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.
Footnotes
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Send reprint requests to: Dr. Yung-Chi Cheng, Department of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. E-mail: cheng@lab{at}yale.edu
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This work was supported by National Institutes of Health Grants CA-63477 and AI-42157.
- Abbreviations:
- HBV
- human hepatitis B virus
- RT
- reverse transcriptase
- dNTPs
- 2′-deoxynucleoside 5′-triphosphates
- β-l-FddCMP
- 5-fluoro-analog of β-l-ddCMP
- β-l-Fd4CMP
- 2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine 5′-monophosphate
- β-l-SddCMP
- β-l-2′,3′-dideoxy-3′-thiocytidine 5′-monophosphate
- β-l-OddCMP
- 2′,3′-dideoxy-3′dioxolane-cytidine 5′-monophosphate
- β-d-d4TMP
- 2′,3′-dideoxy-2′,3′-didehydrothymidine 5′-monophosphate
- PAGE
- polyacrylamide gel electrophoresis
- Received September 30, 1999.
- Accepted January 11, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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