TelO is Essential for the Processing of the Telomere Resolution Site

At very high TelN concentrations (~ 50-fold higher than for telRL), telO is weakly processed suggesting that telO contains all the sequence requirements needed for telomere resolution in vitro (Deneke et al. 2002). For that reason telRL substrates harbouring mutations within telO that had been used for the binding studies were also investigated in terms of recognition and processing by the N15 protelomerase. It turned out that the AT^TA mutation within telO resulted in a complete loss of processing (Fig. 8). Similarly, the substrate containing a second AT^TA mutation, which restored the sequence symmetry of telO, could not be processed. The data make clear that the cen tral palindrome of telRL is of great importance in telomere resolution, and indicate that apparently both the sequence itself and the symmetry of the sequence are crucial for processing. This was confirmed with other substrates in which the sequence of telO was changed (Fig. 8). By stepwise reduction of one end of telO it could be demonstrated that the core of this sequence lies between the base pairs 19 and 38 (Fig. 8).

The importance of the integrity of the central telO sequences for protelom-erase activity was also reported by Huang et al. (2004) who used duplex oligonucleotides described in the previous section. The introduction of a single nick up to position 20 and beyond position 36 on the top strand of telRL did not affect protelomerase activity. By contrast, nicks introduced at locations between nucleotides 22 and 25 and between nucleotides 31 and 33 impeded the processing of the 28-bp (R3) half and 38-bp (L3) half of telRL, respectively, while the respective other half was processed. Interestingly, a nick placed between positions 21 and 22 and at the positions 34 and 35 completely abolished the protelomerase activity, indicating that these linkages are critical for protein-substrate interaction (Fig. 8). The telO sequence of N15 is also present in phage ^KO2, whereas flanking sequences exhibit some mismatches (Fig. 4). Nevertheless, the protelomerases and target sites of these phages are interchangeable, supporting the notion that the 16 nucleotides in the middle of telO are essential for processing. Huang et al. (2004) isolated the protelomerases of both N15 and ^KO2. The TelK enzyme cleaved its own target with the same efficiency as telRL of N15. The telomere resolution site of phage PY54 was not a suitable substrate for TelK. Figure 4 shows that ten nucleotides located around the centre of dyad symmetry within telO are identical in all three palindromes. However, the critical nucleotides mentioned above deviate in PY54. When the positions 15, 16, 27 and 28 of the PY54 palindrome were substituted by nucleotides matching the N15/^KO2 telO core sequence, telRL of PY54 was processed by TelK (Fig. 4). These data unequivocally demonstrate the impact of the central nucleotides of telO for telomere resolution.

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