Interactions of calf thymus DNA polymerase α with primer/templates

The interactions of calf thymus DNA polymerase α (pol α) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primer/template binding or dNTP polymerization (Km, Vmax and processivity). Surprisingly, however, adding a 5′-triphosphate to the primer greatly changed its interactions with pol α (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the ability of pol α to discriminate against nucleotide analogs, it did not compromise the ability of pol α to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect ‘sugar fidelity’, without altering ‘base fidelity’. DNase protection assays showed that pol α strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5′-triphosphate on RNA primers to alter the catalytic properties of pol α.