Reverse transcriptase motifs in the catalytic subunit of telomerase.

Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.

[1]  S. Sarafianos,et al.  Glutamine 151 participates in the substrate dNTP binding function of HIV-1 reverse transcriptase. , 1995, Biochemistry.

[2]  A. Zahler,et al.  Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for replication of the ends of linear DNA molecules. , 1988, Nucleic acids research.

[3]  J E Darnell,et al.  Speculations on the early course of evolution. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[4]  R. Kobayashi,et al.  Purification of tetrahymena telomerase and cloning of genes encoding the two protein components of the enzyme , 1995, Cell.

[5]  J. D. Watson.  Origin of Concatemeric T7DNA , 1972 .

[6]  Lea Harrington,et al.  A Mammalian Telomerase-Associated Protein , 1997, Science.

[7]  Carol W. Greider,et al.  The telomere terminal transferase of tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity , 1987, Cell.

[8]  M. Mann,et al.  Electrospray ionization for mass spectrometry of large biomolecules. , 1989, Science.

[9]  A. Podtelejnikov,et al.  Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  E. Blackburn,et al.  Runaway telomere elongation caused by telomerase RNA gene mutations , 1995, Nature.

[11]  H. Temin.  On the origin of RNA tumor viruses. , 2003, Annual review of genetics.

[12]  J. Lin.  The Saccharomyces CDC13 is a single-strand TG1-3 telomeric DNA-binding protein in vitro that affects telomere in vivo , 1996 .

[13]  D. Baltimore.  Viral RNA-dependent DNA Polymerase: RNA-dependent DNA Polymerase in Virions of RNA Tumour Viruses , 1970, Nature.

[14]  E. Blackburn,et al.  An alternative pathway for yeast telomere maintenance rescues est1− senescence , 1993, Cell.

[15]  D. Morris,et al.  Est1 has the properties of a single-stranded telomere end-binding protein. , 1996, Genes & development.

[16]  K. Lowenhaupt,et al.  Drosophila telomeres: new views on chromosome evolution. , 1996, Trends in genetics : TIG.

[17]  T. Cech,et al.  Purification of telomerase from Euplotes aediculatus: requirement of a primer 3' overhang. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[18]  A M Olovnikov,et al.  A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. , 1973, Journal of theoretical biology.

[19]  D. Gottschling,et al.  TLC1: template RNA component of Saccharomyces cerevisiae telomerase. , 1994, Science.

[20]  A. D. Clark,et al.  Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[21]  A. D. Clark,et al.  Structure of HIV-1 reverse transcriptase/DNA complex at 7 Å resolution showing active site locations , 1992, Nature.

[22]  J. Yudkin.  Sugar and Disease , 1972, Nature.

[23]  B. Schwer,et al.  SLU7 and a novel activity, SSF1, act during the PRP16‐dependent step of yeast pre‐mRNA splicing. , 1995, The EMBO journal.

[24]  E. Blackburn,et al.  Functional evidence for an RNA template in telomerase. , 1990, Science.

[25]  et al.,et al.  The RNA component of human telomerase , 1995, Science.

[26]  T. Lendvay,et al.  Senescence mutants of Saccharomyces cerevisiae with a defect in telomere replication identify three additional EST genes. , 1996, Genetics.

[27]  I Sauvaget,et al.  Identification of four conserved motifs among the RNA‐dependent polymerase encoding elements. , 1989, The EMBO journal.

[28]  J. Steitz,et al.  Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. , 1992, The EMBO journal.

[29]  T. Lange,et al.  Activation of telomerase in a human tumor. , 1994 .

[30]  D M Prescott,et al.  The DNA of ciliated protozoa. , 1994 .

[31]  H. Wang,et al.  The Mauriceville plasmid of Neurospora spp. uses novel mechanisms for initiating reverse transcription in vivo , 1994, Molecular and cellular biology.

[32]  T R Hughes,et al.  Cdc13p: A Single-Strand Telomeric DNA-Binding Protein with a Dual Role in Yeast Telomere Maintenance , 1996, Science.

[33]  T. Cech,et al.  Telomerase and DNA end replication: no longer a lagging strand problem? , 1995, Science.

[34]  E. Blackburn,et al.  Telomerase in yeast. , 1995, Science.

[35]  R. Sousa,et al.  Structural and mechanistic relationships between nucleic acid polymerases. , 1996, Trends in biochemical sciences.

[36]  J. Lin,et al.  The Saccharomyces CDC13 protein is a single-strand TG1-3 telomeric DNA-binding protein in vitro that affects telomere behavior in vivo. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[37]  E. Blackburn,et al.  A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis , 1989, Nature.

[38]  H. Biessmann,et al.  Addition of telomere-associated HeT DNA sequences “heals” broken chromosome ends in Drosophila , 1990, Cell.

[39]  Daniel P. Romero,et al.  A conserved secondary structure for telomerase RNA , 1991, Cell.

[40]  E. Blackburn,et al.  The effects of nucleoside analogs on telomerase and telomeres in Tetrahymena. , 1994, Nucleic acids research.

[41]  E. Blackburn,et al.  Telomerase RNA mutations in Saccharomyces cerevisiae alter telomerase action and reveal nonprocessivity in vivo and in vitro. , 1997, Genes & development.

[42]  N. Lue,et al.  ATP-dependent Processivity of a Telomerase Activity from Saccharomyces cerevisiae(*) , 1995, The Journal of Biological Chemistry.

[43]  T. Steitz,et al.  Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. , 1992, Science.

[44]  A. Shevchenko,et al.  Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry , 1996, Nature.

[45]  M. Wilm,et al.  Error-tolerant identification of peptides in sequence databases by peptide sequence tags. , 1994, Analytical chemistry.

[46]  A. Lambowitz,et al.  A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility , 1995, Cell.

[47]  J. Szostak,et al.  A mutant with a defect in telomere elongation leads to senescence in yeast , 1989, Cell.

[48]  L. Hartwell,et al.  Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint , 1995, Molecular and cellular biology.

[49]  A. Matsuura,et al.  TLP1: A Gene Encoding a Protein Component of Mammalian Telomerase Is a Novel Member of WD Repeats Family , 1997, Cell.

[50]  F. Michel,et al.  Group II self-splicing introns in bacteria , 1993, Nature.

[51]  T. Steitz,et al.  Function and structure relationships in DNA polymerases. , 1994, Annual review of biochemistry.

[52]  A. Lambowitz,et al.  The mauriceville plasmid reverse transcriptase can initiate cDNA synthesis de novo and may be related to reverse transcriptase and DNA polymerase progenitor , 1993, Cell.

[53]  T. Cech,et al.  The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers , 1997, Molecular and cellular biology.

[54]  T. Cech,et al.  Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. , 1994, Genes & development.