Cytarabine-induced destabilization of a model Okazaki fragment

Abstract

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 .

A Novel Role of DNA Polymerase η in Modulating Cellular Sensitivity to Chemotherapeutic Agents

“…It has been reported that internalized AraC or dFdC in DNA changes the structure and stability of DNA (23,24) and inhibits DNA replication (25). It was also shown that AraC within DNA templates can block the Klenow fragment of DNA pol I and T4 DNA polymerase (26).…”

Altered prepulse inhibition in rats treated prenatally with the antimitotic Ara-C: an animal model for sensorimotor gating deficits in schizophrenia

“…The major action of Ara-C is inhibition of DNA synthesis, so it is highly toxic for dividing cells (Sarraf et al 1993;Gmeiner et al 1998;Courtney and Coffey 1999). Although there is an extensive literature on the use of anti-mitotic drugs to study neurodevelopment and neurotoxicology, there are only a limited number of studies that have used anti-mitotics to develop animal models for schizophrenia.…”

Antimetabolite incorporation into DNA: Structural and thermodynamic basis for anticancer activity

“…Thermodynamic studies indicate nucleoside analogs having anticancer activity as a consequence of misincorporation into DNA may destabilize the duplex 2,21,46 while NMR structural studies indicate analog substitution causes local structural perturbations. 2,22,47 These data are useful in rationalizing how nucleoside analogs, such as dFdC, AraC, and FdU, perturb DNA-protein interactions in a way that leads to cell death.…”

“…Over the last several years, there has been increasing knowledge concerning how dFdCTP and AraCTP incorporation into DNA affects DNA structure and stability. 2,21,22 At the same time, the roles of specific proteins that interact with analog-substituted DNA in a different manner than native DNA have been described. 3,23 Collectively, these studies are providing insight into the structure-function relationships that are responsible for the anticancer activities resulting from incorporation of dFdC, AraC, and other antipyrimidines into DNA.…”

“…This model system has also been used to study the effects of AraC substitution on nucleic acid structure and stability. 21,22 The effects of dFdC to the NMR solution structure of the model Okazaki fragment were mainly limited to the site of substitution (dC 20 ). Perhaps the most significant effect of dFdC substitution to the structure of the model Okazaki fragment was the change in sugar pucker for dFdC relative to dC in the control sequence.…”