Publications
2003
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}
}
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}
}
1999
Auxilien S., Keith G., Grice S. F. Le, Darlix J. L.
Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription Article de journal
Dans: J Biol Chem, vol. 274, no. 7, p. 4412-20, 1999, (0021-9258 Journal Article).
Résumé | BibTeX | Étiquettes: *RNA, *Transcription, Acid, Base, Calf, Conformation, Data, DNA, Genetic, Gov't, H, HIV-1, HIV-1/*physiology, Lys/*metabolism, Molecular, Non-U.S., Nucleic, post-transcriptional, Processing, Reverse, Ribonuclease, RNA, Sequence, Support, Templates, Thymus/metabolism, Transcriptase/metabolism, Transfer, Viral/*metabolism, Viral/metabolism
@article{,
title = {Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription},
author = { S. Auxilien and G. Keith and S. F. Le Grice and J. L. Darlix},
year = {1999},
date = {1999-01-01},
journal = {J Biol Chem},
volume = {274},
number = {7},
pages = {4412-20},
abstract = {During HIV reverse transcription, (+) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3' end of tRNALys,3 hybridized to the viral primer binding site (PBS). Before (+) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3' end of primer tRNALys,3. Since the detailed mechanism of (+) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV 5' RNA, natural modified tRNALys,3, synthetic unmodified tRNALys,3 or oligonucleotides (RNA or DNA) complementary to the PBS, as well as the viral proteins RT and nucleocapsid protein (NCp7). Prior to (+) strand DNA transfer, RT stalls at the double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe modified nucleosides of natural tRNALys,3. Modified nucleoside m1A-58 of natural tRNALys,3 is only partially effective as a stop signal, as RT can transcribe as far as the hyper-modified adenosine (ms2t6A-37) in the anticodon loop. m1A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m7G-46, which is sometimes transcribed into T. In contrast, synthetic tRNALys,3, an RNA PBS primer, and a DNA PBS primer are completely reverse-transcribed. In the presence of an acceptor template, (+) strand DNA transfer is efficient only with templates containing natural tRNALys,3 or the RNA PBS primer. Sequence analysis of transfer products revealed frequent errors at the transfer site with synthetic tRNALys,3, not observed with natural tRNALys,3. Thus, modified nucleoside m1A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (+) strand DNA transfer. We show that other factors such as the nature of the (-) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (+) strand DNA transfer.},
note = {0021-9258
Journal Article},
keywords = {*RNA, *Transcription, Acid, Base, Calf, Conformation, Data, DNA, Genetic, Gov't, H, HIV-1, HIV-1/*physiology, Lys/*metabolism, Molecular, Non-U.S., Nucleic, post-transcriptional, Processing, Reverse, Ribonuclease, RNA, Sequence, Support, Templates, Thymus/metabolism, Transcriptase/metabolism, Transfer, Viral/*metabolism, Viral/metabolism},
pubstate = {published},
tppubtype = {article}
}