Telomerase-Null Survivor Screening Identifies Novel Telomere Recombination Regulators

et al. (2013) Telomerase-Null Survivor Screening Identifies Novel Telomere Recombination Regulators. PLoS Genet 9(1): e1003208. doi:10.1371/journal.pgen.1003208 Telomerase-Null Survivor Screening Identifies Novel Telomere Recombination Regulators Yan Hu. 0 Hong-Bo Tang. 0 Ning-Ning Liu. 0 Xia-Jing Tong 0 Wei Dang 0 Yi-Min Duan 0 Xiao-Hong Fu 0 Yang Zhang 0 Jing Peng 0 Fei-Long Meng 0 Jin-Qiu Zhou 0 Michael J. McEachern, University of Georgia, United States of America 0 The State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences , Shanghai , China Telomeres are protein-DNA structures found at the ends of linear chromosomes and are crucial for genome integrity. Telomeric DNA length is primarily maintained by the enzyme telomerase. Cells lacking telomerase will undergo senescence when telomeres become critically short. In Saccharomyces cerevisiae, a very small percentage of cells lacking telomerase can remain viable by lengthening telomeres via two distinct homologous recombination pathways. These ''survivor'' cells are classified as either Type I or Type II, with each class of survivor possessing distinct telomeric DNA structures and genetic requirements. To elucidate the regulatory pathways contributing to survivor generation, we knocked out the telomerase RNA gene TLC1 in 280 telomere-length-maintenance (TLM) gene mutants and examined telomere structures in postsenescent survivors. We uncovered new functional roles for 10 genes that affect the emerging ratio of Type I versus Type II survivors and 22 genes that are required for Type II survivor generation. We further verified that Pif1 helicase was required for Type I recombination and that the INO80 chromatin remodeling complex greatly affected the emerging frequency of Type I survivors. Finally, we found the Rad6-mediated ubiquitination pathway and the KEOPS complex were required for Type II recombination. Our data provide an independent line of evidence supporting the idea that these genes play important roles in telomere dynamics. - . These authors contributed equally to this work. Telomeres are special DNA-protein structures found at the ends of eukaryotic chromosomes. Telomeres are crucial for genome integrity because they prevent chromosome ends from degradation or fusing with each other [1]. In budding yeast Saccharomyces cerevisiae, telomeric DNA consists of ,350 base pairs (bp) of TG13/C13 A repeats with a terminal single-stranded TG13 tract called a G-overhang [2]. Telomeric DNA can be maintained by either telomerase-mediated elongation or homologous recombination [35]. Telomerase is a highly specialized reverse transcriptase that adds telomeric DNA sequences to the 39 G-overhang using its intrinsic RNA template [3]. In Saccharomyces cerevisiae, the core components of telomerase are the catalytic subunit Est2 and its RNA template subunit TLC1 [6,7]. In wildtype yeast cells, the telomerase pathway supercedes the recombination pathway as the predominant mechanism of telomeric DNA elongation [8,9]. In telomerase-null cells, telomeric DNA is maintained via a recombination pathway termed alternative lengthening of telomeres (ALT) [10]. Approximately 85% of immortalized human tumor cells use telomerase to maintain telomeres while 15% apply the ALT mechanism to maintain telomeres [11]. In telomerase-null S. cerevisiae mutants, most cells undergo senescence after about 50100 divisions when telomeres shorten to less than approximately 100 bp [7,12,13]. Surprisingly, a select few of these senescing cells are able to bypass the short telomere survival crisis through lengthening their telomeres via a Rad52dependent recombination pathway [14]. These cells are called post-senescence survivors or survivors for short [14]. Survivors are categorized into two types: Type I and Type II, which possess different telomeric DNA structures and are defined by their dependence on Rad51 or Rad50 respectively [15]. Type I survivors exhibit highly amplified subtelomeric Y elements and short terminal telomeric TG tracts. The formation of Type I survivors depends on the canonical homologous recombination proteins Rad51, Rad54, Rad55 and Rad57 [14]. On the other hand, Type II survivors have long heterogeneous terminal telomeric TG tracts generated by recombination, and their formation depends on the Mre11-Rad50-Xrs2 (MRX) complex and Rad59 [14]. Type II survivors resemble the ALT cells observed in mammals [5]. In S. cerevisiae, about 90% of survivors generated on solid medium are categorized as Type I, while 10% are Type II. Nevertheless, Type II survivors grow at faster rates than Type I survivors, eventually overtaking their counterparts in liquid-grown cultures [14]. Homologous recombination is a means for an organism or a cell to repair damaged DNA in its genome. Eukaryotic chromosomes have a linear configuration with two ends that are special DNAprotein structures called telomeres. Telomeres can be recognized by the cell as DNA doublestrand breaks and subjected to repair by homologous recombination. In the bakers yeast Saccharomyces cerevisiae, cells that lack the enzyme telomerase, which is the primary factor responsible for telomeric DNA elongation, are able to escape senescence and cell death when telomeres undergo repair via homologous recombination. In this study, we have performed genetic screens to identify genes that affect telomeric DNA recombination. By examining the telomere structures in 280 mutants, each of which lacks both a telomere-length-maintenance gene and telomerase RNA gene, we identified 32 genes that were not previously known to be involved in telomere recombination. These genes have functions in a variety of cellular processes, and our work provides new insights into the regulation of telomere recombination in the absence of telomerase. In addition to the proteins in the Rad52 epistasis group, which are well-defined in the canonical survivor formation pathways, other genes involved in survivor formation have sporadically been identified. For example, SGS1, MEC1/TEL1, MDT1, DEF1, CLB2 and SUA5 are required for the generation of Type II survivors, while RIF1 and RIF2 have strong influences toward Type I survivor emerging frequency [1622]. Notably, some of the genes mentioned above appear to contribute to both survivor generation and telomere length regulation. Deletion of RIF1 or RIF2 causes telomere lengthening, while deletion of MRE11, RAD50, XRS2, TEL1, DEF1 or SUA5 results in telomere shortening [16,2325]. These observations suggest that genes involved in telomere recombination pathways and telomere length regulation are in some way linked. So far, there have been 251 telomere length maintenance (TLM) genes identified by genome-wide screens [23,26] and other studies [16,2734]. Furthermore, 29 additional genes previously miss-classified as essent (...truncated)