Abstract

The cytosolic 3′-to-5′ exonuclease from chronic lymphocytic leukemia cells was highly purified, and its ability to remove β-d- and β-l-nucleotide analogs from the 3′-end of DNA was determined. The relative rate of excision of β-d-ddCMP, β-l-ddCMP, β-l-FddCMP, β-l-SddCMP, β-l-Fd4CMP, and β-l-OddCMP from the 3′-end of a single-stranded oligonucleotide primer or a primer annealed with complementary DNA and/or RNA templates was assessed. The rate of excision of β-d-nucleotides from the 3′-end of DNA was higher than that of β-l-nucleotides, which could be partly attributable to the affinity of the enzyme to β-d-nucleotide-terminated DNA being 5-fold higher compared with that of β-l-nucleotide-terminated DNA. The rate of removal of β-l-Fd4CMP and β-l-OddCMP from the 3′-end of DNA was at least 8 to 10 times lower compared with that of β-l-SddCMP. HIV reverse transcriptase could elongate DNA primers after the removal of chain terminators by the cytosolic exonuclease. Concentrations of nucleoside 5′-monophosphate analogs that inhibit the cytosolic exonuclease by 50% were estimated. Among the nucleoside 5′-monophosphate analogs examined, β-l-Fd4CMP appeared to be the most effective inhibitor of the cytosolic exonuclease, with an ID₅₀ value of 38 μM.