Excision of β-D- and β-L-nucleotide analogs from DNA by the human cytosolic 3'-to-5' exonuclease

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.