Summary 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.

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 TG 1-3 repeats was stronger at the terminal tract than at the internal TG 1-3 sequences located between tandem Y elements. We show that the telomeric replication fork pause associated with the terminal TG 1-3 tracts begins ϳ100 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.Replication of the linear DNA of a eukaryotic chromosome imposes a problem of end replication, as originally predicted by Watson (40) and Olovnikov (31). While the synthesis of the leading strand can proceed to the very end of the template, the lagging strand is predicted to shorten upon every round of replication in each cell cycle. Most eukaryotes solve the endreplication problem by maintaining specific repetitive DNA sequences at their chromosome ends, called telomeres, by the enzyme telomerase, which elongates the 3Ј end of the telomeric DNA in a sequence-specific manner. In those rarer situations in which a eukaryote does not have telomerase, other multiple repeats, such as transposable elements in the fruit fly Drosophila melanogaster, are periodically added to their chromosome ends.The yeast Saccharomyces cerevisiae has telomeres containing ϳ250 to 350 bp of TG 1-3 repeats and uses telomerase for their maintenance. About two-thirds of the 32 telomeres in haploid cells carry one or more copies of subtelomeric YЈ elements (see reference 32 for a review). Members of the major class of YЈ elements are 6.7 kb long, and there is a minor 5.2-kb class; they are always arranged in the same orientation, such that multiple YЈ elements form directly repeating arrays separated by short stretches of telomeric TG 1-3 DNA.Replication of eukaryotic chromosomes initiates at autonomously replicating sequence (ARS) elements-origins of replication present at multiple locations on every chromosome. Every YЈ element contains an ARS (4). While many genomic ARSs are fired early in S phase, it has been reported, by using the density transfer method, that YЈ repeats replicate late in S phase (11,28,33,39). Telomeric chromatin has been implicated in determining the timing of activation of subtelomeric ARSs. A...

ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (hsdR, hsdM, and hsdS) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an hsd ؉ bacterium as the result of replication errors or recombinationdependent repair. We show that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive. In such bacteria, HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP. A mutation that blocks only the modification activity of EcoKI, leaving the cell with Ϸ600 unmodified targets, is not lethal provided that ClpXP is present. Our data support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage.Within a bacterium that has a classical restriction and modification (R-M) system, the nucleotide sequences that define the targets for attack by the resident restriction endonuclease are concealed by the modification of appropriate bases within them. For some systems this modification is achieved by the methylation of specific adenine residues, and for others it is achieved by methylation of cytosine residues. The restriction endonuclease has the potential to attack DNA from different strains of the same species because foreign DNA generally lacks the protective imprint of the relevant methyltransferase (for reviews see refs. 1 and 2). Restriction of the host cell's newly synthesized DNA normally is avoided, because the unmethylated strand of each target sequence produced by DNA replication is methylated before the next round of replication. If, however, resident DNA were to acquire unmodified target sequences, would it, like foreign DNA, become a substrate for restriction? In this paper we show that in situations where the modification of the host DNA by a type I R-M system fails, an alternative level of protection impairs the endonuclease activity of the restriction system and the bacteria survive.A type I R-M system is encoded by three genes: hsdR, hsdM, and hsdS. The three polypeptides, HsdR, HsdM, and HsdS, often designated R, M, and S, assemble to give an enzyme (R 2 M 2 S 1 ) that modifies hemimethylated DNA and restricts unmethylated DNA. A smaller complex (M 2 S 1 ) has only the methyltransferase activity. The S subunit confers target specificity; hence, both complexes and both activities respond to the same nucleotide sequence.Type

The response to DNA damage involves regulation of multiple essential processes to maximize the accuracy of DNA damage repair and cell survival 1. 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 a DNA double-strand break (DSB). Using yeast, we show that the major ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. Upon DNA damage, MEC1-RAD53-DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Utilizing a separation of function PIF1 mutation, we show that this phosphorylation is required for the Pif1-mediated telomerase inhibition that takes place specifically at DNA breaks, but not telomeres. Hence DNA damage signalling down-modulates telomerase action at a DNA break via Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a novel regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity.