TelN Binds Covalently to the 3Phosphoryl of the Cleaved Strands

Processing of the telomere resolution site can be divided into three steps: (1) recognition and binding of the substrate, (2) cleavage at the target site, and (3) joining (transesterification) yielding a linear molecule with covalently closed ends. Following cleavage, a transient covalent intermediate between the protelomerase and its target is generated through nucleophilic substitution by the hydroxyl group of an amino acid (probably Tyr-424). Then the new phosphodiester bond with the complementary strand is constituted in a transesterification reaction, catalysed by the TelN-DNA complex.

At which end of the target is TelN bound upon cleavage? To answer this question, Deneke et al. (2002) applied a telRL substrate ((TA)7, see Fig. 8) that was cut but not joined by TelN. One of the products was isolated and labelled at its 3' or 5' ends. After cleavage with a restriction enzyme, the fragments were analysed by autoradiography. The 3' labelling showed only one fragment carrying the label, whereas two fragments were visible by labelling of the 5' ends. It can be concluded that one 3' end was decorated with a TelN molecule which prevented labelling. Huang et al. (2004) used suicide substrates with a nick for labelling. They could demonstrate that one protelomerase molecule is linked to the 3'-phosphoryl of nucleotide 25 on the top strand and a second one to the corresponding residue of nucleotide 32 on the bottom strand. Hairpin formation then occurs by ligation of the 3'-phosphoryl with a 5'-OH created by cleavage by the second protelomerase molecule.

The formation of 3' covalent protein-DNA intermediates is a hallmark of tyrosine recombinases and therefore protelomerases belong to this family of enzymes. The complex of protelomerase and its target site telRL has the potential to form a cruciform structure with two protruding stem-loops in which the cleavage and ligation sites are located at the base of the loops. Alternatively, a duplex structure with DNA in the Z conformation is conceivable (Deneke et al. 2000; Huang et al. 2004) (Fig. 7).

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