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Dorothy E Shippen

Publications and source records attributed to Dorothy E Shippen.

At least 19 recordsLinked to original sources
PubMed2006

Telomere rapid deletion regulates telomere length in Arabidopsis thaliana.

Telomere length is maintained in species-specific equilibrium primarily through a competition between telomerase-mediated elongation and the loss of terminal DNA through the end-replication problem. Recombinational activities are also capable of both lengthening and shortening telomeres. Here we demonstrate that elongated telomeres in Arabidopsis Ku70 mutants reach a new length set point after three generations. Restoration of wild-type Ku70 in these mutants leads to discrete telomere-shortening events consistent with telomere rapid deletion (TRD). These findings imply that the longer telomere length set point is achieved through competition between overactive telomerase and TRD. Surprisingly, in the absence of telomerase, a subset of elongated telomeres was further lengthened, suggesting that in this background a mechanism of telomerase-independent lengthening of telomeres operates. Unexpectedly, we also found that plants possessing wild-type-length telomeres exhibit TRD when telomerase is inactivated. TRD is stochastic, and all chromosome ends appear to be equally susceptible. The frequency of TRD decreases as telomeres shorten; telomeres less than 2 kb in length are rarely subject to TRD. We conclude that TRD functions as a potent force to regulate telomere length in Arabidopsis.

Arabidopsis↗
PubMed2006

The role of the nonhomologous end-joining DNA double-strand break repair pathway in telomere biology.

Double-strand breaks are a cataclysmic threat to genome integrity. In higher eukaryotes the predominant recourse is the nonhomologous end-joining (NHEJ) double-strand break repair pathway. NHEJ is a versatile mechanism employing the Ku heterodimer, ligase IV/XRCC4 and a host of other proteins that juxtapose two free DNA ends for ligation. A critical function of telomeres is their ability to distinguish the ends of linear chromosomes from double-strand breaks, and avoid NHEJ. Telomeres accomplish this feat by forming a unique higher order nucleoprotein structure. Paradoxically, key components of NHEJ associate with normal telomeres and are required for proper length regulation and end protection. Here we review the biochemical mechanism of NHEJ in double-strand break repair, and in the response to dysfunctional telomeres. We discuss the ways in which NHEJ proteins contribute to telomere biology, and highlight how the NHEJ machinery and the telomere complex are evolving to maintain genome stability.

Animals↗
PubMed2005

ATM and ATR make distinct contributions to chromosome end protection and the maintenance of telomeric DNA in Arabidopsis.

Here we examine the function of ATM and ATR at telomeres in Arabidopsis. Although plants lacking ATM or ATR display wild-type telomere length homeostasis, chromosome end protection is compromised in atm atr mutants. Moreover, atm tert Arabidopsis experience an abrupt, early onset of genome instability, arguing that ATM is required for protection of short telomeres. ATR, by contrast, is required for maintenance of telomeric DNA as telomere shortening is dramatically accelerated in atr tert mutants relative to tert plants. Thus, ATM and ATR make essential and distinct contributions to chromosome end protection and telomere maintenance in higher eukaryotes.

Arabidopsis↗
PubMed2005

Telomerase-independent cell survival in Arabidopsis thaliana.

Telomerase is the reverse transcriptase responsible for the maintenance of telomeric repeat sequences in most species that have been studied. Inactivation of telomerase causes telomere shortening and results in the loss of the telomere's protective function, which in mammals leads to cell-cycle arrest and apoptosis. Experiments performed on Arabidopsis thaliana mutants lacking telomerase activity revealed their unusually high tolerance for genome instability. Here we present molecular and cytogenetic analysis of two cell lines (A and B) derived from seeds of late-generation telomerase-deficient A. thaliana. These cultures have survived for about 3 years and are still viable. However, neither culture has adapted mechanisms to maintain terminal telomeric repeats. One culture (B) suffers from severe growth irregularities and a high degree of mortality. Karyological analysis revealed dramatic genomic rearrangements, a large variation in ploidy, and an extremely high percentage of anaphase bridges. The second cell line (A) survived an apparent crisis and phenotypically appears wild-type with respect to growth and morphology. Despite these indications of genome stabilization, a high percentage of anaphase bridges was observed in the A line. We conclude that the restructured chromosome termini provide the A line with partial protection from end-joining repair activities, thus allowing normal growth.

Arabidopsis↗
PubMed2005

The Arabidopsis Pot1 and Pot2 proteins function in telomere length homeostasis and chromosome end protection.

Pot1 (protection of telomeres 1) is a single-stranded telomere binding protein that is essential for chromosome end protection and telomere length homeostasis. Arabidopsis encodes two Pot1-like proteins, dubbed AtPot1 and AtPot2. Here we show that telomeres in transgenic plants expressing a truncated AtPot1 allele lacking the N-terminal oligonucleotide/oligosaccharide binding fold (P1DeltaN) are 1 to 1.5 kb shorter than in the wild type, suggesting that AtPot1 contributes to the positive regulation of telomere length control. In contrast, telomere length is unperturbed in plants expressing the analogous region of AtPot2. A strikingly different phenotype is observed in plants overexpressing the AtPot2 N terminus (P2DeltaC) but not the corresponding region in AtPot1. Although bulk telomeres in P2DeltaC mutants are 1 to 2 kb shorter than in the wild type, these plants resemble late-generation telomerase-deficient mutants with severe growth defects, sterility, and massive genome instability, including bridged chromosomes and aneuploidy. The genome instability associated with P2DeltaC mutants implies that AtPot2 contributes to chromosome end protection. Thus, Arabidopsis has evolved two Pot genes that function differently in telomere biology. These findings provide unanticipated information about the evolution of single-stranded telomere binding proteins.

Amino Acid Sequence↗
PubMed2004

TELOMERASE ACTIVATOR1 induces telomerase activity and potentiates responses to auxin in Arabidopsis.

Telomerase activity is highly regulated, abundant in rapidly dividing cells and reproductive organs, but undetectable in most other differentiated tissues. Little is known about mechanisms that regulate telomerase. Here, we used a biochemical assay to screen activation-tagged lines of Arabidopsis thaliana for mutants that ectopically express this enzyme in their leaves. In one such mutant, a previously uncharacterized zinc-finger protein we designate TELOMERASE ACTIVATOR1 (TAC1) is overexpressed and induces telomerase in fully differentiated leaves without stimulating progression through the cell cycle. Reducing endogenous concentrations of auxin in the mutant blocks the ability of TAC1 to induce telomerase. This result, along with other phenotypes of the mutant, such as the ability of cells to grow in culture without exogenous auxin and increased sensitivity of primary root growth to exogenous auxin, indicates that TAC1 not only is part of the previously reported link between auxin and telomerase expression but also potentiates other classic responses to this phytohormone.

Amino Acid Sequence↗
PubMed2004

A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis.

Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis.

Amino Acid Sequence↗
PubMed2004

Length regulation and dynamics of individual telomere tracts in wild-type Arabidopsis.

Although length of the telomeric DNA tract varies widely across evolution, a species-specific set point is established and maintained by unknown mechanisms. To investigate how telomere length is controlled in Arabidopsis thaliana, we analyzed bulk telomere length in 14 wild-type accessions. We found that telomere tracts in Arabidopsis are fairly uniformly distributed throughout a size range of 2 to 9 kb. Unexpectedly, telomeres in plants of the Wassilewskija ecotype displayed a bimodal size distribution, with some individuals harboring telomeres of 2 to 5 kb and others telomeres of 4 to 9 kb. F1 and F2 progeny of a cross between long and short telomere parents had intermediate telomeres, implying that telomere length in Arabidopsis is not controlled by a single genetic factor. We provide evidence that although global telomere length is strictly regulated within an ecotype-specific range, telomere tracts on individual chromosome ends do not occupy a predetermined length territory. We also demonstrate that individual telomere tracts on homologous chromosomes are coordinately regulated throughout development and that telomerase acts preferentially on the shortest telomeres. We propose that an optimal size for telomere tracts is established and maintained for each Arabidopsis ecotype.

Animals↗
PubMed2004

Molecular analysis of telomere fusions in Arabidopsis: multiple pathways for chromosome end-joining.

End-to-end fusion of critically shortened telomeres in higher eucaryotes is presumed to be mediated by nonhomologous end-joining (NHEJ). Here we describe two PCR-based methods to monitor telomere length and examine the fate of dysfunctional telomeres in Arabidopsis lacking the catalytic subunit of telomerase (TERT) and the DNA repair proteins Ku70 and Mre11. Primer extension telomere repeat amplification relies on the presence of an intact G-overhang, and thus measures functional telomere length. The minimum functional telomere length detected was 300-400 bp. PCR amplification and sequence analysis of chromosome fusion junctions revealed exonucleolytic digestion of dysfunctional ends prior to fusion. In ku70 tert mutants, there was a greater incidence of microhomology at the fusion junction than in tert mutants. In triple ku70 tert mre11 mutants, chromosome fusions were still detected, but microhomology at the junction was no longer favored. These data indicate that both Ku70 and Mre11 contribute to fusion of critically shortened telomeres in higher eucaryotes. Furthermore, Arabidopsis processes critically shortened telomeres as double-strand breaks, using a variety of end-joining pathways.

Arabidopsis↗
PubMed2003

Rearrangements of ribosomal DNA clusters in late generation telomerase-deficient Arabidopsis.

The ends of eukaryotic chromosomes are capped with special nucleoprotein structures called telomeres. Telomere shortening due to telomerase inactivation may result in fusion of homologous or heterologous chromosomes, leading to their successive breakage during anaphase movement, followed by fusion of broken ends in the next cell cycle, i.e. the breakage-fusion-bridge (BFB) cycle. Using fluorescence in situ hybridization (FISH) with 25S rDNA and specific bacterial artificial chromosome (BAC) probes we demonstrate participation of chromosomes 2 and 4 of Arabidopsis thaliana AtTERT null plants in the formation of anaphase bridges. Both homologous and non-homologous chromosomes formed transient anaphase bridges whose breakage and unequal separation led to genome rearrangement, including non-reciprocal translocations and aneuploidy. The 45S rDNA regions located at the ends of chromosomes 2 and 4 were observed in chromosome bridges at a frequency approximately ten times higher than expected in the case of random fusion events. This outcome could result from a functional association of rDNA repeats at nucleoli. We also describe increased variation in the number of nucleoli in some interphase cells with supernumerary rDNA FISH signals. These data indicate that dysfunctional telomeres in Arabidopsis lead to massive genome instability, which is induced by multiple rounds of the BFB mechanism.

Anaphase↗
PubMed2003

Developmentally programmed gene elimination in Euplotes crassus facilitates a switch in the telomerase catalytic subunit.

The primary function of telomerase is to maintain preexisting telomere tracts. In the ciliate Euplotes crassus, however, telomerase RNP structure and substrate recognition are altered during macronuclear development to facilitate de novo telomere addition. We found that E. crassus harbors three TERT genes encoding the telomerase catalytic subunit that not only vary in their nucleotide and predicted protein sequences, but also in their expression profiles. Expression of EcTERT-1 and -3 correlates with the requirement for telomere maintenance, while that of EcTERT-2 correlates with de novo telomere synthesis. All three genes appear to require ribosomal frameshifting for expression of catalytically active protein. The transcriptionally active form of EcTERT-2 exists only transiently in mated cells and is absent from the vegetative macronucleus. Thus, telomerase expression in Euplotes is controlled by unique regulatory mechanisms that culminate in a developmental switch to a different catalytic subunit with properties suited to de novo telomere addition.

Amino Acid Sequence↗
PubMed2003

Ku is required for telomeric C-rich strand maintenance but not for end-to-end chromosome fusions in Arabidopsis.

Telomere dysfunction arising from mutations in telomerase or in telomere capping proteins leads to end-to-end chromosome fusions. Paradoxically, the Ku7080 heterodimer, essential for nonhomologous end-joining double-strand break repair, is also found at telomeres, and in mammals it is required to prevent telomere fusion. Previously, we showed that inactivation of Ku70 in Arabidopsis results in telomere lengthening. Here, we have demonstrated that this telomere elongation is telomerase dependent. Further, we found that the terminal 3' G overhang was significantly extended in ku70 mutants and in plants deficient in both Ku70 and the catalytic subunit of telomerase (TERT), implying that Ku is needed for proper maintenance of the telomeric C-rich strand. Consistent with inefficient C-strand maintenance, telomere shortening was accelerated in ku70 tert double mutants, and the onset of a terminal sterile phenotype was reached two to three times faster than in tert single mutants. Unexpectedly, abundant anaphase bridges were found in terminal plants harboring critically shortened telomeres, indicating that Ku is not required for the formation of end-to-end chromosome fusions in telomerase-deficient Arabidopsis. Together, these findings define Ku70 as a gene in higher eukaryotes required for maintenance of the telomeric C-rich strand and underscore the complexity and diversity of molecular interactions at telomeres.

Arabidopsis↗
PubMed2003

Telomere structure, function and maintenance in Arabidopsis.

The stability of eukaryotic genomes is provided in part by the integrity of telomeres, the nucleoprotein caps on the ends of chromosome. Recent studies reveal that proper telomere architecture is required for long-term proliferation capacity. Here we describe molecular mechanisms that protect and maintain chromosome ends and discuss why Arabidopsis is emerging as a powerful new model for elucidating fundamental aspects of telomere biology.

Arabidopsis↗
PubMed2003

Surprise ending.

Explore the source record for details and available documents.

Alleles↗
PubMed2002

Oligomerization of the telomerase reverse transcriptase from Euplotes crassus.

The telomerase ribonucleoprotein reverse transcriptase uses its RNA subunit as a template to synthesize telomeric repeats and maintain telomere tracts on chromosome ends. In the ciliate Euplotes crassus, the core telomerase ribonucleoprotein particle undergoes a developmentally programmed assembly into three higher order complexes after mating. Here, we provide evidence using oligonucleotide-directed affinity purification that all of the E.crassus telomerase complexes contain at least two enzyme active sites. Furthermore, we show using co-immunoprecipitation experiments that EcTERT, the telomerase catalytic subunit, undergoes multimerization in vitro. Two independent interaction domains were identified in EcTERT, one at the N-terminus that spans amino acids 186-354 and one at the C-terminus that spans amino acids 755-857. Unexpectedly, we found that TERT can form head-to-head, tail-to-tail and head-to-tail oligomers in vitro, implying that E.crassus telomerase has the potential to assume different conformations in vivo. Together, these data indicate that oligomerization is a conserved feature of telomerase and that the minimal functional unit of the enzyme is a dimer.

Animals↗
PubMed2002

Telomere length deregulation and enhanced sensitivity to genotoxic stress in Arabidopsis mutants deficient in Ku70.

The Ku70/80 heterodimer is a critical component of the non-homologous end-joining (NHEJ) pathway and of the telomere cap in yeast and mammals. We report the molecular characterization of the KU70 and KU80 genes in Arabidopsis and describe the consequences of a Ku70 deficiency. Arabidopsis KU70/80 genes are ubiquitously expressed and their products form stable heterodimers in vitro. Plants harboring a T-DNA insertion in KU70 exhibit no growth or developmental defects under standard growth conditions. However, mutant seedlings are hypersensitive to gamma-irradiation-induced double-strand breaks. Unexpectedly, we found that mutants are hypersensitive to methyl methanosulfonate during seed germination, but lose this sensitivity in seedlings, implying that the requirement for NHEJ varies during plant development. Lack of Ku70 results in a dramatic deregulation of telomere length control, with mutant telomeres expanding to more than twice the size of wild type by the second generation. Furthermore, in contrast to the situation in mammals, chromosome fusions are not associated with a Ku deficiency in Arabidopsis. These findings imply that Ku may play a different role in capping plant and animal telomeres.

Alleles↗
PubMed2002

Telomeres, telomerase, and stability of the plant genome.

Telomeres, the complex nucleoprotein structures at the ends of linear eukaryotic chromosomes, along with telomerase, the enzyme that synthesizes telomeric DNA, are required to maintain a stable genome. Together, the enzyme and substrate perform this essential service by protecting chromosomes from exonucleolytic degradation and end-to-end fusions and by compensating for the inability of conventional DNA replication machinery to completely duplicate the ends of linear chromosomes. Telomeres are also important for chromosome organization within the nucleus, especially during mitosis and meiosis. The contributions of telomeres and telomerases to plant genome stability have been confirmed by analysis of Arabidopsis mutants that lack telomerase activity. These mutants have unstable genomes, but manage to survive up to ten generations with increasingly shortened telomeres and cytogenetic abnormalities. Comparisons between telomerase-deficient Arabidopsis and telomerase-deficient mice reveal distinct differences in the consequences of massive genome damage, probably reflecting the greater developmental and genomic plasticity of plants.

Genome, Plant↗