Top1 and Top2 promote replication fork arrest at a programmed pause site.

Programmed fork pausing is a complex process allowing cells to arrest replication forks at specific loci in a polar manner. Studies in budding yeast and other model organisms indicate that such replication fork barriers do not act as roadblocks passively impeding fork progression but rather elicit complex interactions between fork and barrier components. In this issue of Genes & Development, Shyian and colleagues (pp. 87-98) show that in budding yeast, the fork protection complex Tof1-Csm3 interacts physically with DNA topoisomerase I (Top1) at replication forks through the C-terminal domain of Tof1. Fork pausing at the ribosomal DNA (rDNA) replication fork barrier (RFB) is impaired in the absence of Top1 or in a tof1 mutant that does not bind Top1, but the function of Top1 can be partially compensated for by Top2. Together, these data indicate that topoisomerases play an unexpected role in the regulation of programmed fork pausing in Saccharomyces cerevisiae.

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

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.