Fig 4. Pif1 preferentially binds to long telomeres.

Panels A-D show results from ChIP experiments on two diploid strains that had WT length telomeres but were heterozygous at the PIF1 locus (PIF1/pif1-K264A). The only difference between the two strains is that one expressed Pif1-K264A-Myc and the other expressed Yku80-Myc. DNA was either prepared directly from the two strains (Input) or after immuno-precipitation with anti-Myc antibody (ChIP). Both input and ChIP samples were C-tailed and amplified by PCR using primers for telomere VI-R or XV-L and then separated on 1.8% agarose gels. Input and ChIP amplified DNA after gel purification from telomere VI-R (A) or XV-L (B). (C) Graphical presentation of the average difference in bps in lengths of DNA in ChIP and Input samples from the two strains (Pif1 and YKu80) and at both tested telomeres (VI-R and XV-L). Error bars represent one standard deviation from the average for four independent experiments. Using a student’s t-test, the differences between the average telomere lengths of the Input versus ChIP samples were significant for cells expressing Pif1-K264A-Myc (p<0.0001 for both VI-R and XV-L) but not for cells expressing Yku80-Myc (p = 0.9 for VI-R; p = 0.8 for XV-L). (D) The first row shows the average length of telomeres in the two samples (ChIP sample/input sample) for both telomeres in the two strains. The second row shows the average difference in telomere length (length in ChIP minus length in input samples) for the two strains and telomeres. (E and F) Pif1 binding to WT and short telomeres in the inducible VII-L short telomere (panel E) and control WT length VII-L (panel F) strains. Both strains express Pif1-Myc. Both panels show averages ± SD from three experiments. Data are presented as average binding in G1 phase (time points 0, 15 and 30 min; see Fig 3), S phase (time points 45 and 60), and G2 phase (time points 75 and 90). In the experimental strain (panel E), Pif1 binding in S phase was twice as high to the WT VI-R telomere than to the short VII-L in the same cells (p = 0.007; two-tailed unpaired t-tests). In the control strain (panel F), Pif1 bound equally well in S phase to WT length VII-L and WT VI-R telomeres (p = 0.5; two-tailed unpaired t-tests). In both strains, Pif1 binding to WT length telomeres was significantly higher in S phase than in G1 or G2 (p <0.009; two-tailed unpaired t-tests).