2--deoxy-7-deazaguanosine-triphosphate and 2--deoxyadenosine-triphosphate

DNA polymerases recognize their substrates with exceptionally high specificity, restricting the use of unnatural nucleotides and the applications they enable. We describe a strategy to expand the substrate range of polymerases. By selecting for the extension of distorting 3' mismatches, we obtained mutants of Taq DNA polymerase that not only promiscuously extended mismatches, but had acquired a generic ability to process a diverse range of noncanonical substrates while maintaining high catalytic turnover, processivity and fidelity. Unlike the wild-type enzyme, they bypassed blocking lesions such as an abasic site, a thymidine dimer or the base analog 5-nitroindol and performed PCR amplification with complete substitution of all four nucleotide triphosphates with phosphorothioates or the substitution of one with the equivalent fluorescent dye-labeled nucleotide triphosphate. Such 'unfussy' polymerases have immediate utility, as we demonstrate by the generation of microarray probes with up to 20-fold brighter fluorescence.

Topics: Base Pair Mismatch; Biotin; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Deoxyribose; Deoxyuracil Nucleotides; Directed Molecular Evolution; DNA; DNA Mutational Analysis; DNA Probes; Fluorescein-5-isothiocyanate; Indoles; Kinetics; Microarray Analysis; Models, Molecular; Mutation; Point Mutation; Polymerase Chain Reaction; Pyrimidine Dimers; Rhodamines; Sequence Analysis, DNA; Substrate Specificity; Taq Polymerase; Thionucleotides

Telomeres play an important role in chromosome organization and stability. Human telomerase is a terminal transferase that adds TTAGGG units onto the telomere end. In general, telomerase activity is not detected in normal somatic cells but is present in immortalized cells. Consequently, telomerase might be a selective target for cancer chemotherapy. Using cell-free biochemical telomerase assay, we have found that 7-deaza-2'-deoxyguanosine-5'-triphosphate (7-deaza-dGTP) and 7-deaza-2'-deoxyadenosine-5'-triphosphate (7-deaza-dATP) were potent telomerase inhibitors. The concentrations of inhibitors in which 50% of the telomerase activity was inhibited (IC50 values) were 11 and 8 microM for 7-deaza-dGTP and 7-deaza-dATP, respectively. Additional studies show that both 7-deaza-dGTP and 7-deaza-dATP were also incorporated into telomeric DNA by telomerase. However, incorporation of 7-deaza-dATP or 7-deaza-dGTP results in a telomeric ladder that is prematurely shortened. No difference in the number or position of pause sites were observed when 7-deaza-dATP was compared to dATP as substrates. On the other hand, both a shift and an increase in pause sites was observed when dGTP was replaced by 7-deaza-dGTP. Incorporation of 7-deaza nucleotides by telomerase may be used as a tool for the study of telomerase mechanism and function. In addition, this may be a novel approach in the design of new telomerase inhibitors.

Topics: Cells, Cultured; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA Primers; Humans; Kidney; Oligodeoxyribonucleotides; Purine Nucleotides; Ribonuclease, Pancreatic; Telomerase