Chromatin immunoprecipitation confirms structural defects.

<p>(A) The immunoprecipitation of Rap1 following cross-linking should be associated with subtelomeric sequences at natural telomere 6R if the fold-back structure is intact (upper diagram); however the subtelomeric ChIP will be lost upon loop opening (lower diagram). (B) Upon Rap1 ChIP from exponentially growing cells, a subtelomeric signal was detected up to 1 kb away from the base of the telomeric repeats in wild type cells, whereas the signal was largely diminished in <i>hda1Δ</i>, <i>sin3Δ</i> and <i>sir4Δ</i> mutants. DNA stemming from the actin locus (<i>ACT1</i>) was not detected following Rap1 ChIP and was used as a background control. Error bars represent SD from three independent experiments. (C) Cdc13-TAP (13) was also able to precipitate subtelomeric DNA up to 1 kb away from the start of the telomeric sequence at telomere 6R following cross-linking (n = 3, error as SD) in comparison to wild type (non-tagged controls). This ChIP signal at -1000 was reduced to that of non-tagged controls in the <i>sin3Δ</i> strain. The difference in ChIP signal distribution between Rap1 (B) and Cdc13-TAP (C) is likely due to the different positioning of the two proteins on the telomere (compare diagrams in A and C for explanation). For all experiments above error bars represent SD of the mean from at least 3 independent experiments and * indicates statistically significant differences as determined through unpaired student's t-tests whereby * = p<0.05, ** = p<0.01, *** = p<0.001.