Abstract
Genome-wide systematic screens in yeast have uncovered a large gene network (the telomere length maintenance network or TLM), encompassing more than 400 genes, which acts coordinatively to maintain telomere length. Identifying the genes was an important first stage; the next challenge is to decipher their mechanism of action and to organize then into functional groups or pathways. Here we present a new telomere-length measuring program, TelQuant, and a novel assay, telomere length kinetics assay, and use them to organize tlm mutants into functional classes. Our results show that a mutant defective for the relatively unknown MET7 gene has the same telomeric kinetics as mutants defective for the ribonucleotide reductase subunit Rnr1, in charge of the limiting step in dNTP synthesis, or for the Ku heterodimer, a well-established telomere complex. We confirm the epistatic relationship between the mutants and show that physical interactions exist between Rnr1 and Met7. We also show that Met7 and the Ku heterodimer affect dNTP formation, and play a role in non-homologous end joining. Thus, our telomere kinetics assay uncovers new functional groups, as well as complex genetic interactions between tlm mutants.
Highlights
Telomeres are the specialized DNA–protein structures at the ends of eukaryotic chromosomes
We have used a new software, TelQuant, to accuretly measure telomere length, and we have developed a new method, TELKA, to cluster tlm mutants according to the dynamics of telomere length change
We demonstrate the usefulness of TELKA by describing a new telomere pathway composed of the Rnr1 protein, with a role in dNTP synthesis, together with the Met7 folylpolyglutamate synthetase (FPGS) and the Ku heterodimer
Summary
Telomeres are the specialized DNA–protein structures at the ends of eukaryotic chromosomes. In most eukaryotes the telomeric DNA consists of tracts of tandemly repeated sequences whose overall length is highly regulated [2]. Telomeric DNA is synthesized by the enzyme telomerase, which copies a short template sequence within its own RNA moiety [3]. Conventional DNA polymerases are unable to replicate the very ends of chromosomes due to their primer dependency; as a result, telomeres shorten with replicative age in vitro [4,5]. It has been shown that replenishing telomeres by an activated telomerase or by recombination (“Alternative Lengthening of Telomeres” or ALT) is one of the few essential steps that a normal human fibroblast cell must take on its way to become malignant [7]. Understanding how telomere length is monitored has significant medical implications especially in the fields of aging and cancer
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