EXCISION OF 2',2'-DIFLUORODEOXYCYTIDINE (GEMCITABINE) MONOPHOSPHATE RESIDUES FROM DNA

The activity of gemcitabine (dFdC), an effective agent against solid t umors, depends on the incorporation of its triphosphate into DNA. In v itro investigations demonstrated that, depending on the sequence of te mplate DNA, polymerases may pause after incorporation of gemcitabine n ucleotide at either the 3'-terminal or 3'-penultimate position. Proofr eading enzymes such as 3'-->5' exonucleases, which are associated with DNA polymerases, can excise mismatched deoxynucleotides from DNA. To model this reaction, we evaluated excision of the gemcitabine nucleoti de from oligodeoxynucleotide (19-mer) containing 3'-penultimate dFdC m onophosphate (dFdCMP) or dCMP by the 3'-->5' exonuclease of the Klenow fragment. The rate of excision of the 3'-terminal deoxynucleotide was similar, with both primers resulting in formation of primers with ter minal dCMP or dFdCMP. The primer containing dCMP was further excised, and by 40 min, more than 75% of total radioactivity was in excision pr oducts smaller than 18-mer. In contrast, most of the primers (90%) wit h terminal dFdCMP were unexcised. When primers terminated with either dFdCMP or dCMP were used as substrates, normal primer was hydrolyzed a lmost completely by 20 min; however, only 40% of primers containing dF dCMP had excision of dFdCMP molecule. Kinetic studies demonstrated tha t the enzyme had similar affinity for primers containing penultimate o r terminal dPdCMP, but the apparent V-max for excision was 4-5-fold gr eater for removal of a 3'-terminal deoxynucleotide than for cleavage o f a dFdCMP molecule. Reaction conditions that permitted polymerization of one deoxynucleotide to primers containing either 3'-penultimate dC MP or dFdCMP were used to evaluate excision during DNA synthesis. The excised primers could not be extended because the reaction lacked the requisite deoxynucleotide triphosphate. After 5 min, more than one-hal f of the dCMP primers were extended, whereas only 15% had been excised . In comparison, 30% of the analogue-containing primers lost the termi nal deoxynucleotide, with a proportional lower incidence of extension (30%). Lesser excision of dFdCMP-containing substrate was observed in reactions containing deoxynucleotide triphosphates required to make fu ll-length products. Consistent with this result, in the absence of 3'- ->5' exonuclease activity, both primers were extended similarly by the polymerization unit of the Klenow fragment. Taken together, these dat a demonstrate that dFdCMP residues are difficult to excise from DNA, a nd DNA polymerase can extend primers with 3'-dFdCMP. This results in t he internal incorporation of dFdCMP into DNA, as observed in whole cel ls.