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The proper maintenance of genetic material is essential for the survival of living organisms. One of the main safeguards of genome stability is homologous recombination involved in the faithful repair of DNA double-strand breaks, the restoration of collapsed replication forks, and the bypass of replication barriers. Homologous recombination relies on the formation of Rad51 nucleoprotein filaments which are responsible for the homology-based interactions between DNA strands. Here, we demonstrate that without the regulation of these filaments by Srs2 and Rad54, which are known to remove Rad51 from single-stranded and double-stranded DNA, respectively, the filaments strongly inhibit damage-associated DNA synthesis during DNA repair. Furthermore, this regulation is essential for cell survival under normal growth conditions, as in the srs2Δ rad54Δ mutants, unregulated Rad51 nucleoprotein filaments cause activation of the DNA damage checkpoint, formation of mitotic bridges, and loss of genetic material. These genome instability features may stem from the problems at stalled replication forks as the lack of Srs2 and Rad54 in the presence of Rad51 nucleoprotein filaments impedes cell recovery from replication stress. This study demonstrates that the timely and efficient disassembly of recombination machinery is essential for genome maintenance and cell survival. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.11.027 Byline: Shang Li (1), Svetlana Makovets (1), Tetsuya Matsuguchi (1), Justin D. Blethrow (2), Kevan M. Shokat (2), Elizabeth H. Blackburn (1) Keywords: CELLCYCLE; DNA; RNA Abstract: Elongation of telomeres by telomerase replenishes the loss of terminal telomeric DNA repeats during each cell cycle. In budding yeast, Cdc13 plays an essential role in telomere length homeostasis, partly through its interactions with both the telomerase complex and the competing Stn1-Ten1 complex. Previous studies in yeast have shown that telomere elongation by telomerase is cell cycle dependent, but the mechanism underlying this dependence is unclear. In S. cerevisiae, a single cyclin-dependent kinase Cdk1 (Cdc28) coordinates the serial events required for the cell division cycle, but no Cdk1 substrate has been identified among telomerase and telomere-associated factors. Here we show that Cdk1-dependent phosphorylation of Cdc13 is essential for efficient recruitment of the yeast telomerase complex to telomeres by favoring the interaction of Cdc13 with Est1 rather than the competing Stn1-Ten1 complex. These results provide a direct mechanistic link between coordination of telomere elongation and cell-cycle progression in vivo. Author Affiliation: (1) Department of Biochemistry and Biophysics, University of California, San Francisco, Box 2200, San Francisco, CA 94143-2200, USA (2) Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143-2200, USA Article History: Received 2 June 2008; Revised 11 September 2008; Accepted 20 November 2008 Article Note: (miscellaneous) Published: January 8, 2009

Telomeres are an unusual component of the genome because they do not encode genes, but their structure and cellular maintenance machinery (which we define as 'telotype') are essential for chromosome stability. Cells can switch between different phenotypic states. One such example is when they switch from maintenance mediated by telomerase (TERT telotype) to one of the two alternative mechanisms of telomere preservation (ALT I and ALT II telotype). The nature of this switch is largely unknown. Reintroduction of telomerase into ALT II, but not ALT I, yeast led to the loss of their ability to survive a second round of telomerase withdrawal. Mating-based genetic analysis of ALT I and II revealed that both types of telomerase-independent telomere maintenance are inherited as a non-Mendelian trait dominant over senescence (SEN telotype). Additionally, inheritance of ALT I and ALT II did not depend on either the mitochondrial genome or a prion-based mechanism. Type I, but not type II, survivor cells exhibited impaired gene silencing, potentially connecting the switch to the ALT telotype epigenetic changes. These data provide evidence that nonprion epigenetic-like mechanisms confer flexibility on cells as a population to adjust to the life-threatening situation of telomerase loss, allowing cells to switch from TERT to ALT telotypes that can sustain viable populations.

The ability of the Tel2 yeast present in Saccharomyces cerevisiae to activate and localize the ATM and ATR kinase to a double-strand break is discussed. Findings demonstrate the general strategy that is used by different proteins to interact with their binding partners.

Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway – Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening. [ABSTRACT FROM AUTHOR]

The response to DNA damage involves regulation of several essential processes to maximize the accuracy of DNA damage repair and cell survival. Telomerase has the potential to interfere with repair by inappropriately adding telomeres to DNA breaks. It was unknown whether cells modulate telomerase in response to DNA damage to increase the accuracy of repair. Here, we report that telomerase action is regulated as a part of the cellular response to DNA double-strand breaks (DSBs). Using yeast, we show that the main ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. After DNA damage, MEC1–RAD53–DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Using a separation of function PIF1 mutation, we show that this phosphorylation is specifically required for the Pif1-mediated telomerase inhibition that takes place at DNA breaks, but not for that at telomeres. Hence DNA damage signalling down-modulates telomerase action at DNA breaks through Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a new regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity. [ABSTRACT FROM AUTHOR]

Replication initiation and replication fork movement in the subtelomeric and telomeric DNA of native Y' telomeres of yeast were analyzed using two-dimensional gel electrophoresis techniques. Replication origins (ARSs) at internal Y' elements were found to fire in early-mid-S phase, while ARSs at the terminal Y' elements were confirmed to fire late. An unfired Y' ARS, an inserted foreign (bacterial) sequence, and, as previously reported, telomeric DNA each were shown to impose a replication fork pause, and pausing is relieved by the Rrm3p helicase. The pause at telomeric sequence TG1-3 repeats was stronger at the terminal tract than at the internal TG1-3 sequences located between tandem Y' elements. We show that the telomeric replication fork pause associated with the terminal TG1-3 tracts begins ∼100 bp upstream of the telomeric repeat tract sequence. Telomeric pause strength was dependent upon telomere length per se and did not require the presence of a variety of factors implicated in telomere metabolism and/or known to cause telomere shortening. The telomeric replication fork pause was specific to yeast telomeric sequence and was independent of the Sir and Rif proteins, major known components of yeast telomeric heterochromatin. [ABSTRACT FROM AUTHOR]

Telomerase maintains ends of eukaryotic chromosomes, telomeres. Telomerase loss results in replicative senescence and a switch to recombination-dependent telomere maintenance. Telomerase insufficiency in humans leads to telomere syndromes associated with premature ageing and cancer predisposition. Here we use yeast to show that the survival of telomerase insufficiency differs from the survival of telomerase loss and occurs through aneuploidy. In yeast grown at elevated temperatures, telomerase activity becomes limiting: haploid cell populations senesce and generate aneuploid survivors-near diploids monosomic for chromosome VIII. This aneuploidy results in increased levels of the telomerase components TLC1, Est1 and Est3, and is accompanied by decreased abundance of ribosomal proteins. We propose that aneuploidy suppresses telomerase insufficiency through redistribution of cellular resources away from ribosome synthesis towards production of telomerase components and other non-ribosomal proteins. The aneuploidy-induced re-balance of the proteome via modulation of ribosome biogenesis may be a general adaptive response to overcome functional insufficiencies. [ABSTRACT FROM AUTHOR]