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Effect of a Template-located 2',2'-Difluorodeoxycytidine on the Kinetics and Fidelity of Base Insertion by Klenow (3'5'Exonuclease^-) Fragment¹

Carcinogenesis and Mutagenesis Laboratory, Toxicology Research Laboratories, Greenfield, Indiana 46140 [W. E. S., F. C. R.]; Cancer Research Division, Lilly Research Laboratories, a Division of Eli Lilly and Company, Indianapolis, Indiana [L. W. H., J. S. K.]; and Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-1340 [L. B. B., M, F. G.]

² To whom requests for reprints should be addressed.

Gemcitabine [2',2'-difluorodeoxycytidine (dFdCyd)], a potent antitumor agent, inhibits DNA synthesis and is incorporated internally into DNA. The effect of a template-incorporated dFdCyd molecule (dFdCyd-) on DNA polymerase function was examined. Two 25-base deoxyoligo-nucleotides were synthesized with either a single dFdCyd- or template-incorporated deoxycytidine molecule (dCyd-) at the same position. Each was annealed separately to an identical complementary 5'-³²P-labeled primer and extended by the Klenow fragment (3'5' exo^-) of DNA polymerase I. "Correct" insertion of dGMP was 80-fold less efficient opposite dFdCyd- than dCyd-. A comparison of misinsertion efficiencies opposite template dFdCyd gave values of 2.7 x 10^-2 for dAMP insertion, 1.1 x 10^-3 for dTMP insertion, and 5.9 x 10~4 for dCMP insertion. A similar measurement opposite template dC gave values of 1.8 x 10^-4, 1.7 x 10^-4, and 2.9 x 10^-6 for dAMP, dTMP, and dCMP insertion, respectively. Thus, the presence of dFdCyd on the template strand inhibited "normal" DNA synthesis and increased deoxyribonucleotide misinsertion frequencies. Pausing during DNA synthesis occurred directly opposite template dFd-Cyd suggesting that dFdC·dG base pairs might be less stable than normal dC·dG pairs, resulting in a decreased rate of primer extension beyond this site. Consistent with kinetic data, thermal denaturation measurements using comparable surrounding sequences showed that dFdC·dG "correct" pairs were less stable than dC·dG base pairs. Measurements on base mispairs showed that dFdC·dC was more stable than dC·dC, while no measurable T_m differences were found between polymers containing dFdC·dA and dC·dA or dFdC·dT, and dC·dT.

¹ This work was supported in part by NIH Grant GM21422.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3/15/93. Accepted 7/19/93.

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