Reverse transcriptases (RTs) of retroviruses and long terminal repeat (LTR)-retrotransposons possess DNA polymerase and RNase H activities. During reverse transcription these activities are necessary for the programmed sequence of events that include template switching and primer processing. Integrase then inserts the completed cDNA into the genome of the host cell. The RT of the LTR-retrotransposon Tf1 was subjected to random mutagenesis, and the resulting transposons were screened with genetic assays to test which mutations reduced reverse transcription and which inhibited integration. We identified a cluster of mutations in the RNase H domain of RT that were surprising because they blocked integration without reducing cDNA levels. The results of immunoblots demonstrated that these mutations did not reduce levels of RT or integrase. DNA blots showed that the mutations did not lower the amounts of full-length cDNA. The sequences of the 3 ends of the cDNA revealed that mutations within the cluster in RNase H specifically reduced the removal of the polypurine tract (PPT) primer from the ends of the cDNA. These results indicate that primer removal is not a necessary component of reverse transcription. The residues mutated in Tf1 RNase H are conserved in human immunodeficiency virus type 1 and make direct contact with DNA opposite the PPT. Thus, our results identify a conserved element in RT that contacts the PPT and is specifically required for PPT removal.Reverse transcription of retroviruses requires a sequence of programmed steps that include highly specific switching of templates and precise cleavages of RNA primers. The enzyme responsible for these processes, reverse transcriptase (RT), possesses DNA polymerase activity and an RNase H activity that degrades RNA annealed to DNA. The RNase H activity is critical because it degrades RNA templates, allowing strongstop intermediates to transfer to new templates. RNase H activity is also responsible for precise cleavages flanking the polypurine tract (PPT) that "select" the plus-strand primer of reverse transcription. After plus-strand priming has occurred, RNase H removes the PPT. In addition, the RNase H also removes the minus-strand primer. The highly precise cleavages that remove the primers are absolutely critical because ultimately they define the sequence at the ends of the cDNA. For integrase (IN) to complete insertion, the highly conserved CA dinucleotide must be positioned at the 3Ј ends (32).Long terminal repeat (LTR) retrotransposons are closely related to retroviruses and use the same multistep process of reverse transcription to copy their mRNA into full-length double-stranded DNA (32). As a result, retrotransposons are excellent models for the reverse transcription of retroviruses. Previous work revealed that the Tf1 transposon of Schizosaccharomyces pombe is highly active and that the individual steps of reverse transcription can be studied in S. pombe (14,15,19). The presence of all seven conserved domains in the RT of Tf1 indicates that functional...

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

The long terminal repeat retrotransposon Tf1 of Schizosaccharomyces pombe uses a unique mechanism of self priming to initiate reverse transcription. Instead of using a tRNA, Tf1 primes minus-strand synthesis with an 11-nucleotide RNA removed from the 5 end of its own transcript. We tested whether the self primer of Tf1 was similar to tRNA primers in being removed from the cDNA by RNase H. Our analysis of Tf1 cDNA extracted from virus-like particles revealed the surprising observation that the dominant species of cDNA retained the self primer. This suggests that integration of the cDNA relies on mechanisms other than reverse transcription to remove the primer.The reverse transcription of retroviruses and long terminal repeat (LTR) retrotransposons is primed by specific tRNA species for minus-strand initiation and by polypurine tracts (PPT) for plus-strand initiation. These primers play a critical role in defining the ends of the cDNA such that the "CA" dinucleotides required for integration are positioned at the termini (23). During reverse transcription the RNase H activity of reverse transcriptase (RT) removes the tRNA and PPT primers from the 5Ј ends of the cDNA so that their sequences are not copied into the 3Ј termini of the complementary strand of cDNA. This is a critical feature of reverse transcription because addition of these nucleotides after the conserved "CA" at the 3Ј ends of the cDNA would block integration.The LTR retrotransposon Tf1 of Schizosaccharomyces pombe uses a unique mechanism of self priming to initiate reverse transcription (11)(12)(13)15). Instead of using a tRNA, Tf1 primes minus-strand synthesis with an 11-nucleotide RNA removed from the 5Ј end of its own transcript. An increasing number of LTR elements in eukaryotes from yeast to vertebrates are found to use this self-priming mechanism (3, 16).A recent study of mutations in the RT of Tf1 revealed that RNase H removes the PPT from the 5Ј end of the plus-strand cDNA (2). Random mutagenesis of RT resulted in a cluster of mutations in RNase H that inhibited the removal of the PPT without reducing the amount of cDNA produced. That RNase H was responsible for primer removal was not surprising because the PPT of Tf1 is similar to those of other LTR elements. However, because of its unique nature, it is not known whether the self primer of Tf1 is also removed by RNase H.To determine whether the self primer of Tf1 was removed during reverse transcription, we sequenced the ends of cDNA produced by the plasmid copy of Tf1 (Tf1-neoAI) used for transposition assays (1, 11). Virus-like particles were isolated from cultures of S. pombe (YHL6742) and purified on sucrose gradients, and cDNA was extracted (11,14). We determined the sequence of the cDNA at the 3Ј end of the plus strand by ligating an oligonucleotide to the cDNA and using a complementary oligonucleotide to amplify by PCR the terminal sequences (Fig. 1). A ligation bias that could alter the sequence data obtained with this technique has not been observed (18). The ligation-mediated PCR ...