Processing of nontelomeric 3' ends by telomerase: default template alignment and endonucleolytic cleavage.
Telomerase is a specialized reverse transcriptase that maintains telomeres at chromosome ends by extending preexisting tracts of telomeric DNA and forming telomeres de novo on broken chromosomes. Whereas the interaction of telomerase with telomeric DNA has been studied in some detail, relatively little is known about how this enzyme processes nontelomeric DNA. In this study we recruited the Euplotes telomerase to nontelomeric 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 pathways. First, nontelomeric 3' ends could be elongated directly by positioning a primer terminus at a specific site on the RNA template. Delivery to this default site was precise, always resulting in the addition of 4 dG residues to the non-telomeric 3' ends. These same residues initiate new telomeres formed in vivo. Alternatively, 3' nontelomeric nucleotides were removed from primers prior to initiating the first elongation cycle. As with default positioning of 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 observations suggest a mechanism for the precision of developmentally regulated de novo telomere formation and expand our understanding of the enzymatic properties of telomerase.
Melek
M
M
Department of Biochemistry and Biophysics, Texas A&M University, College Station, 77843-2128, USA.
Greene
E C
EC
Shippen
D E
DE
D000818:Animals; D001483:Base Sequence; D002875:Chromosomes / Q000737:chemistry / Q000378:metabolism*; D016054:DNA, Protozoan / Q000096:biosynthesis* / Q000737:chemistry; D004720:Endonucleases / Q000378:metabolism; D016812:Euplotes / Q000235:genetics*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000737:chemistry / Q000378:metabolism*; D013698:Templates, Genetic
D016054:DNA, Protozoan; D009838:Oligodeoxyribonucleotides; D019098:Telomerase; D004720:Endonucleases