The medical importance of retroviruses such as HIV-1 has intensified the need to understand the molecular details of reverse transcription and integration. As a result of their similarity to retroviruses, LTR-retrotransposons in simple eukaryotes are a powerful model. The fundamental information about LTR-retrotransposons may lead to the identification of new antiviral stradegies or targets that could be used to combat the spread of HIV-1. The retrotransposon we study is the Tf1 element of the fission yeast, Schizosaccharomyces pombe. One of our objectives is to map the residues of RT that mediate individual steps of reverse transcription. Another important objective of our research is to identify the mechanisms responsible for the insertion preference of Tf1 at pol II promoters. This work is largely motivated by similar preferences that have been reported for the integration of HIV-1 and Murine Leukemia Virus. The reverse transcription of retroviruses and LTR-retrotransposons is a complex sequence of reactions that produce several critical intermediate products. The synthesis of intermediates requires both the DNA polymerization and RNase H activities of RT. The RNase H domain must degrade the RNA once it is used as template, recognize and preserve the plus strand primer of reverse transcription (PPT), and remove the PPT once it has primed plus strand synthesis. Although much is known about the amino acids that catylize the DNA synthesis and RNA degradation, much less is known about which residues and structures are required for the recognition and removal of the PPT. To identify which residues of RT mediate the interactions with the PPT we generated a collection of 3,000 strains that were unable to support transposition and screened these with a genetic assay that detects intermediates of reverse transcription. We found 35 strains that despite there defects in transposition produced what appeared to be normal levels of full-length, double-stranded cDNA. The remaining elements produced an interesting array of incomplete products. The focus of our experiments became a cluster of mutations in RNase H . The significance of this domain was demonstrated by a crystalographic study of HIV RT that showed it is a ?primer grip? and interacts directly with the nucleotides of the PPT adjacent to the position that is cleaved by RNase H to create and then remove the plus strand primer. Both the position and the sequence of the primer grip residues in HIV corresponded well to the cluster of mutations we identified in the RNase H of Tf1. These observations led us to propose that the mutations in the RNase H of Tf1 that result in full-length cDNA are defective for transposition because the recognition of the PPT was altered. A defect of a few nucleotides in the cleavage of the PPT from the 5? end of the plus strand would have a drastic impact on the ability of IN to catalyze strand transfer. To test the hypothesis that the cluster of mutants in RNase H altered the cleavages of the PPT we characterized the sequences of the 3? end of the minus strand cDNA. RNA ligase was used to attach an oligo to the 3? ends of the cDNA to amply by PCR a product containing the junction of the 3? end. These products were inserted into a vector and 150 to 200 independent junctions were sequenced for each mutant RT. The mutations showed a significant drop in the full-length species that correlated with an increase in 3?ends that retained the PPT sequence. Thus, our screen for mutations in RT that make cDNA products identified the primer grip of RNase H as contributing to the removal of the PPT RNA from the end of the LTR. Surprisingly, this ability to process the PPT is specific and is not required for the other steps in the pathway of reverse transcription. The integration of HIV-1 cDNA shows a significant preference for actively transcribed genes. Similarly, the insertion of murine leukemia virus shows a strong preference for sites within 5kb of transcription initiation. Very little is known about how these viruses interact with the structures of chromatin and recognize their target sites. The study of the retrotransposons Ty1 and Ty3 of S. cerevisiae has demonstrated quite clearly that much has been learned about integration directed to the promoters of pol III transcribed genes. However, our recent observation that the integration of Tf1 occurs specifically at pol II promoters presents the real opportunity to study in S. pombe an integration mechanism that parallels that of retroviruses. The complete DNA sequence of the genome of S. pombe provides the opportunity to investigate the entire complement of transposable elements (TEs), their association with specific sequences, their chromosomal distribution, and their evolution. The sequences of Tf elements used in this analysis were identified through homology-based searches. Only two families of LTR retrotransposons, Tf1 and Tf2, are known to exist in S. pombe. They are closely related elements that differ only in their sequence of Gag and the LTRs. The nucleotide sequences of Tf1 and Tf2 were used as query sequences to BLAST against the entire genome of the laboratory strain of S. pombe, 972. In this analysis, we confirmed that there were no full-length Tf1 elements within the laboratory strain 972. There were however 13 full-length elements of Tf2. The full-length Tf2 elements are a very homogeneous group having an average pairwise DNA sequence identity of 99.7%. The high level of sequence identity among the Tf2 elements indicates that they have all transposed very recently. The solo LTRs found in the genome of S. pombe can be classified into at least three large groups as follows: (1) those that are closely related to Tf2, (2) those that are closely related to Tf1 and (3) many small families of LTRs that are more distantly related to Tf1 and Tf2. These designations are derived from a complete phylogenetic characterization of the LTRs using DNA distance values from comparisons with LTRs of full-length Tf1 and Tf2 elements. The short distances of many of the terminal branches of Tf1 and Tf2 indicate they represent the largest number of recently active elements within the genome. To determine whether preferences for integration sites existed during the insertion of the 186 Tf sequences, we compared the locations of solo LTRs and full-length Tf2s to the positions of all 4,984 predicted ORFs of S. pombe. We found that all insertions were located exclusively in intergenic regions of the genome. These inserts were only found in intergenic spaces that contained pol II promoters. In addition, the LTRs were clustered within 300 nulceotides of the 5? end of ORFs. These specific positions could be the result of selective pressures, either positive or negative, that favor populations of S. pombe with each of the patterns observed. Alternatively, the patterns of the Tf sequences could be strictly the result of biochemical mechanisms of integration that caused the patterns we observed. Each of the biases in the position of Tf sequences described above were very similar in pattern and magnitude to the positions of insertions resulting from the induction of Tf1 transposition. These extensive similarities argue strongly that the biases in the position of Tf sequences as observed in the genome of S. pombe are the result of biochemical preferences of integration for specific sites. These data indicate that Tf elements recognize and insert upstream of RNA polymerase II promoters.
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