Nucleic Acids Research, Vol 26, Issue 10 2359-2365, Copyright © 1998 by Oxford University Press
WH Gmeiner, A Skradis, RT Pon and J Liu
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 .
ARTICLES
Cytarabine-induced destabilization of a model Okazaki fragment
Eppley Institute and Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA. bgmeiner@unmc.edu
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