Publications
2003
Heyman T., Wilhelm M., Wilhelm F. X.
The central PPT of the yeast retrotransposon Ty1 is not essential for transposition Article de journal
Dans: J Mol Biol, vol. 331, no. 2, p. 315-20, 2003, (0022-2836 Journal Article).
Résumé | BibTeX | Étiquettes: Base, cerevisiae/genetics, Data, DNA/*biosynthesis, Genetic, Gov't, Models, Molecular, Mutation, Non-U.S., Purines/*chemistry, Retroelements/*genetics, Saccharomyces, Sequence, Support
@article{,
title = {The central PPT of the yeast retrotransposon Ty1 is not essential for transposition},
author = { T. Heyman and M. Wilhelm and F. X. Wilhelm},
year = {2003},
date = {2003-01-01},
journal = {J Mol Biol},
volume = {331},
number = {2},
pages = {315-20},
abstract = {The yeast retrotransposon Ty1 has structural and functional similarities to retroviruses. We report here that, as in retroviruses, the plus-strand DNA of Ty1 is synthesized as two segments. A central DNA flap is formed during reverse transcription consecutive to elongation (with strand displacement) of the upstream segment beyond the central polypurine tract (cPPT) until the replication machinery is stopped at the central termination sequence. Comparison of wild-type and cPPT-mutant Ty1 elements shows that the mutant element lacking the central DNA flap is only twofold defective in transposition.},
note = {0022-2836
Journal Article},
keywords = {Base, cerevisiae/genetics, Data, DNA/*biosynthesis, Genetic, Gov't, Models, Molecular, Mutation, Non-U.S., Purines/*chemistry, Retroelements/*genetics, Saccharomyces, Sequence, Support},
pubstate = {published},
tppubtype = {article}
}
Wilhelm F. X., Wilhelm M., Gabriel A.
Extension and cleavage of the polypurine tract plus-strand primer by Ty1 reverse transcriptase Article de journal
Dans: J Biol Chem, vol. 278, no. 48, p. 47678-84, 2003, (0021-9258 Journal Article).
Résumé | BibTeX | Étiquettes: Base, Calf, Data, DNA, DNA/chemistry, Genetic, Gov't, H, Messenger/metabolism, Models, Molecular, Non-U.S., P.H.S., Polymerase/*chemistry, Primers, Proteins/chemistry, Purines/*chemistry, Recombinant, Replication, Retroelements/*genetics, Ribonuclease, RNA, RNA-Directed, RNA/chemistry, Sequence, Support, Templates, Thymus/chemistry, U.S., Viral
@article{,
title = {Extension and cleavage of the polypurine tract plus-strand primer by Ty1 reverse transcriptase},
author = { F. X. Wilhelm and M. Wilhelm and A. Gabriel},
year = {2003},
date = {2003-01-01},
journal = {J Biol Chem},
volume = {278},
number = {48},
pages = {47678-84},
abstract = {Using hybrid RNA/DNA substrates containing the polypurine tract (PPT) plus-strand primer, we have examined the interaction between the Ty1 reverse transcriptase (RT) and the plus-strand initiation complex. We show here that, although the PPT sequence is relatively resistant to RNase H cleavage, it can be cleaved internally by the polymerase-independent RNase H activity of Ty1 RT. Alternatively, this PPT can be used to initiate plus-strand DNA synthesis. We demonstrate that cleavage at the PPT/DNA junction occurs only after at least 9 nucleotides are extended. Cleavage leaves a nick between the RNA primer and the nascent plus-strand DNA. We show that Ty1 RT has a strand displacement activity beyond a gap but that the PPT is not efficiently re-utilized in vitro for another round of DNA synthesis after a first plus-strand DNA has been synthesized and cleaved at the PPT/U3 junction.},
note = {0021-9258
Journal Article},
keywords = {Base, Calf, Data, DNA, DNA/chemistry, Genetic, Gov't, H, Messenger/metabolism, Models, Molecular, Non-U.S., P.H.S., Polymerase/*chemistry, Primers, Proteins/chemistry, Purines/*chemistry, Recombinant, Replication, Retroelements/*genetics, Ribonuclease, RNA, RNA-Directed, RNA/chemistry, Sequence, Support, Templates, Thymus/chemistry, U.S., Viral},
pubstate = {published},
tppubtype = {article}
}
2002
Cristofari G., Bampi C., Wilhelm M., Wilhelm F. X., Darlix J. L.
A 5'-3' long-range interaction in Ty1 RNA controls its reverse transcription and retrotransposition Article de journal
Dans: EMBO J, vol. 21, no. 16, p. 4368-79, 2002, (0261-4189 Journal Article).
Résumé | BibTeX | Étiquettes: *Gene, *Transcription, Acid, cerevisiae/*genetics, Complementary/biosynthesis, Conformation, DNA, Expression, Fungal, Fungal/chemistry/*metabolism, Genetic, Gov't, in, Messenger/chemistry/*metabolism, Non-U.S., Nucleic, Phylogeny, Regulation, Retroelements/*genetics, RNA, Saccharomyces, Support, vitro
@article{,
title = {A 5'-3' long-range interaction in Ty1 RNA controls its reverse transcription and retrotransposition},
author = { G. Cristofari and C. Bampi and M. Wilhelm and F. X. Wilhelm and J. L. Darlix},
year = {2002},
date = {2002-01-01},
journal = {EMBO J},
volume = {21},
number = {16},
pages = {4368-79},
abstract = {LTR-retrotransposons are abundant components of all eukaryotic genomes and appear to be key players in their evolution. They share with retroviruses a reverse transcription step during their replication cycle. To better understand the replication of retrotransposons as well as their similarities to and differences from retroviruses, we set up an in vitro model system to examine minus-strand cDNA synthesis of the yeast Ty1 LTR-retrotransposon. Results show that the 5' and 3' ends of Ty1 genomic RNA interact through 14 nucleotide 5'-3' complementary sequences (CYC sequences). This 5'-3' base pairing results in an efficient initiation of reverse transcription in vitro. Transposition of a marked Ty1 element and Ty1 cDNA synthesis in yeast rely on the ability of the CYC sequences to base pair. This 5'-3' interaction is also supported by phylogenic analysis of all full-length Ty1 and Ty2 elements present in the Saccharomyces cerevisiae genome. These novel findings lead us to propose that circularization of the Ty1 genomic RNA controls initiation of reverse transcription and may limit reverse transcription of defective retroelements.},
note = {0261-4189
Journal Article},
keywords = {*Gene, *Transcription, Acid, cerevisiae/*genetics, Complementary/biosynthesis, Conformation, DNA, Expression, Fungal, Fungal/chemistry/*metabolism, Genetic, Gov't, in, Messenger/chemistry/*metabolism, Non-U.S., Nucleic, Phylogeny, Regulation, Retroelements/*genetics, RNA, Saccharomyces, Support, vitro},
pubstate = {published},
tppubtype = {article}
}
2001
Boutabout M., Wilhelm M., Wilhelm F. X.
DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1 Article de journal
Dans: Nucleic Acids Res, vol. 29, no. 11, p. 2217-22, 2001, (1362-4962 Journal Article).
Résumé | BibTeX | Étiquettes: cerevisiae/*genetics/metabolism, DNA, Fungal/genetics, Fungal/genetics/*metabolism, Genetic, Gov't, Kinetics, Non-U.S., Nucleotides/genetics/metabolism, Polymerase/*metabolism, Retroelements/*genetics, RNA, RNA-Directed, Saccharomyces, Support, Templates
@article{,
title = {DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1},
author = { M. Boutabout and M. Wilhelm and F. X. Wilhelm},
year = {2001},
date = {2001-01-01},
journal = {Nucleic Acids Res},
volume = {29},
number = {11},
pages = {2217-22},
abstract = {The fidelity of the yeast retrotransposon Ty1 reverse transcriptase (RT) was determined by an assay based on gel electrophoresis. Steady-state kinetics analyses of deoxyribonucleotide (dNTP) incorporation at a defined primer-template site indicate that Ty1 RT misincorporates dNTP at a frequency of 0.45 x 10(-5) for the A(t):A mispair in which dATP is misincorporated opposite a template A to 6.27 x 10(-5) for the C(t):A mispair. The G(t):G and T(t):T mispairs are formed with very low efficiency. The fidelity parameters of Ty1 RT do not depend on whether RNA or DNA are copied. Relative to lentiviral RTs (HIV-1, HIV-2 or EIAV) Ty1 RT is approximately 10-fold less error prone. Our data also show that the Ty1 RT is able to recapitulate two error-generating mechanisms: extension of mismatches and non-templated addition of nucleotides at the end of a blunt-end primer-template.},
note = {1362-4962
Journal Article},
keywords = {cerevisiae/*genetics/metabolism, DNA, Fungal/genetics, Fungal/genetics/*metabolism, Genetic, Gov't, Kinetics, Non-U.S., Nucleotides/genetics/metabolism, Polymerase/*metabolism, Retroelements/*genetics, RNA, RNA-Directed, Saccharomyces, Support, Templates},
pubstate = {published},
tppubtype = {article}
}
2000
Wilhelm M., Boutabout M., Wilhelm F. X.
Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities Article de journal
Dans: Biochem J, vol. 348, no. Pt 2, p. 337-42, 2000, (0264-6021 Journal Article).
Résumé | BibTeX | Étiquettes: &, Acid, affinity, Alignment, Amino, Calf, cerevisiae/*enzymology/*genetics, Chromatography, Cloning, Codon, coli, Comparative, Data, DNA, DNA/metabolism, Escherichia, Frames, Fusion, Genetic, Gov't, H, Heteroduplexes/metabolism, HIV-1, Homology, Integrases/chemistry/metabolism, Kinetics, Molecular, Non-U.S., Nucleic, Open, Polymerase/chemistry/isolation, Proteins/chemistry/isolation, purification/*metabolism, purification/metabolism, Reading, Recombinant, Retroelements/*genetics, Reverse, Ribonuclease, RNA-Directed, RNA/metabolism, Saccharomyces, Sequence, Study, Support, Templates, Terminator, Thymus/isolation, Transcriptase/chemistry
@article{,
title = {Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities},
author = { M. Wilhelm and M. Boutabout and F. X. Wilhelm},
year = {2000},
date = {2000-01-01},
journal = {Biochem J},
volume = {348},
number = {Pt 2},
pages = {337-42},
abstract = {Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.},
note = {0264-6021
Journal Article},
keywords = {&, Acid, affinity, Alignment, Amino, Calf, cerevisiae/*enzymology/*genetics, Chromatography, Cloning, Codon, coli, Comparative, Data, DNA, DNA/metabolism, Escherichia, Frames, Fusion, Genetic, Gov't, H, Heteroduplexes/metabolism, HIV-1, Homology, Integrases/chemistry/metabolism, Kinetics, Molecular, Non-U.S., Nucleic, Open, Polymerase/chemistry/isolation, Proteins/chemistry/isolation, purification/*metabolism, purification/metabolism, Reading, Recombinant, Retroelements/*genetics, Reverse, Ribonuclease, RNA-Directed, RNA/metabolism, Saccharomyces, Sequence, Study, Support, Templates, Terminator, Thymus/isolation, Transcriptase/chemistry},
pubstate = {published},
tppubtype = {article}
}
1997
Wilhelm M., Heyman T., Friant S., Wilhelm F. X.
Heterogeneous terminal structure of Ty1 and Ty3 reverse transcripts Article de journal
Dans: Nucleic Acids Res, vol. 25, no. 11, p. 2161-6, 1997, (0305-1048 Journal Article).
Résumé | BibTeX | Étiquettes: *Nucleic, *Transcription, Acid, Calf, Chain, Conformation, DNA, Fungal/*chemistry/metabolism, Genetic, Gov't, H, Hybridization, Non-U.S., Nucleic, Plasmids/chemistry/genetics/metabolism, Polymerase, Reaction, Replication, Retroelements/*genetics, Ribonuclease, RNA, Support, Thymus/metabolism, Transfer/chemistry
@article{,
title = {Heterogeneous terminal structure of Ty1 and Ty3 reverse transcripts},
author = { M. Wilhelm and T. Heyman and S. Friant and F. X. Wilhelm},
year = {1997},
date = {1997-01-01},
journal = {Nucleic Acids Res},
volume = {25},
number = {11},
pages = {2161-6},
abstract = {A specific terminal structure of preintegrative DNA is required for transposition of retroviruses and LTR-retrotransposons. We have used an anchored PCR technique to map the 3'ends of DNA intermediates synthesized inside yeast Ty1 and Ty3 retrotransposon virus-like particles. We find that, unlike retroviruses, Ty1 replicated DNA does not have two extra base pairs at its 3'ends. In contrast some Ty3 preintegrative DNA molecules have two extra nucleotides at the 3'end of upstream and downstream long terminal repeats. Moreover we find that some molecules of replicated Ty3 DNA have more than two extra nucleotides at the 3'end of the upstream LTR. This observation could be accounted for by imprecise RNAse H cutting of the PPT sequence. The site of Ty1 and Ty3 plus-strand strong-stop DNA termination was also examined. Our results confirm that the prominent Ty1 and Ty3 plus-strand strong-stop molecules harbor 12 tRNA templated bases but also show that some Ty1 and Ty3 plus-strand strong-stop DNA molecules harbor less tRNA templated bases. We propose that these less than full length plus-strand molecules could be active intermediates in Ty retrotransposon replication.},
note = {0305-1048
Journal Article},
keywords = {*Nucleic, *Transcription, Acid, Calf, Chain, Conformation, DNA, Fungal/*chemistry/metabolism, Genetic, Gov't, H, Hybridization, Non-U.S., Nucleic, Plasmids/chemistry/genetics/metabolism, Polymerase, Reaction, Replication, Retroelements/*genetics, Ribonuclease, RNA, Support, Thymus/metabolism, Transfer/chemistry},
pubstate = {published},
tppubtype = {article}
}
1996
Friant S., Heyman T., Wilhelm M. L., Wilhelm F. X.
Extended interactions between the primer tRNAi(Met) and genomic RNA of the yeast Ty1 retrotransposon Article de journal
Dans: Nucleic Acids Res, vol. 24, no. 3, p. 441-9, 1996, (0305-1048 Journal Article).
Résumé | BibTeX | Étiquettes: Acid, Base, cerevisiae, Conformation, Data, Gov't, Met/genetics/*metabolism, Molecular, Mutation, Non-U.S., Nucleic, Retroelements/*genetics, RNA, RNA/genetics/*metabolism, Saccharomyces, Sequence, structure, Support, Transfer
@article{,
title = {Extended interactions between the primer tRNAi(Met) and genomic RNA of the yeast Ty1 retrotransposon},
author = { S. Friant and T. Heyman and M. L. Wilhelm and F. X. Wilhelm},
year = {1996},
date = {1996-01-01},
journal = {Nucleic Acids Res},
volume = {24},
number = {3},
pages = {441-9},
abstract = {Reverse transcription of the yeast Ty1 retrotransposon is primed by tRNAi(Met) base paired to the primer binding site near the 5'-end of Ty1 genomic RNA. To understand the molecular basis of the tRNAi(Met)-Ty1 RNA interaction the secondary structure of the binary complex was analysed. Enzymatic probes were used to test the conformation of tRNAi(Met) and of Ty1 RNA in the free form and in the complex. A secondary structure model of the tRNAi(Met) Ty1 RNA complex consistent with the probing data was constructed with the help of a computer program. The model shows that besides interactions between the primer binding site and the last 10 nt at the 3'-end of tRNAi(Met), three short regions of Ty1 RNA named boxes 0, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Mutations were made in the boxes or in the complementary sequences of tRNAi(Met) to study the contribution of these sequences to formation of the complex. We find that interaction with at least one of the two boxes 0 or 1 is absolutely required for efficient annealing of the two RNAs. Sequence comparison showing that the primary sequence of the boxes is strictly conserved in Ty1 and Ty2 elements and previously published in vivo results underline the functional importance of the primary sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primary tRNAi(Met) play a role in the reverse transcription pathway.},
note = {0305-1048
Journal Article},
keywords = {Acid, Base, cerevisiae, Conformation, Data, Gov't, Met/genetics/*metabolism, Molecular, Mutation, Non-U.S., Nucleic, Retroelements/*genetics, RNA, RNA/genetics/*metabolism, Saccharomyces, Sequence, structure, Support, Transfer},
pubstate = {published},
tppubtype = {article}
}