Telomere Dynamics and End Processing in Mammalian Cells
Sfeir, Agnel J.
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Telomeres are repetitive DNA sequences that end in single-stranded 3' overhangs. With each cell division, normal human cells lose a small amount of telomeric DNA due to the end-replication problem and the action of an unidentified nuclease. In order for tumor cells to divide indefinitely, they maintain telomere length by expressing the enzyme telomerase. The end structure of mammalian telomeres is not very well understood. Two assays were developed using ligation and PCR amplification to identify the terminal nucleotides of both the C-rich and G-rich telomeric strands in human cells. The results showed that ~ 80 % of the C-strands terminate precisely in ATC-5', demonstrating that the nuclease resection of the C-strand post replication is specific for a single nucleotide. In contrast, the last base of the G-strand in normal human cells was less precise with 70% of the ends being TAG-3', TTA-3' or GTT-3'. An enrichment for the TAG-3' end was noted in cells that express telomerase. A series of nucleases were tested for their involvement in specifying the last base of C-strands and the results indicated that none of those nucleases were responsible for telomere-end resection. Inhibiting the normal function of most telomere binding proteins altered normal telomere function, however only one protein (POT-1) influenced last base specificity. Knocking down POT-1 in normal and tumor cells randomized the last base of the C-strand. These finding have important implications for the processing events that act on the telomere ends and they will help identify the nuclease that resects the chromosome ends. In the second part of this study, the dynamics of telomerase action in mammalian cells was examined. Using a PCR-based, single telomere-length measurement assay (STELA) we showed that telomerase adds an average of 250-nucleotides per end in one replication cycle. Cell cycle studies showed that while the telomeres on the Xp chromosome replicated in early S-phase their elongation by telomerase took place during late S/early G2 phase. Therefore, in mammalian cells telomerase action is not coupled to DNA replication. These studies will provide much needed information for exploiting our knowledge of telomere biology for telomerase-based therapeutic purposes.