Evidence That a RecQ Helicase Slows Senescence by Resolving Recombining Telomeres

Citation: Lee JY, Kozak M, Martin JD, Pennock E, Johnson FB ( Evidence That a RecQ Helicase Slows Senescence by Resolving Recombining Telomeres Julia Y. Lee 0 1 2 Marina Kozak 0 1 2 Joel D. Martin 0 1 2 Erin Pennock 0 1 2 F. Brad Johnson 0 1 2 0 Current address: Department of Biology, Saint Joseph's University , Philadelphia, Pennsylvania , United States of America 1 Academic Editor: Titia De Lange, Rockefeller University , United States of America 2 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine , Philadelphia, Pennsylvania , United States of America RecQ helicases, including Saccharomyces cerevisiae Sgs1p and the human Werner syndrome protein, are important for telomere maintenance in cells lacking telomerase activity. How maintenance is accomplished is only partly understood, although there is evidence that RecQ helicases function in telomere replication and recombination. Here we use twodimensional gel electrophoresis (2DGE) and telomere sequence analysis to explore why cells lacking telomerase and Sgs1p (tlc1 sgs1 mutants) senesce more rapidly than tlc1 mutants with functional Sgs1p. We find that apparent Xshaped structures accumulate at telomeres in senescing tlc1 sgs1 mutants in a RAD52- and RAD53-dependent fashion. The X-structures are neither Holliday junctions nor convergent replication forks, but instead may be recombination intermediates related to hemicatenanes. Direct sequencing of examples of telomere I-L in senescing cells reveals a reduced recombination frequency in tlc1 sgs1 compared with tlc1 mutants, indicating that Sgs1p is needed for tlc1 mutants to complete telomere recombination. The reduction in recombinants is most prominent at longer telomeres, consistent with a requirement for Sgs1p to generate viable progeny following telomere recombination. We therefore suggest that Sgs1p may be required for efficient resolution of telomere recombination intermediates, and that resolution failure contributes to the premature senescence of tlc1 sgs1 mutants. - Telomeres are critical for genome stability and normal cell physiology because they cap the ends of chromosomes; if uncapped, telomeres behave as DNA breaks and thus elicit damage responses and are subject to nucleolytic degradation and recombination [1,2]. Capping depends on telomere architecture, which is mediated by chromatin factors, and on telomere length. The enzyme telomerase can counteract the shortening of telomeres that accompanies DNA replication or DNA damage, but dividing cells lacking sufficient telomerase can develop critically short, uncapped telomeres that signal cell cycle arrest (cell senescence) or death. Some cells bypass these barriers by up-regulating telomerase expression and thus elongating telomeres. In other cases, bypass involves the use of recombination to maintain telomere length. Examples of the latter case are so-called survivors of telomerase deletion in Saccharomyces cerevisiae and alternative lengthening of telomeres (ALT) cells in mammals [3,4]. A growing number of proteins are recognized as participating in telomere maintenance [2]. Among these are members of the RecQ family of DNA helicases [5], including the human Werner syndrome (WS) and Bloom syndrome proteins (WRN and BLM, respectively) and S. cerevisiae Sgs1p. Deficiencies in these helicases lead to genome instability caused by defects in recombinational repair of DNA damage, replication fork stability, and checkpoint signaling, and can lead to the premature onset of cancer and age-related pathologies [5,6]. The precise mechanisms by which RecQ helicases help maintain telomeres are not yet clear, but there is evidence that they are important for telomere replication, repair, and recombination [718]. A well-characterized function of RecQ helicases throughout the genome is the regulation of homologous recombination, by which they facilitate resolution of recombination intermediates and perhaps avoid the initiation of inappropriate recombination events [5]. Yeast survivors of telomerase deletion and mammalian ALT cells are two settings in which RecQ helicases are important in recombination-dependent telomere maintenance. For example, Sgs1p is required for emergence of type II survivors, which depend on recombination among telomere repeat sequences [1517]; the Schizosaccharomyces pombe RecQ homolog SPAC212.11 similarly facilitates survivor emergence [7], and WRN regulates the generation of ALT cells from murine telomerase knockout cells [19]. In addition to their roles in survivors and in ALT cells, RecQ helicases function in telomere maintenance in primary cells that have little or no telomerase activity. For example, human WS fibroblasts suffer occasional complete loss of a telomere, which occurs preferentially at the guaninerich telomere strand, which is replicated by lagging-strand synthesis [11,20]. These loss events presumably contribute to Because telomeres are situated at the ends of chromosomes, they are both essential for chromosome integrity and particularly susceptible to processes that lead to loss of their own DNA sequences. The enzyme telomerase can counter these losses, but there are also other means of telomere maintenance, some of which depend on DNA recombination. The RecQ family of DNA helicases process DNA recombination intermediates and also help ensure telomere integrity, but the relationship between these activities is poorly understood. Family members include yeast Sgs1p and human WRN and BLM, which are deficient in the Werner premature aging syndrome and the Bloom cancer predisposition syndrome, respectively. We have found that the telomeres of yeast cells lacking both telomerase and Sgs1p accumulate structures that resemble recombination intermediates. Further, we provide evidence that the inability of cells lacking Sgs1p to process these telomere recombination intermediates leads to the premature arrest of cell division. We predict that similar defects in the processing of recombination intermediates may contribute to telomere defects in human Werner and Bloom syndrome cells. the premature senescence of cultured WS cells and their arrest at longer mean telomere lengths than control cells [21]; even though the shortening of most telomeres may be normal in WS cells, the increased frequency of occasional and critically shortened telomeres could signal senescence. Further, mutations in Wrn or Blm synergize with short telomeres in telomerase knockout mice to cause several degenerative pathologies, indicating that the helicases play important roles in telomere maintenance [10,12]. And in yeast, although sgs1 mutants maintain telomeres of normal length in the presence of telomerase, tlc1 sgs1 mutants senesce faster than tlc1 mutants [15,17]. The rapid senescence of tlc1 sgs1 mutants is due to an increased propensity of cells lacking Sgs1p to suffer G2/M arrest at a given average extent of telomere shortening; this suggests a role for S (...truncated)