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.

Figure 1.

Preferential telomerase recruitment at short TG tracts. (A) Schematic representation of the modified subtelomeric regions of Chr. VII-L and Chr. V-R. (B) Analysis by ChIP of the binding of Est1-Myc and Est2-Myc after galactose induction of a DSB. See Materials and Methods for details.

Figure 2.

Recruitment of Cdc13 and Mre11 is strongly reduced at long TG tracts. Analysis by ChIP of the binding of Cdc13-Myc in wild-type and est2-D670A strains (A) and of Mre11-Myc (B) in wild-type strains after galactose induction of a DSB.

Figure 3.

5′ end resection is inhibited at long TG arrays. (Top panel) Southern blots monitoring resection of the C_1–3A strand at TG-250 in a wild-type strain and TG-80 repeats in wild-type, est2-D670A, and mre11-Δ strains. (INT) The internal loading control (band of 165 bp specific for a region on Chr. XV); (CUT) a band of 418 bp or 239 bp, corresponding to the C_1–3A strand of the long or short telomeric tracts, respectively. (Bottom panel) Quantification of the relative signal for the C_1–3A strand obtained by measuring the amount of the “CUT” band relative to the internal control “INT,” normalized to the efficiency of cleavage.

Figure 4.

Multiple Rap1 binding suppresses Mre11 and Cdc13 recruitment at long TG tracts. (A) Analysis by ChIP of the binding of Mre11-Myc and Cdc13-Myc after galactose induction of a DSB in wild-type and rap1-17 strains. (B) Southern blots monitoring cleavage at the HO site in a rap1-17 strain. (INT) The internal loading control; (U) an uncut fragment; (C) the fragment resulting from “U” after induction of the HO cut. (C) Analysis by ChIP of the binding of Mre11-Myc and Cdc13-Myc after galactose induction of a DSB adjacent to 16× and 4× wild-type and mutant Rap1 site arrays.

Figure 5.

Long TG tracts fail to induce a strong checkpoint response. Analysis by ChIP of the binding of Rpa1-Myc and Mec1-Myc after galactose induction of a DSB in wild-type strains.

Figure 6.

Binding of yKu at long TG tracts does not inhibit resection. Analysis by ChIP after galactose induction of a DSB of the binding of yKu70-Myc in a wild-type strain (A) and of the binding of Mre11-Myc and Cdc13-Myc in wild-type and yku70-Δ strains (B).

Figure 7.

Limited role for Cdc13 in capping long TG-tract ends. Detection of single-stranded DNA at the YER188W locus in cdc13-1 mutant or CDC13⁺ cells following HO cutting adjacent to either TG-80 or TG-250 tracts (as indicated) at Chr. V-R. (YER188W is 1130 or 1309 bp centromere-proximal to HO cut site at the short or long TG tracts, respectively). Cells were first grown for 3 h at 24°C in YPLG, then transferred to fresh medium containing galactose (2%) at 36°C. The QAOS assay was used to measure single-stranded DNA levels on the TG strand at the indicated times following galactose induction of the DSB. See Materials and Methods for additional details.