat) values for phosphodiester bond hydrolysis of 3'-terminal nucleotides are not very different when comparing intact T4 DNA polymerase with the 27-kDa fra gment derived from it. Thus, participation of residues distal to 331 a re not required for catalysis, but only for binding, and, based on the similarity of k(cat) values, the geometry of the residues responsible for catalysis are preserved even in the absence of the carboxyl-termi nal 567 residues.