PROCESSING OF NONTELOMERIC 3' ENDS BY TELOMERASE - DEFAULT TEMPLATE ALIGNMENT AND ENDONUCLEOLYTIC CLEAVAGE

Telomerase is a specialized reverse transcriptase that maintains telom eres at chromosome ends by extending preexisting tracts of telomeric D NA and forming telomeres de novo an broken chromosomes. Whereas the in teraction of telomerase with telomeric DNA has been studied in some de tail, relatively little is known about how this enzyme processes nonte lomeric DNA. In this study we recruited the Euplotes telomerase to non telomeric 3' termini in vitro using chimeric DNA primers that carried one repeat of a telomeric sequence at various positions upstream of a nontelomeric 3' end. Such primers were processed in two distinct pathw ays. First, nontelomeric 3' ends could be elongated directly by positi oning a primer terminus at a specific site on the RNA template. Delive ry to this default site was precise, always resulting in the addition of 4 dG residues' to the nontelomeric 3' ends. These same residues ini tiate new telomeres formed in vivo. Alternatively, 3' nontelomeric nuc leotides were removed from primers prior to initiating the first elong ation cycle. As with default positioning df nontelomeric 3' ends, the cleavage event was' extremely precise and was followed by the addition of dG residues to the primer 3' ends. The specificity of the cleavage reaction was mediated by primer interaction with the RNA template and , remarkably, proceeded by an endonucleolytic mechanism. These observa tions suggest a mechanism for the precision of developmentally regulat ed de nova telomere formation and expand our understanding bf the enzy matic properties of telomerase.