ENZYMOLOGIC MECHANISM OF CALF THYMUS DNA POLYMERASE-DELTA

The catalytic core protomer of calf thymus DNA polymerase-delta (pol-delta) 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-alpha (pol-alpha), in the absence of a primer, pol-delta was able to bind single-stranded but not double-stranded DNA. This, in conjunction with the observation of induced substrate (dNTP) inhibition of pol-delta in the presence of a correctly base-paired 2',3'-dideoxyribonucleotide-terminated primer, suggests that pol-delta follows an ordered sequential ter-reactant mechanism of substrate recognition and binding similar to that elucidated for pol-alpha. Pol-delta binds template first followed by primer and then template-directed dNTP. With suitable substrates, addition to incubations of proliferating cell nuclear antigen, the pol-delta 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-delta by increasing both the residence time of pol-delta on the DNA template-primer and the rate at which individual nucleotides are incorporated.