Inhibition of DNA primase by 9-beta-D-arabinofuranosyladenosine triphosphate.
9-beta-D-Arabinofuranosyladenosine triphosphate (araATP) is a potent inhibitor of DNA primase. Primase readily incorporates araATP into primers, and primers containing araAMP are then elongated by DNA polymerase alpha (pol alpha) upon addition of dNTPs. AraATP did not inhibit utilization of primers under conditions where the ability of pol alpha to elongate primers was independent of the dATP concentration. The fraction of primers elongated by pol alpha was reduced by araATP only when elongation was dependent upon the dATP concentration. When the Ki for primase was measured in terms of the inhibition of the synthesis of primers that can be utilized by pol alpha, we obtained Ki = 2.7 microM (37 degrees C) and 2.0 microM (25 degrees C). Inhibition was competitive with ATP. Inhibition of pol alpha activity by araATP was measured under conditions where primase-catalyzed primer synthesis was required for the pol alpha activity. The decreased pol alpha activity was due to primase inhibition, and at constant dATP, araATP inhibition was competitive with ATP and gave Ki = 1.2 microM, similar to the Ki for primase alone. Increasing the dATP concentration had no effect on inhibition. In combination with previously reported in vivo data, we conclude that DNA primase is the primary in vivo target of the arabinofuranosyl nucleotides, not pol alpha.
Kuchta
R D
RD
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Willhelm
L
L
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002417:Cattle; D004247:DNA / Q000096:biosynthesis*; D004257:DNA Polymerase II / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication / Q000187:drug effects*; D008969:Molecular Sequence Data; D012316:RNA Nucleotidyltransferases / Q000037:antagonists & inhibitors*; D013950:Thymus Gland / Q000201:enzymology; D001084:Vidarabine Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology
D001084:Vidarabine Phosphate; C000101:ara-ATP; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II