Cytarabine is a potent anticancer drug that interferes with elongation of the 
lagging strand at the replication fork during DNA synthesis. The effects of 
cytarabine substitution on the structural and thermodynamic properties of a 
model Okazaki fragment were investigated using UV hyperchromicity and 1H NMR 
spectroscopy to determine how cytarabine alters the physicochemical properties 
of Okazaki fragments that are intermediates during DNA replication. Two model 
Okazaki fragments were prepared 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 with 
its complementary (DNA) strand. The second model Okazaki fragment was identical 
to the first with the exception of cytarabine substitution for deoxycytidine at 
the third DNA nucleotide of the hybrid strand. Thermodynamic parameters for the 
duplex to single strand transition for each model Okazaki fragment were 
calculated from the concentration dependence of the T m at 260 nm. Cytarabine 
significantly decreased the stability of this model Okazaki fragment, decreasing 
the melting temperature 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 fragment relative 
to the control at 37 degrees C. Analysis of the temperature dependence of the 
imino1H resonances for the two duplexes demonstrated that cytarabine 
specifically destabilized the DNA:DNA duplex portion 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 Okazaki fragments in vivo .