Inhibition of DNA primase by nucleoside triphosphates and their arabinofuranosyl analogs

Abstract

DNA primase (EC 2.7.7.6) produces an RNA oligomer of approximately 10 bases, which is required by DNA polymerase α (EC 2.7.7.7) for the initiation of DNA synthesis. We partially purified DNA primase from acute lymphocytic leukemia cells from patients using several chromatography columns. Poly(dT) and poly(dC), but not poly(dA) or poly(dG), were good templates for ribonucleoside triphosphate (rNTP)-dependent DNA synthesis (i.e., DNA primase activity), and they were used in the study of the effect of natural and arabinosylfuranosyl nucleoside triphosphates on DNA primase activity. The K(m) for GTP in the poly(dC) primase assay was approximately 175 μM. All noncomplementary natural rNTPs and deoxyribonucleoside triphosphates (dNTPs) inhibited poly(dC) primase activity to a similar extent (K(i) values of ATP and CTP were 610 and 517 μM, respectively). 1-β-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) and 9-β-D-arabinofuranosyladenine 5'-triphosphate (araATP) were more potent inhibitors of poly(dC) primase activity than were CTP and ATP (K(i) values were approximately 125 μM). araCTP, araATP, CTP, and ATP inhibited DNA primase activity in a manner competitive with GTP. The concentration required to inhibit poly(dC) DNA primase activity by 50% was determined for a number of arabinofuranosyl nucleoside triphosphate analogs, and the relative potency of inhibition of DNA primase activity was as follows: rNTP = dNTP = 5-aza-dCTP < ara-5-azaCTP = araTTP = araATP = araCTP < 2-fluoro-araATP = 2'-azido-2'-deoxy-araCTP < 2'-fluoro-araTTP = 2'-fluoro-5-iodo-araCTP = 2'-fluoro-5-methyl-araCTP. In the poly(dT) primase assay ATP did not follow classic Michaelis-Menten kinetics (ATP exhibited positive cooperativity with a Hill coefficient of 2.0). However, this assay was very sensitive to araCTP (apparent K(i) of 25 μM). In summary, these experiments suggested that DNA primase is controlled by the levels of ribonucleoside triphosphates, and that the perturbation of these pools by any agent could lead to the inhibition of DNA primase and thereby inhibit DNA synthesis. Furthermore, aranucleoside triphosphate analogs directly inhibited DNA primase, and it is possible that this effect may contribute to the cytotoxicity of these compounds.