Biomedical subjects

Marcelle Wilhelm

Publications and source records attributed to Marcelle Wilhelm.

3 recordsLinked to original sources

PubMed2006

Cooperation between reverse transcriptase and integrase during reverse transcription and formation of the preintegrative complex of Ty1.

Reverse transcriptase (RT) and integrase (IN) play a central role in the replication and transposition of retroelements. Increasing evidence suggests that the interaction between these two enzymes is functional and plays an important role in replication. In the yeast Saccharomyces cerevisiae retrotransposon Ty1, the interaction of IN with RT is critical for the formation of an active conformation of RT. We show here that the RT associated with VLPs is active only if it is in close interaction with IN. To probe the IN-RT cis-trans relationship, we have used a complementation assay based on coexpressing two transposons. We show that IN acts in cis to activate RT and that a functional integrase provided in trans is not able to complement replication and transposition defects of IN deletion or IN active-site mutant elements. Our data support a model in which IN not only interacts closely with RT during reverse transcription but also remains associated with RT during the formation of the preintegrative complex.

Binding Sites↗

PubMed2003

Extension and cleavage of the polypurine tract plus-strand primer by Ty1 reverse transcriptase.

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.

Base Sequence↗

PubMed2002

A 5'-3' long-range interaction in Ty1 RNA controls its reverse transcription and retrotransposition.

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.

DNA, Complementary↗