Human DNA Polymerase ε: Enzymologic Mechanism and Gap-Filling Synthesis

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Abstract
DNA polymerase ε (pol ε) was purified to apparent homogeneity from human placentas. The purified enzyme contains a single polypeptide of approximately 170 kDa (apparent mass) and has both DNA polymerase and 3‘−5‘-exonuclease activities. Competitive inhibition studies indicate that like DNA polymerases α and δ (pol α and pol δ, respectively), free pol ε binds single-stranded but not double-stranded DNA. This conclusion was confirmed by sedimentation binding analysis. Also like pol α and pol δ, pol ε exhibits induced dNTP inhibition in the presence of template annealed to complementary primer containing a 2‘,3‘-H (dideoxy)-terminus. Together, these data suggest that pol ε follows an ordered sequential ter-reactant mechanism of substrate recognition and binding; it binds template first followed by annealed primer and then template-specified dNTP. Enzymologic studies suggest that in contrast to both pol α and pol δ, pol ε functions more efficiently as gap size decreases. This observation is consistent with a specific role for pol ε in gap-filling invivo. Gap-filling is essential for both replication and repair.

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