A quantitative assay for telomere protection in Saccharomyces cerevisiae.
Telomeres are the protective ends of linear chromosomes. Telomeric components have been identified and described by their abilities to bind telomeric DNA, affect telomere repeat length, participate in telomeric DNA replication, or modulate transcriptional silencing of telomere-adjacent genes; however, their roles in chromosome end protection are not as well defined. We have developed a genetic, quantitative assay in Saccharomyces cerevisiae to measure whether various telomeric components protect chromosome ends from homologous recombination. This "chromosomal cap" assay has revealed that the telomeric end-binding proteins, Cdc13p and Ku, both protect the chromosome end from homologous recombination, as does the ATM-related kinase, Tel1p. We propose that Cdc13p and Ku structurally inhibit recombination at telomeres and that Tel1p regulates the chromosomal cap, acting through Cdc13p. Analysis with recombination mutants indicated that telomeric homologous recombination events proceeded by different mechanisms, depending on which capping component was compromised. Furthermore, we found that neither telomere repeat length nor telomeric silencing correlated with chromosomal capping efficiency. This capping assay provides a sensitive in vivo approach for identifying the components of chromosome ends and the mechanisms by which they are protected.
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, Seattle, WA 98109-1024, USA.
Michelle L
ML
DuBois
Zara W
ZW
Haimberger
Martin W
MW
McIntosh
Daniel E
DE
Gottschling
D015825:Chromosomes, Fungal / Q000235:genetics; D017931:DNA Primers; D004271:DNA, Fungal / Q000235:genetics; D005838:Genotype; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016615:Telomere / Q000235:genetics*
D017931:DNA Primers; D004271:DNA, Fungal