scholarly journals Uncapping and Deregulation of Telomeres Lead to Detrimental Cellular Consequences in Yeast

1999 ◽  
Vol 145 (2) ◽  
pp. 203-214 ◽  
Author(s):  
Christopher D. Smith ◽  
Elizabeth H. Blackburn
Keyword(s):  
Dna Sequences ◽  
Tandem Repeats ◽  
Function Analysis ◽  
Kluyveromyces Lactis ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Function ◽  
Morphological Defects ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Structures

Telomeres are the protein–nucleic acid structures at the ends of eukaryote chromosomes. Tandem repeats of telomeric DNA are templated by the RNA component (TER1) of the ribonucleoprotein telomerase. These repeats are bound by telomere binding proteins, which are thought to interact with other factors to create a higher-order cap complex that stabilizes the chromosome end. In the budding yeast Kluyveromyces lactis, the incorporation of certain mutant DNA sequences into telomeres leads to uncapping of telomeres, manifested by dramatic telomere elongation and increased length heterogeneity (telomere deregulation). Here we show that telomere deregulation leads to enlarged, misshapen “monster” cells with increased DNA content and apparent defects in cell division. However, such deregulated telomeres became stabilized at their elongated lengths upon addition of only a few functionally wild-type telomeric repeats to their ends, after which the frequency of monster cells decreased to wild-type levels. These results provide evidence for the importance of the most terminal repeats at the telomere in maintaining the cap complex essential for normal telomere function. Analysis of uncapped and capped telomeres also show that it is the deregulation resulting from telomere uncapping, rather than excessive telomere length per se, that is associated with DNA aberrations and morphological defects.

Dynamics of Telomeric DNA Turnover in Yeast

Genetics ◽  
2002 ◽  
Vol 160 (1) ◽  
pp. 63-73
Author(s):  
Michael J McEachern ◽  
Dana Hager Underwood ◽  
Elizabeth H Blackburn
Keyword(s):  
Kluyveromyces Lactis ◽  
Mutant Gene ◽  
Dna Repeats ◽  
Wild Type ◽  
Telomeric Dna ◽  
Cell Divisions ◽  
Dna Turnover ◽  
Length Regulation ◽  
Turn Over

Abstract Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.

scholarly journals Genetic Dissection of the Kluyveromyces lactis Telomere and Evidence for Telomere Capping Defects in TER1 Mutants with Long Telomeres

2004 ◽  
Vol 3 (2) ◽  
pp. 369-384 ◽  
Author(s):  
Dana H. Underwood ◽  
Coleen Carroll ◽  
Michael J. McEachern
Keyword(s):  
Binding Site ◽  
Telomere Length ◽  
Kluyveromyces Lactis ◽  
Genetic Dissection ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
Functional Regions ◽  
Telomere Capping ◽  
Terminal Repeats

ABSTRACT In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3′ of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3′ terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.

scholarly journals Telomeres and telomerase

2004 ◽  
Vol 359 (1441) ◽  
pp. 109-122 ◽  
Author(s):  
Simon R. W. L. Chan ◽  
Elizabeth H. Blackburn
Keyword(s):  
Reverse Transcriptase ◽  
Tandem Repeats ◽  
Protein Complexes ◽  
Genomic Stability ◽  
Telomeric Dna ◽  
Catastrophic Event ◽  
Template Sequence ◽  
Telomere Function ◽  
Dna Strand

Telomeres are the protective DNA–protein complexes found at the ends of eukaryotic chromosomes. Telomeric DNA consists of tandem repeats of a simple, often G–rich, sequence specified by the action of telomerase, and complete replication of telomeric DNA requires telomerase. Telomerase is a specialized cellular ribonucleoprotein reverse transcriptase. By copying a short template sequence within its intrinsic RNA moiety, telomerase synthesizes the telomeric DNA strand running 5' to 3' towards the distal end of the chromosome, thus extending it. Fusion of a telomere, either with another telomere or with a broken DNA end, generally constitutes a catastrophic event for genomic stability. Telomerase acts to prevent such fusions. The molecular consequences of telomere failure, and the molecular contributors to telomere function, with an emphasis on telomerase, are discussed here.

scholarly journals Characterization of γ-Butyrolactone Autoregulatory Signaling Gene Homologs in the Angucyclinone Polyketide WS5995B Producer Streptomyces acidiscabies

2009 ◽  
Vol 191 (15) ◽  
pp. 4786-4797 ◽  
Author(s):  
Frank G. Healy ◽  
Kevin P. Eaton ◽  
Prajit Limsirichai ◽  
Joel F. Aldrich ◽  
Alaina K. Plowman ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Type Strain ◽  
Wild Type Strain ◽  
Morphological Differentiation ◽  
Pcr Analysis ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Morphological Defects ◽  
In The Wild ◽  
Streptomyces Acidiscabies

ABSTRACT Organisms belonging to the genus Streptomyces produce numerous important secondary metabolites and undergo a sophisticated morphological differentiation program. In many instances these processes are under the control of γ-butyrolactone (GBL) autoregulatory systems. Streptomyces acidiscabies strain 84.104 produces the secondary metabolite aromatic angucyclinone polyketide WS5995B. In order to explore the role of GBL regulatory circuitry in WS5995B production and morphogenesis in S. acidiscabies, a gene cluster encoding GBL autoregulatory signaling homologs was identified and characterized. Two GBL receptor homologs, sabR and sabS, were found flanking a GBL synthase homolog sabA. Strains carrying mutations in sabS produced elevated levels of WS5995B and displayed conditional morphological defects reminiscent of defects seen in Streptomyces bldA mutants. Notably, sabS possesses a TTA codon predicted to be recognized by tRNAleu. sabA mutants produced higher levels of WS5995B than the wild-type strain but to a lesser extent than the levels of WS5995B seen in sabS mutants. Purified recombinant SabR and SabS were tested for their abilities to bind predicted AT-rich autoregulatory element (ARE) boxes within the sabRAS region. SabS did not bind any DNA sequences in this region, while SabR bound an ARE box in the region upstream of sabS. Quantitative reverse transcription-PCR analysis revealed higher levels of sabS transcript in sabR mutants than in the wild-type strain, suggesting that sabS expression is repressed by SabR. Based on these data, we propose that the S. acidiscabies sabRAS genes encode components of a signaling pathway which participates in the regulation of WS5995B production and morphogenesis.

scholarly journals Telomere Instability in Lynch Syndrome Families Leads to Some Shorter Telomeres in MSH2+/- Carriers

Life ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 265
Author(s):  
M. Carmen Garrido-Navas ◽  
Frances Tippins ◽  
Julian Barwell ◽  
Jonathan Hoffman ◽  
Veryan Codd ◽  
...  
Keyword(s):  
Lynch Syndrome ◽  
Telomere Length ◽  
Short Tandem Repeats ◽  
Tandem Repeats ◽  
Cell Turnover ◽  
Wild Type ◽  
Telomeric Dna ◽  
Dna Mismatch ◽  
And Control ◽  
Short Tandem

Lynch syndrome (LS) is an inherited predisposition to early onset of various cancers, caused by mutation in a DNA mismatch repair (MMR) gene. In heterozygous MMR+/− carriers, somatic mutation, loss or silencing of the wild type allele increases the mutation rate, facilitating the initiation of MMR-defective cancers. These cancers are characterized by instability at short tandem repeats (STRs) and in telomeric DNA. We have investigated telomere length in saliva DNA from LS and control families, using single telomere analysis at XpYp and 12q and by qPCR to measure total telomeric DNA. Single telomere analysis showed a trend for shorter XpYp telomeres in MSH2+/− carriers compared to MLH1+/− carriers or controls, but this was masked in the comparative analysis of total telomeric DNA. Comparison of age-adjusted telomere length within families showed that neither MSH2+/− or MLH1+/− children had consistently shorter or longer telomeres than their MMR+/− parent, indicating the absence of an inter-generational effect on telomere length. Unexpectedly however, wildtype children in families with MSH2 mutations, had significantly longer XpYp telomeres than their MMR+/− parent. Altogether our data suggest that MMR insufficiency, particularly in MSH2+/− carriers, increases telomere instability and somatic cell turnover during the lifetime of LS mutation carriers but has minimal consequences for telomere length in the germline.

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

scholarly journals Involvement of Very Short DNA Tandem Repeats and the Influence of the RAD52 Gene on the Occurrence of Deletions in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 549-557 ◽  
Author(s):  
Anne J Welcker ◽  
Jacky de Montigny ◽  
Serge Potier ◽  
Jean-Luc Souciet
Keyword(s):  
Fusion Proteins ◽  
Tandem Repeats ◽  
Chromosomal Rearrangements ◽  
Reversion Rate ◽  
Wild Type ◽  
Recombination Mechanism ◽  
Nonhomologous Recombination ◽  
Deletion Rate ◽  
Dna Tandem Repeats

Abstract Chromosomal rearrangements, such as deletions, duplications, or Ty transposition, are rare events. We devised a method to select for such events as Ura+ revertants of a particular ura2 mutant. Among 133 Ura+ revertants, 14 were identified as the result of a deletion in URA2. Of seven classes of deletions, six had very short regions of identity at their junctions (from 7 to 13 bp long). This strongly suggests a nonhomologous recombination mechanism for the formation of these deletions. The total Ura+ reversion rate was increased 4.2-fold in a rad52Δ strain compared to the wild type, and the deletion rate was significantly increased. All the deletions selected in the rad52Δ context had microhomologies at their junctions. We propose two mechanisms to explain the occurrence of these deletions and discuss the role of microhomology stretches in the formation of fusion proteins.

scholarly journals Telomeric DNA sequences in beetle taxa vary with species richness

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniela Prušáková ◽  
Vratislav Peska ◽  
Stano Pekár ◽  
Michal Bubeník ◽  
Lukáš Čížek ◽  
...  
Keyword(s):  
Species Richness ◽  
Dna Sequences ◽  
Genome Instability ◽  
Telomeric Sequence ◽  
Dna Repeats ◽  
Telomeric Dna ◽  
Common Pattern ◽  
Telomeric Sequences ◽  
Genomic Changes ◽  
Protective Structures

AbstractTelomeres are protective structures at the ends of eukaryotic chromosomes, and disruption of their nucleoprotein composition usually results in genome instability and cell death. Telomeric DNA sequences have generally been found to be exceptionally conserved in evolution, and the most common pattern of telomeric sequences across eukaryotes is (TxAyGz)n maintained by telomerase. However, telomerase-added DNA repeats in some insect taxa frequently vary, show unusual features, and can even be absent. It has been speculated about factors that might allow frequent changes in telomere composition in Insecta. Coleoptera (beetles) is the largest of all insect orders and based on previously available data, it seemed that the telomeric sequence of beetles varies to a great extent. We performed an extensive mapping of the (TTAGG)n sequence, the ancestral telomeric sequence in Insects, across the main branches of Coleoptera. Our study indicates that the (TTAGG)n sequence has been repeatedly or completely lost in more than half of the tested beetle superfamilies. Although the exact telomeric motif in most of the (TTAGG)n-negative beetles is unknown, we found that the (TTAGG)n sequence has been replaced by two alternative telomeric motifs, the (TCAGG)n and (TTAGGG)n, in at least three superfamilies of Coleoptera. The diversity of the telomeric motifs was positively related to the species richness of taxa, regardless of the age of the taxa. The presence/absence of the (TTAGG)n sequence highly varied within the Curculionoidea, Chrysomeloidea, and Staphylinoidea, which are the three most diverse superfamilies within Metazoa. Our data supports the hypothesis that telomere dysfunctions can initiate rapid genomic changes that lead to reproductive isolation and speciation.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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