Figure 5

Mutation of the Est1 TPR does not interfere with recruitment to telomeric chromatin. Chromatin immunoprecipitation of FLAG-tagged Est1 proteins (in pRS426) was performed as described in Methods. Enrichment of telomeric DNA was determined by comparing the intensity of PCR products generated using primer pairs matching a subtelomeric locus on chromosome X (tel XR) or another single-copy genomic locus (ARS607) and normalising to the values corresponding to input DNA. Data represent mean ± S.D. for n = 4 independent samples. Significant differences relative to the vector control (indicated by asterisks; * p < 0.01, *** p < 0.0001) were determined by one-way ANOVA and the Tukey post-hoc test. Nomenclature: vector, pRS426 alone; Est1, untagged; Est1-FLAG; KWQ, Est1(K84A/W87A/Q89A)-FLAG; EQW, Est1(E92A/Q96A/W97A)-FLAG; NN, Est1(N277A/N278A)-FLAG. (B) TRF analysis of the same strains as in (A); lane 10, S288C (WT). DNA markers at left in kbp. Note that average telomere lengths are longer in rad52Δ strains (lanes 1-3) compared with WT (lane 10), as expected [15, 16]. All samples were resolved on the same gel, and irrelevant or blank lanes between lanes 3, 4 and 9, 10 were removed.