Mutually Exclusive Binding of Telomerase RNA and DNA by Ku Alters Telomerase Recruitment Model

• Published in: Cell Vol. 148; no. 5; pp. 922 - 932
• Main Authors: Pfingsten, Jennifer S., Goodrich, Karen J., Taabazuing, Cornelius, Ouenzar, Faissal, Chartrand, Pascal, Cech, Thomas R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.03.2012
• Subjects: Base Sequence; Brain-derived neurotrophic factor; Chromatin; Chromosomes; Data processing; Dendrites; DNA; DNA End-Joining Repair; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Gene deletion; Gene silencing; miRNA; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; mutants; Neurons; Non-homologous end joining; Post-transcription; Protein biosynthesis; RNA; RNA - metabolism; RNA processing; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Sequence Deletion; Telomerase; Telomerase - chemistry; Telomerase - metabolism; Telomere - genetics; Telomere - metabolism; Telomeres; Transcription; Translation
• ISSN: 0092-8674; 1097-4172; 1097-4172
• DOI: 10.1016/j.cell.2012.01.033

In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes Ku's telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt nonhomologous end-joining (NHEJ), telomeric gene silencing, and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in vitro and in vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. [Display omitted] [Display omitted] ► Yeast Ku's telomeric functions require its DNA-binding ring ► Ku mutation that inhibits DNA binding unexpectedly inhibits telomerase RNA binding ► Competition experiments show mutually exclusive binding of DNA and RNA to Ku ► Supports revision of the current model for recruitment of telomerase to telomeres by Ku The DNA-repair protein Ku binds to the telomerase RNA and helps recruit telomerase to chromosome ends; however, Ku cannot bind DNA and telomerase RNA simultaneously. DNA displaces Ku from telomerase, suggesting a reconsideration of the current model for Ku recruitment of telomerase.