Human telomerase catalyzes nucleolytic primer cleavage.
Telomerase is a reverse transcriptase that uses an integral RNA molecule to add de novo G-rich repeats onto telomeric DNA, or onto nontelomeric DNA generated during chromosome fragmentation and breakage events. A telomerase-mediated DNA substrate cleavage activity has been reported in ciliates and yeasts. Nucleolytic cleavage may serve a proofreading function, enhance processivity or ensure that nontemplate telomerase RNA sequences are not copied into DNA. We identified and characterized a human telomerase-mediated nucleolytic cleavage activity using enzyme reconstituted in a rabbit reticulocyte lysate in vitro transcription/translation system and native enzyme extracted from cells. We found that telomerase catalyzed the removal of nucleotides from DNA substrates including those that can form a mismatch with the RNA template or that contain nontelomeric sequences located 3' to a telomeric sequence. Unlike Tetrahymena telomerase, human telomerase catalyzed the removal of more than one nucleotide (up to 13) from telomeric primers. DNA substrates predicted to align at the 3'-end of the RNA template were not cleaved, consistent with cleavage being dictated by the template 5'-end. We also found some differences in the nuclease activity between RRL-reconstituted human telomerase and native enzyme.
Huard
Sylvain
S
Department of Anatomy and Cell Biology, McGill University, Montréal, Québec H3A 2B4, Canada.
Autexier
Chantal
C
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D002474:Cell-Free System; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009711:Nucleotides / Q000378:metabolism*; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity; D019098:Telomerase / Q000302:isolation & purification / Q000378:metabolism*; D016615:Telomere / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D017931:DNA Primers; D009711:Nucleotides; D012313:RNA; D004247:DNA; D019098:Telomerase