Telomere formation by Rap1p binding site arrays reveals end-specific length regulation requirements and active telomeric recombination

Rap1p, the major telomere repeat binding protein in yeast, has been implica ted in both de novo telomere formation and telomere length regulation. To c haracterize the role of Rap1p in these processes in more detail, we studied the generation of telomeres in vivo from linear DNA substrates containing defined arrays of Rap1p binding sites. Consistent with previous work, our r esults indicate that synthetic Rap1p binding sites within the internal half of a telomeric array are recognized as an integral part of the telomere co mplex in an orientation-independent manner that is largely insensitive to t he precise spacing between adjacent sites. By extending the lengths of thes e constructs, we found that several different Rap1p site arrays could never be found at the very distal end of a telomere, even when correctly oriente d. Instead, these synthetic arrays were always followed by a short (approxi mate to 100-bp) "cap" of genuine TG repeat sequence, indicating a remarkabl y strict sequence requirement for an end-specific function(s) of the telome re. Despite this fact, even misoriented Rap1p site arrays promote telomere formation when they are placed at the distal end of a telomere-healing subs trate, provided that at least a single correctly oriented site is present w ithin the array. Surprisingly, these heterogeneous arrays of Rap1p binding sites generate telomeres through a RAD52-dependent fusion resolution reacti on that results in an inversion of the original array. Our results provide new insights into the nature of telomere end capping and reveal one way by which recombination can resolve a defect in this process.