Incorporation of the anticancer
drug fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate;
F-ara-AMP) into the 3'-end of
DNA during replication causes termination of
DNA strand elongation and is strongly correlated with loss of clonogenicity. Because the proofreading mechanisms that remove 3'-F-ara-AMP from
DNA represent a possible means of resistance to the
drug, the present study investigated the excision of incorporated
F-ara-AMP from
DNA by the 3' -->
5'-exonuclease activity of
DNA polymerase epsilon from human
leukemia CEM cells. Using the
drug-containing and normal deoxynucleotide oligomers (21-base) annealed to M13mp18(+)
DNA as the excision substrates, we demonstrated that
DNA polymerase epsilon was unable to effectively remove
F-ara-AMP from the 3'-end of the oligomer. However, 3'-terminal dAMP and subsequently other deoxynucleotides were readily excised from
DNA in a distributive fashion. Kinetic evaluation demonstrated that although
DNA polymerase epsilon has a higher affinity for
F-ara-AMP-terminated
DNA (Km = 7.1 pM) than for dAMP-terminated
DNA of otherwise identical sequence (Km = 265 pM), excision of
F-ara-AMP proceeded at a substantially slower rate (Vmax = 0.053 pmol/min/mg) than for 3'-terminal dAMP (Vmax = 1.96 pmol/min/mg). When the 3'-5' phosphodiester bond between
F-ara-AMP at the 3'-terminus and the adjacent normal deoxynucleotide was cleaved by
DNA polymerase epsilon, the reaction products appeared to remain associated with the
enzyme but without the formation of a covalent bond. No further excision of the remaining oligomers was observed after the addition of fresh
DNA polymerase epsilon to the reaction. Furthermore, the addition of
DNA polymerase alpha and deoxynucleoside triphosphates to the excision reaction failed to extend the oligomers. After
DNA polymerase epsilon had been incubated with 3'-F-ara-AMP-21-mer for 10 min, the
enzyme was no longer able to excise 3'-terminal dAMP from a freshly added normal 21-mer annealed to M13mp18(+) template. We conclude that the
3' --> 5' exonuclease of human
DNA polymerase epsilon can remove 3'-terminal
F-ara-AMP from
DNA with difficulty and that this excision results in a mechanism-mediated formation of "dead end complex."