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Effects of telomerase on DNA replication
Update time:2018-09-13 02:24:24   【 Font: Large  Medium Small

    Telomerases have been most thoroughly studied in ciliated protozoa, which have macronuclei containing large numbers of telomerase molecules. However, the ciliates have the disadvantage of not being easy to manipulate genetically. In contrast, S. cerevisiae have low levels of telomerase activity but are amenable to genetic studies.

    Telomerases have been purified from two ciliates, Tetrabymena thermopbilia and Euplotes aediculatus. The enzyme is assayed by incubating a synthetic DNA primer such as d (TTGGGG)3 to mimic the end of a chromosome with [ 32p] GTP and TTP. The progress of the reaction can be monitored by following radioactive incorporation into acid precipitable material. Alternatively, labeled strands can be separated by gelelectrophoresis. When this separation is performed, hundreds of d(TTGGGG) repeats are added to the primer.

    Telomerase cannot extend a DNA chain from a blunt end, creating a problem for leading strand replication. It shows one hypothesis that has been proposed to solve the leading strand problem. According to this hypothesis, (1) a strand-specific 5'→3'exonuclease removes nucleotides from the 5'-end of the C-rich strand to generate a G-rich 3'-overhang; (2) telomerase extends the 3'-overhang generated by the exonuclease (3) the newly synthesized region serves as a template for standard semiconservative replication. restoring the 5'-end of C-rich strand.

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