INHIBITION OF DNA PRIMASE AND POLYMERASE-ALPHA BY ARABINOFURANOSYLNUCLEOSIDE TRIPHOSPHATES AND RELATED-COMPOUNDS

Inhibition of DNA primase and polymerase-alpha from calf thymus was examined. DNA primase requires a 3'-hydroxyl on the incoming NTP in order to polymerize it, while the 2'-hydroxyl is advantageous, but not essential. Amazingly, primase prefers to polymerize araATP rather than ATP by 4-fold (k(cat)/K(M)). However, after incorporation of an araNMP into the growing primer, further synthesis is abolished. The 2'- and 3'-hydroxyls of the incoming nucleotide appear relatively unimportant for nucleotide binding to primase. Polymerization of nucleoside triphosphates by DNA polymerase-alpha onto a DNA primer was similarly analyzed. Removing the 3'-hydroxyl of the incoming triphosphate decreases the polymerization rate > 1000-fold (k(cat)/K(M)), while a 2'-hydroxyl in the ribo configuration abolishes polymerization. If the 2'-hydroxyl is in the ara configuration, there is almost no effect on polymerization. An araCMP or ddCMP at the 3'-terminus of a DNA primer slightly decreased DNA binding as well as binding of the next correct 2'-dNTP. Changing the primer from DNA to RNA dramatically and unpredictably altered the interactions of pol-alpha with araNTPs and ddNTPs. Compared to the identical DNA primer, pol-alpha discriminated 4-fold better against araCTP polymerization when the primer was RNA, but 85-fold worse against ddCTP polymerization. Additionally, pol-alpha elongated RNA primers containing 3'-terminal araNMPs more efficiently than the identical DNA substrate.