Telomere length kinetics assay (TELKA) sorts the telomere length maintenance (tlm) mutants into functional groups

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.

Figure 1.

TelQuant. The TelQuant program scans teloblots and determines the median telomere length of each sample. The program automatically detects size markers and the terminal telomeric fragment, calculates telomere size and exports the results to an Excel sheet for additional processing.

Figure 2.

TELKA divides 30 tlm mutants into functional categories. (A) The tlm mutants can be clustered into four functional groups. A kinetic graph for each group is displayed. The X-axis represents the number of streaks (approximately 25 generations/streak) and the Y-axis represents telomere length relative to the wild-type length. (B) Mean of the kinetics of shortening of each group.

Figure 3.

Telomeric phenotype of met7Δ, rnr1Δ, yku70Δ and yku80Δ mutants. (A) Teloblots of single, double and triple mutants. (B) Left panel: Ethidium bromide-stained gel. Right panel: In-gel Southern blot that hybridizes to the G-rich ssDNA telomeric overhang.

Figure 4.

Role of MET7 in NHEJ and co-IP of Met7 and Rnr1. (A) A plasmid digested with a linearizing restriction enzyme or an uncut plasmid were electroporated into different strains and their ability to repair by NHEJ was measured by counting colonies. The graph shows the ratio of transformants with the cut plasmid relative to the uncut plasmid. (B) Co-immunoprecipitation of Met7 and Rnr1. A strain carrying a TAP (Tandem Affinity Purification)-tagged Met7, an untagged wt strain (BY4741), a control strain deleted for RNR1 and the same strain carrying a TAP-tagged Met7 were submitted to western blot analysis, before or after being immunoprecipitated with antibodies specific for the TAP tag, or anti-Rnr1. Rnr1 can be detected after anti-TAP immunoprecipitation, and Met7-TAP is detected upon immunoprecipitation of Rnr1.

Figure 5.

RNR1, YKU70 and MET7 affect dNTP synthesis. The relative levels of dNTPs are shown in single, double and triple mutants, compared to those of the wt.