DNA breaks are masked by multiple Rap1 binding in yeast: implications for telomere capping and telomerase regulation

Abstract

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks by packaging them in a protective structure referred to as the telomere "cap." Here we investigate the nature of the telomere cap by examining events at DNA breaks generated adjacent to either natural telomeric sequences (TG repeats) or arrays of Rap1-binding sites that vary in length. Although DNA breaks adjacent to either short or long telomeric sequences are efficiently converted into stable telomeres, they elicit very different initial responses. Short telomeric sequences (80 base pair [bp]) are avidly bound by Mre11, as well as the telomere capping protein Cdc13 and telomerase enzyme, consistent with their rapid telomerase-dependent elongation. Surprisingly, little or no Mre11 binding is detected at long telomere tracts (250 bp), and this is correlated with reduced Cdc13 and telomerase binding. Consistent with these observations, ends with long telomere tracts undergo strongly reduced exonucleolytic resection and display limited binding by both Rpa1 and Mec1, suggesting that they fail to elicit a checkpoint response. Rap1 binding is required for end concealment at long tracts, but Rif proteins, yKu, and Cdc13 are not. These results shed light on the nature of the telomere cap and mechanisms that regulate telomerase access at chromosome ends.[Keywords: DNA double-strand break; telomere capping; telomerase; DNA damage checkpoint; telomere length regulation] Supplemental material is available at http://www.genesdev.org.

Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

“…28 These data provide evidence that the catalytic subunit of telomerase directly or indirectly influences the association of Rap1p with telomeric DNA in a manner that is independent of the telomere sequence, suggesting that telomerase plays roles upstream and downstream of Rap1p in the regulation of telomere length. Mechanisms of telomere overelongation by est2 alleles.…”

“…The deletion of RIF1 or RIF2 increases the frequency of telomere elongation during a single cell cycle, suggesting that the Rap1p/ Rif1p/Rif2p complex modulates telomerase access in a manner responsive to telomere length (39). Rap1p also mediates the silencing of genes near telomeres and has been implicated in the repression of nonhomologous end joining and telomere resection near a double-strand break (27)(28)(29).…”

Characterization of the Yeast Telomere Nucleoprotein Core

“…Alternatively, Rap1 binding to DNA could exhibit length-dependent cooperativity in vivo that is not observed here in vitro. The telomere repeat length at which telomeres convert from "closed" to "open" is different for the different actions specified above: effective telomerase processivity is increased at a repeat length of 100 -125 (six or seven Rap1 binding sites), whereas Rap1-mediated inhibition of NHEJ may require two to four Rap1 binding sites (24,26,48). Because of the different lengths at which these different "conversions" occur, we favor a model for telomere structure in which Rap1 binds independently to each repeat element in a manner governed by affinity and that the interaction of secondary proteins with Rap1 changes as different numbers of Rap1 molecules are telomere-bound.…”

“…This irregular array complicates the assessment of binding independence. Therefore, we first examined this question by using defined arrays of equally spaced Rap1 recognition sites, which have previously been shown to mimic the action of telomeres in vivo by their capacity to both control telomere length maintenance when placed proximal to a short stretch of native telomere sequence and to prevent NHEJ (19,(25)(26)(27). Each synthetic telomere array contained a known number of identically oriented Rap1 binding sites, each separated by a defined spacing between adjacent sites.…”

Opening the DNA repair toolbox: Localization of DNA double strand breaks to the nuclear periphery

“…28 appears to play a key role in de novo telomere formation at DSBs that lack extensive TG repeats. 16,17 Persistent DSBs are Targeted to the Nuclear Periphery How does the cell respond when a DSB remains un-repaired for a long period of time? In yeast, cells can eventually inactivate the cell cycle checkpoint response (a process termed checkpoint adaptation) and proceed through mitosis with a broken chromosome, but adaptation occurs only after ∼15 hours of failed DSB repair.…”