Abstract
5'-Triphosphates of beta-D and beta-L-enantiomers of 2',3'-dideoxycytidine (ddC), 2',3'-dideoxy-5-fluorocytidine (FddC), 1,3-dioxolane-cytidine (OddC), and 1,3-dioxolane-5-fluorocytidine (FOddC) were evaluated as inhibitors and substrates for human DNA polymerases alpha, beta, gamma, delta, and epsilon. L-ddCTP was not a substrate or inhibitor for any DNA polymerase studied; L-FddCTP was not an inhibitor or substrate for replicative DNA polymerases and was a less potent inhibitor of DNA polymerases gamma and beta than its D-enantiomer by 2 orders of magnitude. In contrast, all L-dioxolane analogs were potent inhibitors and chain terminators for all cellular DNA polymerases studied. The Ki values of their 5'-triphosphates for DNA polymerase gamma were found to be in the following order: D-ddC < D-FddC L-OddC D-FOddC < L-FOddC << L-FddC. The Ki values of L-OddCTP for the reactions catalyzed by DNA polymerases alpha, delta, epsilon, beta, and gamma were 6.0, 1.9, 0.4, 3.0, and 0.014 microM, respectively, and those of L-FOddCTP were 6.5, 1.9, 0.7, 19, and 0.06 microM, respectively. The Km values for incorporation of L-OddCTP into the standing points of primer extension were also evaluated and determined to be 1.3, 3.5, 1.5, 2.8, and 0.7 microM for DNA polymerases alpha, delta, epsilon, beta, and gamma, respectively. The incorporation of dioxolane analogs into DNA by replicative DNA polymerases could explain their potent cellular toxicity.
Highlights
5-Triphosphates of -D and -L-enantiomers of 2,3dideoxycytidine, 2,3-dideoxy-5-fluorocytidine (FddC), 1,3-dioxolane-cytidine (OddC), and 1,3-dioxolane-5-fluorocytidine (FOddC) were evaluated as inhibitors and substrates for human DNA polymerases ␣, , ␥, ␦, and ⑀
The results obtained for D-ddCTP and D-FddCTP were similar to those published previously [29, 30]. pol ␥ was much more sensitive than any other polymerase to all compounds tested with the exception of L-ddCTP
The results obtained for D-ddCTP were similar to those previously described [23]
Summary
Materials and Compounds—L- and D-stereoisomers of ddC and OddC and their 5-fluoro-derivatives were synthesized as described previously [11, 13]. The primer oligonucleotides were labeled at the 5Ј position with T4 polynucleotide kinase using [␥-32P]ATP, annealed to M13 phage ssDNA as described in Ref. 16, purified on a Sephadex G-25 column, and used as substrates for elongation reactions. The ability to use poly(rA)1⁄7oligo(dT) as template and primer in the presence of 80 mM KCl at pH 7.5– 8.0 and high sensitivity to ddNTP indicated that the DNA polymerase activity was that of pol ␥ [17,18,19]. Pol ␣ was very sensitive to the inhibitor BuAdATP, displaying 25% residual activity in the presence of 5 M of this analog. Incorporation of one template-complementary nucleotide residue into the 3Ј-terminus of the primer was carried out in 8 l of a mixture containing the appropriate buffer solution as described above, enzyme, 0.01 M primer-template complex, and the analog under investigation. Complexes of M13 phage DNA and primer used in this study were as follows
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