Recruitment of Rad51 and Rad52 to Short Telomeres Triggers a Mec1-Mediated Hypersensitivity to Double- Stranded DNA Breaks in Senescent Budding Yeast Yi-Hsuan Lin, Chia-Ching Chang, Chui-Wei Wong, Shu-Chun Teng* Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan Abstract Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Here we demonstrated that the budding yeast Saccharomyces cerevisiae type I survivors derived from telomerase-deficient cells were hypersensitive to DNA damaging agents. Assays to track telomere lengths and drug sensitivity of telomerase-deficient cells from spore colonies to survivors suggested a correlation between telomere shortening and bleomycin sensitivity. Our genetic studies demonstrated that this sensitivity depends on Mec1, which signals checkpoint activation, leading to prolonged cell-cycle arrest in senescent budding yeasts. Moreover, we also observed that when cells equipped with short telomeres, recruitments of homologous recombination proteins, Rad51 and Rad52, were reduced at an HO-endonuclease-catalyzed double-strand break (DSB), while their associations were increased at chromosome ends. These results suggested that the sensitive phenotype may be attributed to the sequestration of repair proteins to compromised telomeres, thus limiting the repair capacity at bona fide DSB sites. Citation: Lin Y-H, Chang C-C, Wong C-W, Teng S-C (2009) Recruitment of Rad51 and Rad52 to Short Telomeres Triggers a Mec1-Mediated Hypersensitivity to Double-Stranded DNA Breaks in Senescent Budding Yeast. PLoS ONE 4(12): e8224. doi:10.1371/journal.pone.0008224 Editor: Michael Polymenis, Texas A&M University, United States of America Received October 17, 2009; Accepted November 16, 2009; Published December 14, 2009 Copyright: 2009 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Science Council (NRPGM-98-3112-B-002-039) and National Health Research Institute of Taiwan (NHRI-EX98-9727BI) to SCT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: shuchunteng@ntu.edu.tw Introduction Telomeres are the specialized DNA-proteins complexes at the ends of eukaryotic chromosomes that allow intact ends to be distinguished from broken chromosomes, prevent chromosomes from end-to-end fusion, degradation, genomic instability and the associated risk of cancer. In most organisms, telomeric DNA is composed of a tandem array of short sequences. For example, these repeats in S. cerevisiae consist of ,350675 bp of duplex TG1–3/ C1–3A tracts. Internal to the TG1–3/C1–3A tracts are two classes of middle repetitive DNA elements, called X and Y’ [1]. X elements with heterogeneous sizes ranged from 0.3,3.75 kb exist in all chromosomes of S. cerevisiae [2], whereas one to four copies of Y’-long (6.7 kb) or Y’-short (5.2 kb) are found on about two-thirds of the telomeres. Telomeric DNA forms a heterochromatin structure with the Rap1p complex [3,4]. Loss of telomeric protection can lead to the formation of end-to-end fusions, which directly disrupt the correct structure at telomere termini [5]. In addition to the inhibition of inappropriate repair responses, fusions or recombinations, telomeres need to be fully replicated by the action of telomerase to prevent end-replication problem [6,7,8,9]. In S. cerevisiae, telomerase RNA and reverse transcriptase are encoded by EST2 and TLC1, respectively. Although the majority of telomerase-minus S. cerevisiae cells enter cell cycle arrest eventually following the senescence stage [10,11], survivors arise through RAD52-dependent homologous recombination (HR). Most survivors are type I, which have multiple tandem copies of the subtelomeric Y’ element followed by very short terminal tracts of TG1–3/CA1–3 DNA [10,12]. In a minor fraction of the survivors (type II), the lengths of telomere sequence are increased heterogeneously from several hundred base pairs to 10 kb or longer (Teng and Zakian, 1999). The generation of type I survivors depends on the presence of helicase Rad54, Rad51 that acts in strand invasion, Rad55-Rad57 complex that stabilizes recombi- nation filament [13], and 59-to-39 exonuclease Exo1 [14]. The formation of type II survivors, on the other hand, requires MRX complex (Mre11, Rad50 and Xrs2), Rad59 for strand invasion, and the Sgs1-Top3 complex, which is involved in the process of HR [15,16,17,18,19]. Telomere attrition caused by lack of telomerase in S. cerevisiae has been shown to activate DNA damage checkpoints as they approach senescence, and this activation is also believed to be responsible for the premature growth arrest in these strains [20,21]. The convergence of DNA damage and telomere checkpoints has also been revealed in mammalian cells [5,22,23], suggesting that DSBs and compromised telomere structure both impinge upon the ATM pathway that converges on a critical p53 nodal point. In the telomerase mutant mouse model, lack of p53 allows dysfunctional cells to avoid fates associated with checkpoint activation, including growth arrest and apoptosis [24,25]. These results indicate that in mammals, as in yeast, telomere attrition resulting from lack of telomerase or alterations in telomere structure triggers characteristic DNA damage-initiated checkpoint. Additionally, not only do short telomeres share some of the biological and mechanistic features of DNA damage sites, they also exert effects on cellular response to PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8224