Telomerase counteracts telomere erosion that stems from incomplete chromosome end replication and nucleolytic processing. A precise understanding of telomere length homeostasis has been hampered by the lack of assays that delineate the nonuniform telomere extension events of single chromosome molecules. Here, we measure telomere elongation at nucleotide resolution in Saccharomyces cerevisiae. The number of nucleotides added to a telomere in a single cell cycle varies between a few to more than 100 nucleotides and is independent of telomere length. Telomerase does not act on every telomere in each cell cycle, however. Instead, it exhibits an increasing preference for telomeres as their lengths decline. Deletion of the telomeric proteins Rif1 or Rif2 gives rise to longer telomeres by increasing the frequency of elongation events. Thus, by taking a molecular snapshot of a single round of telomere replication, we demonstrate that telomere length homeostasis is achieved via a switch between telomerase-extendible and -nonextendible states.

Telomerase is the ribonucleoprotein enzyme that elongates telomeres to counteract telomere shortening. The core enzyme consists of a reverse transcriptase protein subunit and an RNA subunit. The RNA subunit contains a short region that is used as a template by the reverse transcriptase to add short, tandem, G-rich repeats to the 3 ends of telomeres. By coexpressing two RNA subunits that differ in the telomeric repeat sequence specified and examining the telomere extensions after one cell cycle, we determined that Saccharomyces cerevisiae telomerase can dissociate and reassociate from a given telomere during one cell cycle. We also confirmed that telomerase is nonprocessive in terms of telomeric repeat addition. However, repeat addition processivity is significantly increased at extremely short telomeres, a process that is dependent on the ATM-ortholog Tel1. We propose that this enhancement of telomerase processivity at short telomeres serves to rapidly elongate critically short telomeres.[Keywords: Telomerase; processivity; Saccharomyces cerevisiae; TLC1; Tel1] Supplemental material is available at http://www.genesdev.org.

Telomerase enables telomere length homeostasis, exhibiting increasing preference for telomeres as their lengths decline. This regulation involves telomere repeat-bound Rap1, which provides a length-dependent negative feedback mechanism, and the Tel1 and Mec1 kinases, which are positive regulators of telomere length. By analysing telomere elongation of wild-type chromosome ends at single-molecule resolution, we show that in tel1D cells the overall frequency of elongation decreases considerably, explaining their short telomere phenotype. At an artificial telomere lacking a subtelomeric region, telomere elongation no longer increases with telomere shortening in tel1D cells. By contrast, a natural telomere, containing subtelomeric sequence, retains a preference for the elongation of short telomeres. Tethering of the subtelomere binding protein Tbf1 to the artificial telomere in tel1D cells restored preferential telomerase action at short telomeres; thus, Tbf1 might function in parallel to Tel1, which has a crucial role in a TG-repeat-controlled pathway for the activation of telomerase at short telomeres.

Homologous recombination (HR) plays a vital role in DNA metabolic processes including meiosis, DNA repair, DNA replication and rDNA homeostasis. HR defects can lead to pathological outcomes, including genetic diseases and cancer. Recent studies suggest that the post-translational modification by the small ubiquitin-like modifier (SUMO) protein plays an important role in mitotic and meiotic recombination. However, the precise role of SUMOylation during recombination is still unclear. Here, we characterize the effect of SUMOylation on the biochemical properties of the Saccharomyces cerevisiae recombination mediator protein Rad52. Interestingly, Rad52 SUMOylation is enhanced by single-stranded DNA, and we show that SUMOylation of Rad52 also inhibits its DNA binding and annealing activities. The biochemical effects of SUMO modification in vitro are accompanied by a shorter duration of spontaneous Rad52 foci in vivo and a shift in spontaneous mitotic recombination from single-strand annealing to gene conversion events in the SUMO-deficient Rad52 mutants. Taken together, our results highlight the importance of Rad52 SUMOylation as part of a ‘quality control’ mechanism regulating the efficiency of recombination and DNA repair.

In most mammals, daily rhythms in physiology are driven by a circadian timing system composed of a master pacemaker in the suprachiasmatic nucleus (SCN) and peripheral oscillators in most body cells. The SCN clock, which is phase-entrained by light-dark cycles, is thought to synchronize subsidiary oscillators in peripheral tissues, mainly by driving cyclic feeding behavior. Here, we examined the expression of circadian clock genes in the SCN and the liver of the common vole Microtus arvalis, a rodent with ultradian activity and feeding rhythms. In these animals, clock-gene mRNAs accumulate with high circadian amplitudes in the SCN but are present at nearly constant levels in the liver. Interestingly, highamplitude circadian liver gene expression can be elicited by subjecting voles to a circadian feeding regimen. Moreover, voles with access to a running wheel display a composite pattern of circadian and ultradian behavior, which correlates with low-amplitude circadian gene expression in the liver. Our data indicate that, in M. arvalis, the amplitude of circadian liver gene expression depends on the contribution of circadian and ultradian components in activity and feeding rhythms.circadian gene expression ͉ circadian rhythm ͉ peripheral clocks ͉ suprachiasmatic nucleus ͉ feeding rhythms A ccording to current belief, molecular circadian rhythms in mammals are generated by two interconnected feedback loops of clock-gene expression (1). In this model, period 1 (PER1), period 2 (PER2), cryptochrome 1 (CRY1), and cryptochrome 2 (CRY2), the members of the negative limb, form heterotypic protein complexes that repress transcription of their own genes by interfering with the activity of the transcription factors CLOCK and BMAL1, the members of the positive limb. The antiphasic circadian transcription cycles of positive-and negative-limb members are interlocked by the orphan nuclear receptor REV-ERB␣, which periodically represses Bmal1 expression. It is not completely understood how the oscillations generated by this molecular clockwork circuitry are translated into overt rhythms in physiology and behavior, but mutations in circadian clock genes lead to behavioral arrhythmicity or periodlength changes (1).The mammalian circadian timing system has a hierarchical structure, in that a central pacemaker in the suprachiasmatic nucleus (SCN) coordinates peripheral clocks in most peripheral cells. Central and peripheral oscillators have a similar molecular makeup (see above) and, accordingly, share many properties. For example, both operate in a cell-autonomous and selfsustained fashion (2-5), and clock-gene mutations affecting period length shorten or lengthen the period of both behavioral cycles (driven by SCN neurons) and circadian gene expression in cultured fibroblast (6, 7). Perhaps the most obvious difference between central and peripheral circadian oscillators lies in the mechanisms by which they are synchronized. Whereas the phase of the SCN master pacemaker is entrained primarily by the photoperiod (8, 9), that of peri...