Biomedical subjects
Publications and source records attributed to Julia Promisel Cooper.
In the early stages of meiosis, all the telomeres in the cell attach to the nuclear envelope and gather near the centrosome. This polarized chromosomal array is known as the bouquet, as the clustered telomeres resemble the gathered stems of a floral arrangement. In this issue of Cell, Chikashige et al. (2006) provide intriguing clues about the molecular details underlying this conserved meiotic event.
Telomere replication is achieved through the combined action of the conventional DNA replication machinery and the reverse transcriptase, telomerase. Telomere-binding proteins have crucial roles in controlling telomerase activity; however, little is known about their role in controlling semi-conservative replication, which synthesizes the bulk of telomeric DNA. Telomere repeats in the fission yeast Schizosaccharomyces pombe are bound by Taz1, a regulator of diverse telomere functions. It is generally assumed that telomere-binding proteins impede replication fork progression. Here we show that, on the contrary, Taz1 is crucial for efficient replication fork progression through the telomere. Using two-dimensional gel electrophoresis, we find that loss of Taz1 leads to stalled replication forks at telomeres and internally placed telomere sequences, regardless of whether the telomeric G-rich strand is replicated by leading- or lagging-strand synthesis. In contrast, the Taz1-interacting protein Rap1 is dispensable for efficient telomeric fork progression. Upon loss of telomerase, taz1Delta telomeres are lost precipitously, suggesting that maintenance of taz1Delta telomere repeats cannot be sustained through semi-conservative replication. As the human telomere proteins TRF1 and TRF2 are Taz1 orthologues, we predict that one or both of the human TRFs may orchestrate fork passage through human telomeres. Stalled forks at dysfunctional human telomeres are likely to accelerate the genomic instability that drives tumorigenesis.
Telomere protection and maintenance are accomplished through the coordinated actions of telomere-specific DNA binding proteins and their interacting partners. The fission yeast ortholog of human TRF1/2, Taz1, binds telomeric DNA and regulates numerous aspects of telomere function. Here, we ask which aspects of Taz1 function are mediated through its interacting proteins, Rap1 and Rif1. We demonstrate that rap1+ deletion phenocopies some, but not all, aspects of taz1Delta telomere dysfunction, while Rif1 exhibits a very different functional spectrum. Rap1 acts in a Taz1-dependent pathway to prevent chromosome end fusions and regulate telomeric 3' overhang formation, while Rif1 is dispensable for these functions. Telomerase inhibition by Taz1 is mediated by two separate pathways, one involving Rap1 and the other involving Rif1. In contrast, Taz1 is uniquely required to prevent chromosomal entanglements and missegregation at cold temperatures. Strikingly, while rap1+ deletion exacerbates the cold sensitivity of taz1Delta cells, rif1+ deletion restores full viability. Thus, Rap1 and Rif1 are each required for a subset of the functions of Taz1, but each acquires Taz1-independent functions in its absence. Furthermore, Taz1 can function independently of its known binding partners.
Several considerations suggest that levels of the two major modes of double-strand break (DSB) repair, homologous recombination (HR), and nonhomologous end joining (NHEJ), are regulated through the cell cycle. However, this idea has not been explicitly tested. In the absence of the telomere-binding protein Taz1, fission yeast undergo lethal telomere fusions via NHEJ. These fusions occur only during periods of nitrogen starvation and fail to accumulate during logarithmic growth, when the majority of cells are in G2. We show that G1 arrest is the specific nitrogen starvation-induced event that promotes NHEJ between taz1(-) telomeres. Furthermore, the general levels of NHEJ and HR are reciprocally regulated through the cell cycle, so that NHEJ is 10-fold higher in early G1 than in other cell cycle stages; the reverse is true for HR. Whereas NHEJ is known to be dispensable for survival of DSBs in cycling cells, we find that it is critical for repair and survival of DSBs arising during G1.
Telomeres share the ability to silence nearby transcription with heterochromatin, but the requirement of heterochromatin proteins for most telomere functions is unknown. The fission yeast Rik1 protein is required for heterochromatin formation at centromeres and the mating-type locus, as it recruits the Clr4 histone methyltransferase, whose modification of histone H3 triggers binding by Swi6, a conserved protein involved in spreading of heterochromatin. Here, we demonstrate that Rik1 and Clr4, but not Swi6, are required along with the telomere protein Taz1 for crucial chromosome movements during meiosis. However, Rik1 is dispensable for the protective roles of telomeres in preventing chromosome end-fusion. Thus, a Swi6-independent heterochromatin function distinct from that at centromeres and the mating-type locus operates at telomeres during sexual differentiation.
The protective "cap" that assembles at chromosome ends recruits and controls an intricate network of biochemical activities, each one critical for telomere structure and the maintenance of genomic stability. Recent studies have uncovered the components of telomere caps and have started to define the pathways that lead from telomere dysfunction to chromosomal catastrophe.
Telomerase regulation is critical to genome maintenance yet remains poorly understood. Without telomerase's ability to synthesize telomere repeats, chromosome ends shorten progressively, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. In Saccharomyces cerevisiae, telomerase activity in vivo absolutely depends on a set of telomerase accessory proteins that includes Est1p, which appears to recruit or activate telomerase at the site of polymerization. Thus, est1Delta cells have the same cellular senescence phenotype as cells lacking either the catalytic protein subunit of telomerase or its template-containing RNA subunit. While the telomerase protein is highly conserved among eukaryotes, the apparent lack of Est1p homologs has frustrated efforts to describe a common mechanism of telomerase recruitment and activation. Here, we describe SpEst1p, a homolog of Est1p from the evolutionarily distant Schizosaccharomyces pombe. Like ScEst1p, SpEst1p is required for telomerase activity in vivo. Coupled with the identification of an orthologous Est1 protein in humans [10], this suggests a much wider conservation of telomerase regulation than was previously known. Strikingly, in cells with compromised telomere function (taz1Delta), SpEst1p loss confers a lethal germination phenotype, while telomerase loss does not, indicating that SpEst1p plays an unexpected additional role in chromosome end protection.
One fundamental function of telomeres is to prevent the ends of chromosomes from being sensed and treated as DNA damage. Here we present evidence for additional roles of telomeres in promoting proper chromosome segregation and DNA repair. We find that the fission yeast telomere protein Taz1p is required for cell cycle progression at 20 degrees C, a temperature at which taz1Delta cells exhibit a G(2)/M DNA damage checkpoint delay, chromosome missegregation, and DNA double-strand breaks (DSBs). Spindle assembly checkpoint components and a checkpoint-independent function of Rad3p are required for taz1Delta cells to survive at 20 degrees C. Disruption of topoisomerase II activity suppresses the cold sensitivity of taz1Delta cells, suggesting a scenario in which telomeric entanglement is the primary defect. Furthermore, hypersensitivity to treatments that induce DSBs suggests that Taz1p is involved in DSB repair. Our observations imply roles for Taz1p-containing telomeres in preventing and repairing DNA breaks throughout the genome.