Properties of the Primer-Binding Site and the Role of Magnesium Ion in Primer-Template Recognition by KB Cell DNA Polymerase α

The results of classical steady-state kinetic analyses and semiquantitative sedimentation binding assays have provided substantial definition of the important step of primerterminus recognition by KB cell DNA polymerase α. The polymerase is capable of binding a base-paired 3'-primer stem following a prerequisite step of binding to single-stranded polydeoxynucleotide template [Fisher, P. A., & Korn, D. (1981) Biochemistry, preceding paper in this issue]. Efficient priming with a bimolecular primer-template requires an octadeoxynucleotide, and this length requirement appears to be independent of both base composition and temperature. Both ribo and deoxyribo termini bearing 3'-hydroxyl groups are recognized with equal facility and support similar rates of catalysis. 2’,3'-Dideoxy termini, while unable to support catalysis, are bound by the polymerase similarly to 3'-hydroxyl termini. By exploiting the phenomenon of dideoxy primer induced dNTP inhibition [Fisher, P. A., & Korn, D. (1981) Biochemistry, preceding paper in this issue], we demonstrate that a single terminally mismatched primer residue blocks detectable binding of the primer stem. Mg²⁺ plays an important role in the interaction of the polymerase with both template and primer. Increasing concentrations of Mg²⁺ lead to dramatic increases in the affinity of polymerase a for pyrimidine deoxynucleotide homopolymer and copolymer templates, as well as for natural single-stranded DNA, but over the same concentration range, Mg²⁺ has little or no effect on the binding of purine polydeoxynucleotide templates. From this result, it has been possible with the hook template-primer (dA)m-(dT)m to demonstrate that free Mg²⁺ produces a highly parabolic pattern of competitive inhibition, and the measured Hill coefficient of 3.9 supports the interpretation that at least four Mg²⁺-binding sites are involved. The effects of Mg²⁺ on the binding of template and primer have been shown by sedimentation binding analyses to be qualitatively independent of the presence or absence of dNTPs and thus to reflect a property of the polymerase-nucleic acid interaction that is independent of catalysis. The combined results are compatible with the proposal that DNA polymerase a binds a terminally base-paired primer stem essentially independently of the nature of the S'-terminal sugar residue and that it does so via a Mg²⁺ chelate of the phosphodiester backbone that involves the participation of four primer-binding subsites.