CYTARABINE-INDUCED DESTABILIZATION OF A MODEL OKAZAKI FRAGMENT

Cytarabine is a potent anticancer drug that interferes with elongation of the lagging strand at the replication fork during DNA synthesis. T he effects of cytarabine substitution on the structural and thermodyna mic properties of a model Okazaki fragment were investigated using UV hyperchromicity and H-1 NMR spectroscopy to determine how cytarabine a lters the physicochemical properties of Okazaki fragments that are int ermediates during DNA repiication. Two model Okazaki fragments were pr epared corresponding to a primary initiation site for DNA replication in the SV40 viral genome. One model Okazaki fragment consisted of five ribo- and seven deoxyribonucleotides on the hybrid strand, together w ith its complementary (DNA) strand. The second model Okazaki fragment was identical to the first with the exception of cytarabine substituti on for deoxycytidine at the third DNA nucleotide of the hybrid strand. Thermodynamic parameters for the duplex to single strand transition f or each model Okazaki fragment were calculated from the concentration dependence of the T-m at 260 nm, Cytarabine significantly decreased th e stability of this model Okazaki fragment, decreasing the melting tem perature from 46.8 to 42.4 degrees C at a concentration of 1.33 x 10(- 5) M. The free energy for the duplex to single strand transition was 1 .2 kcal/mol less favorable for the cytarabine-substituted Okazaki frag ment relative to the control at 37 degrees C, Analysis of the temperat ure dependence of the imino H-1 resonances for the two duplexes demons trated that cytarabine specifically destabilized the DNA:DNA duplex po rtion of the model Okazaki fragment. These results are consistent with inhibition of lagging strand DNA synthesis by cytarabine substitution resulting from destabilization of the DNA:DNA duplex portion of Okaza ki fragments in vivo.