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 (kcat/KM). 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 greater than 1000-fold (kcat/KM), 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.
Kuchta
R D
RD
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Ilsley
D
D
Kravig
K D
KD
Schubert
S
S
Harris
B
B
D000818:Animals; D001085:Arabinofuranosylcytosine Triphosphate / Q000494:pharmacology; D001088:Arabinonucleotides / Q000494:pharmacology*; D001483:Base Sequence / Q000187:drug effects; D002384:Catalysis; D002417:Cattle; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors*; D019915:DNA Primase; D004261:DNA Replication; D008969:Molecular Sequence Data; D012316:RNA Nucleotidyltransferases / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship
D001088:Arabinonucleotides; D001085:Arabinofuranosylcytosine Triphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II