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Specific recognition and cleavage of the plus-strand primer by reverse transcriptase
Angela Atwood-Moore1, Kenechi Ejebe, Henry L Levin
1Section on Eukaryotic Transposable Elements, Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Journal of Virology
|November 12, 2005
Summary
Mutations in the RNase H domain of reverse transcriptase (RT) blocked retrotransposon integration without affecting cDNA synthesis. This suggests polypurine tract (PPT) primer removal is not essential for reverse transcription.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Reverse transcriptases (RTs) are crucial enzymes in retroviruses and retrotransposons, possessing DNA polymerase and RNase H activities.
- These activities facilitate key steps in reverse transcription, including template switching and primer processing, before integrase inserts the cDNA into the host genome.
Purpose of the Study:
- To investigate the role of the RNase H domain in the reverse transcription and integration processes of the LTR-retrotransposon Tf1.
- To identify specific mutations affecting these processes and elucidate their impact on cDNA synthesis and primer removal.
Main Methods:
- Random mutagenesis of the Tf1 RT gene.
- Genetic screening assays to assess reverse transcription and integration efficiency.
- Immunoblotting to quantify RT and integrase levels.
- DNA blotting and sequencing to analyze cDNA ends and primer removal.
Main Results:
- A cluster of mutations in the RNase H domain inhibited integration without reducing cDNA levels.
- These mutations did not affect RT or integrase protein levels or full-length cDNA amounts.
- Mutations specifically impaired the removal of the polypurine tract (PPT) primer from cDNA 3' ends.
Conclusions:
- Primer removal by RNase H is not a necessary step for completing reverse transcription.
- Conserved residues in the RNase H domain contact the PPT and are specifically required for its removal, highlighting a conserved mechanism across different retroelements.