Excision of -D- and -L-Nucleotide Analogs from DNA by the Human Cytosolic 3'-to-5' Exonuclease HELENE PELICANO, MARINA KUKHANOVA, and YUNG-CHI CHENG Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut Received September 30, 1999; accepted January 11, 2000 This paper is available online at http://www.molpharm.org ABSTRACT The cytosolic 3'-to-5' exonuclease from chronic lymphocytic leukemia cells was highly purified, and its ability to remove -D- and -L-nucleotide analogs from the 3'-end of DNA was deter- mined. The relative rate of excision of -D-ddCMP, -L-ddCMP, -L-FddCMP, -L-SddCMP, -L-Fd4CMP, and -L-OddCMP from the 3'-end of a single-stranded oligonucleotide primer or a primer annealed with complementary DNA and/or RNA tem- plates was assessed. The rate of excision of -D-nucleotides from the 3'-end of DNA was higher than that of -L-nucleotides, which could be partly attributable to the affinity of the enzyme to -D-nucleotide-terminated DNA being 5-fold higher com- pared with that of -L-nucleotide-terminated DNA. The rate of removal of -L-Fd4CMP and -L-OddCMP from the 3'-end of DNA was at least 8 to 10 times lower compared with that of -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, -L-Fd4CMP appeared to be the most effective inhibitor of the cytosolic exonuclease, with an ID 50 value of 38 M. Dideoxynucleoside analogs in both -D and -L configura- tion have been shown to possess the activity against HIV and human hepatitis B virus (HBV) (Mitsuya et al., 1985; Belleau et al., 1989; Doong et al., 1991; Chang et al., 1992, 1993; Furman et al., 1992; Lin et al., 1994a,b, 1995; Zhu et al., 1998). The antiviral activity of nucleoside analogs is attrib- utable to the preferential incorporation of their triphosphate forms into the 3'-end of viral DNA by viral reverse transcrip- tase (RT), which results in termination of viral DNA synthe- sis. The incorporation of nucleoside analogs into the 3'-end of DNA is critical for antiviral activity; therefore, the excision of these nucleoside analogs from DNA by 3'-to-5' exonucleases may decrease their potential for chain termination and may reverse the action of these nucleoside analogs. Several years ago, a 3'-to-5' exonuclease from the cytosol of human acute lymphoblastic leukemia H9 cells was partly purified in this laboratory (Skalski et al., 1993, 1995). The removal of nucleotide analogs with the natural -D configu- ration from the 3'-end of DNA was shown to occur much faster than that of the -L-nucleotide analogs. These results could explain in part higher efficiency of action of -L-SddC (3TC), -L-ddC, and -L-FddC against HIV reproduction com- pared with their -D- counterpart. We continue to investigate the human cytosolic 3'-to-5' exonuclease and its role in the removal of -L- and -D- nucleotide analogs from the 3'-end of DNA. Special attention was focused on the excision of -L-Fd4CMP and -L-Odd- CMP. As was shown earlier, both -L-OddC and -L-Fd4C were even more potent inhibitors of HIV and HBV replication compared with -L-SddC in cell culture and in vivo (Lee et al., 1995; Lin et al., 1996; Dutschman et al., 1998). The time to reappearance of HBV DNA after removal of -L-Fd4C from cell culture is longer than that of -L-SddC (Zhu et al., 1998). It was not clear how efficiently -L-Fd4CMP or -L-OddCMP can be removed from the 3'-end of DNA by the cytosolic exonuclease once they were incorporated into the 3' termini. In this study, we have highly purified the 3'-to-5' exonu- clease from acute lymphocytic leukemia cells of patients in leukemic blast crisis after leukophoresis. The relative rate of excision of -L-OddCMP and -L-Fd4CMP from the 3'-end of DNA was compared with that of -D-ddCMP, -L-ddCMP, -L-FddCMP, and -L-SddCMP. We also evaluated the affin- ity of the enzyme to DNA terminated with -L- and -D- nucleotide analogs. In addition, the susceptibility of the ex- onuclease to monophosphate metabolites of -L- and -D- nucleoside analogs was also assessed. 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; -L-FddCMP, 5-fluoro- analog of -L-ddCMP; -L-Fd4CMP, 2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine 5'-monophosphate; -L-SddCMP, -L-2',3'-dideoxy-3'- thiocytidine 5'-monophosphate; -L-OddCMP, 2',3'-dideoxy-3'dioxolane-cytidine 5'-monophosphate; -D-d4TMP, 2',3'-dideoxy-2',3'-didehy- drothymidine 5'-monophosphate; PAGE, polyacrylamide gel electrophoresis. 0026-895X/00/051051-05$3.00/0 MOLECULAR PHARMACOLOGY Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics MOL 57:1051–1055, 2000 /13006/817851 1051 at ASPET Journals on June 16, 2016 molpharm.aspetjournals.org Downloaded from