TY - JOUR
T1 - Inhibition of the 3' → 5' exonuclease of human DNA polymerase ε by fludarabine-terminated DNA
AU - Kamiya, Ken Ichi
AU - Huang, Peng
AU - Plunkett, William
PY - 1996
Y1 - 1996
N2 - Incorporation of the anticancer drug fludarabine (9-β-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 ε 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 ε 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 ε has a higher affinity for F-ara-AMP-terminated DNA (K(m) = 7.1 pM) than for dAMP- terminated DNA of otherwise identical sequence (K(m) = 265 pM), excision of F-ara-AMP proceeded at a substantially slower rate (V(max) = 0.053 pmol/min/mg) than for 3'-terminal dAMP (V(max) = 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 ε, 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 ε to the reaction. Furthermore, the addition of DNA polymerase α and deoxynucleoside triphosphates to the excision reaction failed to extend the oligomers. After DNA polymerase ε 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 ε 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'.
AB - Incorporation of the anticancer drug fludarabine (9-β-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 ε 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 ε 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 ε has a higher affinity for F-ara-AMP-terminated DNA (K(m) = 7.1 pM) than for dAMP- terminated DNA of otherwise identical sequence (K(m) = 265 pM), excision of F-ara-AMP proceeded at a substantially slower rate (V(max) = 0.053 pmol/min/mg) than for 3'-terminal dAMP (V(max) = 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 ε, 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 ε to the reaction. Furthermore, the addition of DNA polymerase α and deoxynucleoside triphosphates to the excision reaction failed to extend the oligomers. After DNA polymerase ε 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 ε 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'.
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U2 - 10.1074/jbc.271.32.19428
DO - 10.1074/jbc.271.32.19428
M3 - Article
C2 - 8702631
AN - SCOPUS:0029666487
SN - 0021-9258
VL - 271
SP - 19428
EP - 19435
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 32
ER -