Ku Binding on Telomeres Occurs at Sites Distal from the Physical Chromosome Ends

Abstract

The Ku complex binds non-specifically to DNA breaks and ensures repair via NHEJ. However, Ku is also known to bind directly to telomeric DNA ends and its presence there is associated with telomere capping, but avoiding NHEJ. How the complex discriminates between a DNA break and a telomeric extremity remains unknown. Our results using a tagged Ku complex, or a chromosome end capturing method, in budding yeast show that yKu association with telomeres can occur at sites distant from the physical end, on sub-telomeric elements, as well as on interstitial telomeric repeats. Consistent with previous studies, our results also show that yKu associates with telomeres in two distinct and independent ways: either via protein-protein interactions between Yku80 and Sir4 or via direct DNA binding. Importantly, yKu associates with the new sites reported here via both modes. Therefore, in sir4Δ cells, telomere bound yKu molecules must have loaded from a DNA-end near the transition of non-telomeric to telomeric repeat sequences. Such ends may have been one sided DNA breaks that occur as a consequence of stalled replication forks on or near telomeric repeat DNA. Altogether, the results predict a new model for yKu function at telomeres that involves yKu binding at one-sided DNA breaks caused by replication stalling. On telomere proximal chromatin, this binding is not followed by initiation of non-homologous end-joining, but rather by break-induced replication or repeat elongation by telomerase. After repair, the yKu-distal portion of telomeres is bound by Rap1, which in turn reduces the potential for yKu to mediate NHEJ. These results thus propose a solution to a long-standing conundrum, namely how to accommodate the apparently conflicting functions of Ku on telomeres.

In vivo chromatin organization on native yeast telomeric regions is independent of a cis-telomere loopback conformation

“…The in vivo ChEC method (for Chromatin Endogenous Cleavage), based on fusions of specific DNA-binding proteins of interest with the catalytic domain of micrococcal nuclease (MNase), was initially developed and applied to map the DNA-binding sites of the certain proteins on genomic sequences [41][42][43]. In short, MNase-dependent DNA cuts are induced in permeabilized yeast cells in vivo by addition of calcium ions.…”

“…Given the absence of known Gal4-binding sites in our regions of interest, i.e., subtelomeres and telomeres, we similarly consider this GBD-MN a non-targeted nuclear protein. In addition, we analyzed MNase-dependent DNA cuts induced by MN-Rap1 [42,43]. Rap1 being an abundant telomeric protein, it will yield a high local concentration of MNase at telomeres and provide a demarcation point for the transition between telomeric repeats bound by Rap1 and subtelomeric sequences [43].…”

“…In addition, we analyzed MNase-dependent DNA cuts induced by MN-Rap1 [42,43]. Rap1 being an abundant telomeric protein, it will yield a high local concentration of MNase at telomeres and provide a demarcation point for the transition between telomeric repeats bound by Rap1 and subtelomeric sequences [43]. Finally, to map subtelomeric nucleosomes on the same regions, we used a previously developed H2A-MN construct [41,45].…”

Telomerase in Space and Time: Regulation of Yeast Telomerase Function at Telomeres and DNA Breaks

“…Apart from the Sir4-bound fraction, Yku also binds to telomeres directly, as it does at DSBs [71][72][73]. In addition to having two different modes of telomere binding, Yku was also found at diverse telomeric loci, including the junction between telomere and subtelomere regions, as well as interstitial telomeric sequences between the subtelomeric repeats [73]. Both populations include Yku directly bound to DNA and Sir4-bound Yku.…”

“…Although in this scenario, telomerase is kept at telomeres in G1, it does not engage in productive interactions with telomeres, possibly due to the competition between Rif and Sir proteins for Rap1 binding. Interestingly, the fact that Sir4-Yku80 complexes locate distally from telomere ends [73], might also ensure that telomerase recruitment to telomeres in G1 remains unproductive.…”