Enzymologic mechanism of calf thymus DNA polymerase δ

The catalytic core protomer of calf thymus DNA polymerase δ (pol δ) was purified to apparent homogeneity by a modified procedure, and its enzymologic mechanism was investigated using a combination of steady-state kinetics and semiquantitative sedimentation binding analyses. Like DNA polymerase α (pol α), in the absence of a primer, pol δ was able to bind single-stranded but not double-stranded DNA. This, in conjunction with the observation of induced substrate (dNTP) inhibition of pol 5 in the presence of a correctly base-paired 2′ ,3′-dideoxyribonucleotide-terminated primer, suggests that pol δ follows an ordered sequential ter-reactant mechanism of substrate recognition and binding similar to that elucidated for pol α. Pol δ binds template first followed by primer and then template-directed dNTP. With suitable substrates, addition to incubations of proliferating cell nuclear antigen, the pol δ auxiliary factor, leads to a reduction in K_m and increase in V_max. This suggests that proliferating cell nuclear antigen enhances the processivity of pol δ by increasing both the residence time of pol δ on the DNA template-primer and the rate at which individual nucleotides are incorporated.