A novel motif in telomerase reverse transcriptase regulates telomere repeat addition rate and processivity

Mingyi Xie, Joshua D. Podlevsky, Xiaodong Qi, Christopher J. Bley, Julian Chen

Research output: Contribution to journalArticle

48 Scopus citations

Abstract

Telomerase is a specialized reverse transcriptase that adds telomeric DNA repeats onto chromosome termini. Here, we characterize a new telomerase-specific motif, called motif 3, in the catalytic domain of telomerase reverse transcriptase, that is crucial for telomerase function and evolutionally conserved between vertebrates and ciliates. Comprehensive mutagenesis of motif 3 identified mutations that remarkably increase the rate or alter the processivity of telomere repeat addition. Notably, the rate and processivity of repeat addition are affected independently by separate motif 3 mutations. The processive telomerase action relies upon a template translocation mechanism whereby the RNA template and the telomeric DNA strand separate and realign between each repeat synthesis. By analyzing the mutant telomerases reconstituted in vitro and in cells, we show that the hyperactive mutants exhibit higher repeat addition rates and faster enzyme turnovers, suggesting higher rates of strand-separation during template translocation. In addition, the strong correlation between the processivity of the motif 3 mutants and their ability to use an 8 nt DNA primer, suggests that motif 3 facilitates realignment between the telomeric DNA and the template RNA following strand-separation. These findings support motif 3 as a key determinant for telomerase activity and processivity.

Original languageEnglish (US)
Article numbergkp1198
Pages (from-to)1982-1996
Number of pages15
JournalNucleic acids research
Volume38
Issue number6
DOIs
StatePublished - Dec 30 2009

ASJC Scopus subject areas

  • Genetics

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