Molecular Basis for Telomere Repeat Divergence in Budding Yeast

ABSTRACT Telomerase is a ribonucleoprotein enzyme that adds repetitive sequences to the ends of linear chromosomes, thereby counteracting nucleotide loss due to incomplete replication. A short region of the telomerase RNA subunit serves as template for nucleotide addition onto the telomere 3′ end. Although Saccharomyces cerevisiaecontains only one telomerase RNA gene, telomere repeat sequences are degenerate in this organism. Based on a detailed analysis of the telomere sequences specified by wild-type and mutant RNA templates in vivo, we show that the divergence of telomere repeats is due to abortive reverse transcription in the 3′ and 5′ regions of the template and due to the alignment of telomeres in multiple registers within the RNA template. Through the interpretation of wild-type telomere sequences, we identify nucleotides in the template that are not accessible for base pairing during substrate annealing. Rather, these positions become available as templates for reverse transcription only after alignment with adjacent nucleotides has occurred, indicating that a conformational change takes place upon substrate binding. We also infer that the central part of the template region is reverse transcribed processively. The inaccessibility of certain template positions for alignment and the processive polymerization of the central template portion may serve to reduce the possible repeat diversification and enhance the incorporation of binding sites for Rap1p, the telomere binding protein of budding yeast.

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Targeting Human Immunodeficiency Virus (HIV) Type 2 Integrase Protein into HIV Type 1

Journal of Virology ◽

10.1128/jvi.73.10.8831-8836.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8831-8836 ◽

Cited By ~ 10

Author(s):

Hongmei Liu ◽

Xiaoyun Wu ◽

Hongling Xiao ◽

John C. Kappes

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcription ◽

Wild Type Virus ◽

Strand Transfer ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell’s chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3′ processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN2) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN2 into IN mutant HIV-1 virions by expression intrans as a Vpr-IN fusion protein.Trans-complementation with IN2 markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous trans-IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126–2135, 1999), cells infected with IN2-complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous trans-IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, thetrans-IN2 protein was shown to support integration up to a level of 55% compared with that of the homologoustrans-IN protein. The delivery of heterologous IN protein into HIV-1 particles in trans offers a novel approach to understand IN protein function in vivo.

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Deletion of Budding Yeast MAD2 Suppresses Clone-to-Clone Differences in Artificial Linear Chromosome Copy Numbers and Gives Rise to Higher Retention Rates

Microorganisms ◽

10.3390/microorganisms8101495 ◽

2020 ◽

Vol 8 (10) ◽

pp. 1495

Author(s):

Scott C. Schuyler ◽

Lin-Ing Wang ◽

Yi-Shan Ding ◽

Yi-Chieh Lee ◽

Hsin-Yu Chen

Keyword(s):

Budding Yeast ◽

Retention Rate ◽

Retention Rates ◽

Yeast Cells ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Linear Chromosome ◽

Copy Numbers ◽

Clone Differences ◽

Linear Chromosomes

Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.

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Telomerase-Associated Protein TEP1 Is Not Essential for Telomerase Activity or Telomere Length Maintenance In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.20.21.8178-8184.2000 ◽

2000 ◽

Vol 20 (21) ◽

pp. 8178-8184 ◽

Cited By ~ 47

Author(s):

Yie Liu ◽

Bryan E. Snow ◽

M. Prakash Hande ◽

Gabriela Baerlocher ◽

Valerie A. Kickhoefer ◽

...

Keyword(s):

Telomere Length ◽

Rna Binding ◽

Es Cells ◽

Embryonic Stem ◽

Telomerase Activity ◽

Telomerase Rna ◽

Wild Type ◽

Rnp Complex ◽

Successive Generations

ABSTRACT TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. ThemTep1-deficient (mTep1 −/−) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues frommTep1 −/− mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1 −/− mice. Telomere length, even in later generations ofmTep1 −/− mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

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Expression of Mutated Paramecium Telomerase RNAs In Vivo Leads to Templating Errors That Resemble Those Made by Retroviral Reverse Transcriptase

Molecular and Cellular Biology ◽

10.1128/mcb.19.4.2887 ◽

1999 ◽

Vol 19 (4) ◽

pp. 2887-2894 ◽

Cited By ~ 5

Author(s):

Amanda J. Ye ◽

W. John Haynes ◽

Daniel P. Romero

Keyword(s):

Reverse Transcriptase ◽

De Novo ◽

Telomerase Rna ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Telomeric Dna ◽

Telomeric Repeats ◽

Immunodeficiency Virus ◽

In The Wild

ABSTRACT Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutatedP. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase.

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Expression of Telomerase RNA Template, but Not Telomerase Reverse Transcriptase, Is Limiting for Telomere Length Maintenance In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.7024-7031.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 7024-7031 ◽

Cited By ~ 76

Author(s):

Y. Jeffrey Chiang ◽

Michael T. Hemann ◽

Karen S. Hathcock ◽

Lino Tessarollo ◽

Lionel Feigenbaum ◽

...

Keyword(s):

Reverse Transcriptase ◽

Telomere Length ◽

Telomerase Activity ◽

Telomere Maintenance ◽

Gene Copy ◽

Telomerase Reverse Transcriptase ◽

Telomerase Rna ◽

Wild Type ◽

Telomere Elongation

ABSTRACT Telomerase consists of two essential components, the telomerase RNA template (TR) and telomerase reverse transcriptase (TERT). The haplo-insufficiency of TR was recently shown to cause one form of human dyskeratosis congenita, an inherited disease marked by abnormal telomere shortening. Consistent with this finding, we recently reported that mice heterozygous for inactivation of mouse TR exhibit a similar haplo-insufficiency and are deficient in the ability to elongate telomeres in vivo. To further assess the genetic regulation of telomerase activity, we have compared the abilities of TR-deficient and TERT-deficient mice to maintain or elongate telomeres in interspecies crosses. Homozygous TERT knockout mice had no telomerase activity and failed to maintain telomere length. In contrast, TERT+/− heterozygotes had no detectable defect in telomere elongation compared to wild-type controls, whereas TR+/− heterozygotes were deficient in telomere elongation. Levels of TERT mRNA in heterozygous mice were one-third to one-half the levels expressed in wild-type mice, similar to the reductions in telomerase RNA observed in TR heterozygotes. These findings indicate that both TR and TERT are essential for telomere maintenance and elongation but that gene copy number and transcriptional regulation of TR, but not TERT, are limiting for telomerase activity under the in vivo conditions analyzed.

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Alternative Lengthening of Telomeres in the Budding Yeast Naumovozyma castellii

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400428 ◽

2019 ◽

Vol 9 (10) ◽

pp. 3345-3358 ◽

Cited By ~ 1

Author(s):

Marita Cohn ◽

Ahu Karademir Andersson ◽

Raquel Quintilla Mateo ◽

Mirja Carlsson Möller

Keyword(s):

Budding Yeast ◽

Telomerase Activity ◽

Telomeric Sequence ◽

Wild Type ◽

Alternative Lengthening Of Telomeres ◽

Initiation Point ◽

Interstitial Telomeric Sequence ◽

Alternative Lengthening ◽

Telomere Function ◽

Linear Chromosomes

The enzyme telomerase ensures the integrity of linear chromosomes by maintaining telomere length. As a hallmark of cancer, cell immortalization and unlimited proliferation is gained by reactivation of telomerase. However, a significant fraction of cancer cells instead uses alternative telomere lengthening mechanisms to ensure telomere function, collectively known as Alternative Lengthening of Telomeres (ALT). Although the budding yeast Naumovozyma castellii (Saccharomyces castellii) has a proficient telomerase activity, we demonstrate here that telomeres in N. castellii are efficiently maintained by a novel ALT mechanism after telomerase knockout. Remarkably, telomerase-negative cells proliferate indefinitely without any major growth crisis and display wild-type colony morphology. Moreover, ALT cells maintain linear chromosomes and preserve a wild-type DNA organization at the chromosome termini, including a short stretch of terminal telomeric sequence. Notably, ALT telomeres are elongated by the addition of ∼275 bp repeats containing a short telomeric sequence and the subtelomeric DNA located just internally (TelKO element). Although telomeres may be elongated by several TelKO repeats, no dramatic genome-wide amplification occurs, thus indicating that the repeat addition may be regulated. Intriguingly, a short interstitial telomeric sequence (ITS) functions as the initiation point for the addition of the TelKO element. This implies that N. castellii telomeres are structurally predisposed to efficiently switch to the ALT mechanism as a response to telomerase dysfunction.

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The Role of Nucleocapsid and U5 Stem/A-Rich Loop Sequences in tRNA3Lys Genomic Placement and Initiation of Reverse Transcription in Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.72.5.3907-3915.1998 ◽

1998 ◽

Vol 72 (5) ◽

pp. 3907-3915 ◽

Cited By ~ 35

Author(s):

Yue Huang ◽

Ahmad Khorchid ◽

Juliana Gabor ◽

Jing Wang ◽

Xuguang Li ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Wild Type ◽

Extracellular Virus ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We have studied the effect of mutations in the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) sequence on tRNA3 Lys genomic placement, i.e., the in vivo placement of primer tRNA3 Lys on the HIV-1 primer binding site (PBS). HIV-1 produced from COS cells transfected with wild-type or mutant proviral DNA was used in this study. We have found that mutations in the amino acid sequences flanking the first Cys-His box in the NC sequence produce the maximum inhibition of genomic placement. A similar finding was obtained when the NC-facilitated annealing of primer tRNA3 Lys to the HIV PBS in vitro was studied. However, since the genomic placement of tRNA3 Lys occurs independently of precursor protein processing, the NC mutations studied here have probably exerted their effect through one or both of the precursor proteins, Pr55 gag and/or Pr160 gag-pol . One mutation in the linker region between the two Cys-His boxes, P31L, prevented packaging of both Pr160 gag-pol and tRNA3 Lys and prevented the genomic placement of tRNA3 Lys. Both packaging and genomic placement were rescued by cotransfection with a plasmid coding for wild-type Pr160 gag-pol . For other linker mutations [R7R10K11 S, R32G, and S3(32-34)], packaging of Pr160 gag-pol and tRNA3 Lyswas not affected, but genomic placement was, and placement could not be rescued by cotransfection with plasmids coding for either Pr55 gag or Pr160 gag-pol . After placement, the initiation of reverse transcription within extracellular virions is characterized by a 2-base DNA extension of the placed tRNA3 Lys. This process requires precursor processing, and those NC mutations which showed the most inhibition of initiation were in either of the two NC Cys-His boxes. Destabilization of a U5 stem-A-rich loop immediately upstream of the PBS (through deletion of four consecutive A’s in the loop) did not affect the in vivo genomic placement of tRNA3 Lys but resulted in the presence in the extracellular virus of longer cDNA extensions of tRNA3 Lys, with a corresponding decrease in the presence of unextended and 2-base-extended tRNA3 Lys.

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A Quantitative Assay for Telomere Protection in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/161.3.995 ◽

2002 ◽

Vol 161 (3) ◽

pp. 995-1013 ◽

Cited By ~ 2

Author(s):

Michelle L DuBois ◽

Zara W Haimberger ◽

Martin W McIntosh ◽

Daniel E Gottschling

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Transcriptional Silencing ◽

Repeat Length ◽

Quantitative Assay ◽

Telomeric Dna ◽

Telomere Repeat ◽

Telomere Protection ◽

Linear Chromosomes

Abstract Telomeres are the protective ends of linear chromosomes. Telomeric components have been identified and described by their abilities to bind telomeric DNA, affect telomere repeat length, participate in telomeric DNA replication, or modulate transcriptional silencing of telomere-adjacent genes; however, their roles in chromosome end protection are not as well defined. We have developed a genetic, quantitative assay in Saccharomyces cerevisiae to measure whether various telomeric components protect chromosome ends from homologous recombination. This “chromosomal cap” assay has revealed that the telomeric end-binding proteins, Cdc13p and Ku, both protect the chromosome end from homologous recombination, as does the ATM-related kinase, Tel1p. We propose that Cdc13p and Ku structurally inhibit recombination at telomeres and that Tel1p regulates the chromosomal cap, acting through Cdc13p. Analysis with recombination mutants indicated that telomeric homologous recombination events proceeded by different mechanisms, depending on which capping component was compromised. Furthermore, we found that neither telomere repeat length nor telomeric silencing correlated with chromosomal capping efficiency. This capping assay provides a sensitive in vivo approach for identifying the components of chromosome ends and the mechanisms by which they are protected.

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Tetrahymena Mutants With Short Telomeres

Genetics ◽

10.1093/genetics/150.2.643 ◽

1998 ◽

Vol 150 (2) ◽

pp. 643-650

Author(s):

Shawn Ahmed ◽

Hong Sheng ◽

Luming Niu ◽

Eric Henderson

Keyword(s):

Growth Rate ◽

Telomere Length ◽

Genetic Defect ◽

Telomerase Rna ◽

Wild Type ◽

Cell Type ◽

Variant Cell ◽

Length Regulation ◽

Telomere Length Regulation

Abstract Telomere length is dynamic in many organisms. Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo. In Tetrahymena thermophila, telomeres become long at 30°, and growth rate slows. A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres and a rapid-doubling rate. Here we show that this variant cell type with short telomeres is in fact a mutant with a genetic defect in telomere length regulation. One of these telomere growth inhibited forever (tgi) mutants was heterozygous for a telomerase RNA mutation, and this mutant telomerase RNA caused telomere shortening when overexpressed in wild-type cells. Several other tgi mutants were also likely to be heterozygous at their mutant loci, since they reverted to wild type when selective pressure for short telomeres was removed. These results illustrate that telomere length can regulate growth rate in Tetrahymena and that this phenomenon can be exploited to identify genes involved in telomere length regulation.

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Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase.

Molecular and Cellular Biology ◽

10.1128/mcb.16.1.66 ◽

1996 ◽

Vol 16 (1) ◽

pp. 66-75 ◽

Cited By ~ 52

Author(s):

D Gilley ◽

E H Blackburn

Keyword(s):

Active Site ◽

Tetrahymena Thermophila ◽

Telomerase Rna ◽

Specific Interactions ◽

Wild Type ◽

Specific Primer ◽

Telomeric Dna ◽

Template Sequence ◽

New Evidence

The ribonucleoprotein enzyme telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA by copying a template sequence within the telomerase RNA. Here we analyze the actions of telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-type telomerase RNA to define further the roles of particular telomerase RNA residues involved in essential enzymatic functions: templating, substrate alignment, and promotion of polymerization. Position 49 of the telomerase RNA defined the 3' templating residue boundary, demonstrating that seven positions, residues 43 to 49, are capable of acting as templating residues. We demonstrate directly that positioning of the primer substrate involves Watson-Crick base pairing between the primer with telomerase RNA residues. Unexpectedly, formation of a Watson-Crick base pair specifically between the primer DNA and telomerase RNA residue 50 is critical in promoting primer elongation. In contrast, mutant telomerase with the cytosine at position 49 mutated to a G exhibited efficient 3' mispair extension. This work provides new evidence for specific primer-telomerase interactions, as well as base-specific interactions involving the telomerase RNA, playing roles in essential active-site functions of telomerase.

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