Excision of beta-D- and beta-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 beta-D- and beta-L-nucleotide analogs from the 3'-end of DNA was determined. The relative rate of excision of beta-D-ddCMP, beta-L-ddCMP, beta-L-FddCMP, beta-L-SddCMP, beta-L-Fd4CMP, and beta-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 beta-D-nucleotides from the 3'-end of DNA was higher than that of beta-L-nucleotides, which could be partly attributable to the affinity of the enzyme to beta-D-nucleotide-terminated DNA being 5-fold higher compared with that of beta-L-nucleotide-terminated DNA. The rate of removal of beta-L-Fd4CMP and beta-L-OddCMP from the 3'-end of DNA was at least 8 to 10 times lower compared with that of beta-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, beta-L-Fd4CMP appeared to be the most effective inhibitor of the cytosolic exonuclease, with an ID(50) value of 38 microM.
Pelicano
H
H
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA.
Kukhanova
M
M
Cheng
Y C
YC
D004247:DNA / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D054306:Dideoxynucleotides; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D006801:Humans; D009711:Nucleotides / Q000378:metabolism; D018119:Stavudine / Q000031:analogs & derivatives / Q000494:pharmacology; D013379:Substrate Specificity
C113124:2',3'-dideoxy-2',3'-didehydrothymidine monophosphate; D017931:DNA Primers; D054306:Dideoxynucleotides; D009711:Nucleotides; D004247:DNA; D018119:Stavudine; D054303:HIV Reverse Transcriptase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V