Nonproteolyzed Form of DNA-Polymerase ε from T-Cell Spontaneous Lymphoma of Sprague-Dawley Inbred Rat: Isolation and Characterization

Abstract

Using a simple isolation procedure and selective assay for the determination of enzyme activity the nonproteolyzed and proteolyzed form of DNA-polymerase ε (pol ε and pol ε*) from the lymphoma of Sprague-Dawley inbred rats were purified. Nonproteolyzed pol ε is composed of two subunits (240 000 and 50 000) with sedimentation coefficient 10.5 S, while the subunit composition of pol ε* was 145 000 and 73 000. Estimated K_m values for dATP and dGTP as well as K_i values for acyclic nucleotide analogs (PMEApp, HPMPApp and PMEDAPpp) in pol ε and pol ε* catalyzed reactions have shown that a proteolysis probably does not affect pol ε binding site for dNTPs. Both enzymes (pol ε and pol ε*) possess 3'-5'-exonuclease activity with different K_m for 3'-OH end of template poly dA-oligo dT₁₈ (1.6 μmol/l and 0.36 μmol/l, respectively).