The Most Efficient Binding Substrate for TelN is tos

Telomere resolution can be dissected into several steps. Prior to processing, the protelomerases have to recognize and bind their target DNAs. In order to study the recognition and binding characteristics of TelN, the binding and processing steps were uncoupled by use of a TelN mutant protein (TelNY424F), in which the proposed active tyrosyl residue Tyr-424 was replaced by a phenylalanine (Deneke et al. 2002). Preceding experiments had shown that TelNY424F lost its cleaving-joining activity but retained its specific DNA binding ability (Deneke et al. 2000). The modified TelN protein was incubated with either tos, telRL or telO followed by electron microscopic analysis of the complexes. Site-specific complex formation was observed with tos and telRL only while telO failed to yield specific complexes. Symmetric stepwise reduction of the 56-bp telRL sequence resulted in a gradual loss of DNA binding and at a substrate size of 36 bp, specific binding was no longer observed. Furthermore, the removal of one arm (R3) of the inverted repeat flanking telO abolished the sequence-specific binding (Fig. 4). Thus, the repetitive sequences L3 and R3 surrounding the telO palindrome in telRL are essential for TelN binding. This was confirmed by fragment retention experiments. On polyacrylamide gels, target fragments containing tos or telRL were bound by TelN resulting in decreased electrophoretic mobility. By contrast, no retention was observed with telO. The apparent equilibrium dissociation constant value (kd(app)) for telO was the same as that for vector DNA. Besides the specific binding capacity, TelN also binds DNA in a non-sequence-specific way.

Interestingly, there was an almost fivefold increase in stability of the TelN-tos complexes compared with telRL complexes. This finding indicates that the additional repeats L1/R1 and L2/R2 present in tos are not vital for binding and for processing of the substrate in vitro but enhance the stability of the complexes (Fig. 4). The authors of this study speculate that the aforementioned repeats might serve as association points where TelN preferably binds and slides along the DNA until it hits telRL, where stable complex formation takes place. The sets of repeats within tos may also favour a structural alteration at telRL that improves DNA-TelN interactions to yield more stable complexes. Hertwig et al. (2003a) studied the processing of the PY54 palindrome in Yersinia. They ascertained that a plasmid harbouring the 42-bp palindrome was not linearized upon infection with PY54. However, a construct comprising the palindrome and flanking sequences including the 15-bp inverted repeat was clearly processed (Figs. 4 and 6). This observation suggests that the additional 15-bp repeat is essential for processing under in vivo conditions, and raises the question whether the repeats L1/R1 and L2/R2 present in tos are also required for telomere resolution in vivo.

Fig. 6 In vivo linearization of the PY54 telRL site occurs only in the presence of its flanking inverted repeat (IR). Y. enterocolitica strains harbouring only pBR329 or pBR329 with either telRL or telRL and IR were infected by PY54. Plasmids were analysed on a 0.8% agarose gel after 10 min, 1 h or 6 h of infection. Lanes 1 and 19, size marker (X Eco130I); lane 2, pBR329/HindIII; lanes 3-5, pBR329 isolated from an infected Yersinia strain; lanes 6-8, pBR329 with telRL isolated from a non-infected Yersinia strain; lanes 9-11, same as before, but from an infected Yersinia strain; lanes 12-14, pBR329 with telRL and IR isolated from a non-infected Yersinia strain; lanes 15-17, same as before, but from an infected Yersinia strain; lane 18, same construct as before, digested with HindIII. ccc, covalently closed circle; oc, open circle (Hertwig et al. 2003)

Fig. 6 In vivo linearization of the PY54 telRL site occurs only in the presence of its flanking inverted repeat (IR). Y. enterocolitica strains harbouring only pBR329 or pBR329 with either telRL or telRL and IR were infected by PY54. Plasmids were analysed on a 0.8% agarose gel after 10 min, 1 h or 6 h of infection. Lanes 1 and 19, size marker (X Eco130I); lane 2, pBR329/HindIII; lanes 3-5, pBR329 isolated from an infected Yersinia strain; lanes 6-8, pBR329 with telRL isolated from a non-infected Yersinia strain; lanes 9-11, same as before, but from an infected Yersinia strain; lanes 12-14, pBR329 with telRL and IR isolated from a non-infected Yersinia strain; lanes 15-17, same as before, but from an infected Yersinia strain; lane 18, same construct as before, digested with HindIII. ccc, covalently closed circle; oc, open circle (Hertwig et al. 2003)

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