The retrovirus HIV is the major disease causing member of the long terminal repeat (LTR)-containing retroelements. The development of new antiviral strategies requires an increased understanding of retrovirus biology. To understand the mechanisms that retroviruses use to propagate, we study LTR-retrotransposons, a closely related variety of retroelement that exists in model organisms such as yeast. The LTR-retrotransposon we study is the Tf1 element of the fission yeast, Schizosaccharomyces pombe. The focus of our efforts is to understand the molecular details of mechanisms such as reverse transcription, transport of Tf1 into the nucleus, and the integration of Tf1 cDNA. The process of reverse transcription is a complex set of reactions mediated by reverse transcriptase (RT), the polymerase that uses an RNA template to synthesize a full-length, double-stranded DNA copy of the element. RT requires specialized primers to initiate the synthesis of minus and plus strand DNA. During this process RT also mediates the transfer of DNA intermediates to specific segments of the transposon template. A key function of RT required for the production of mature cDNA is its RNase H domain, the enzyme that degrades the RNA templates and primers once they have annealed to DNA. Although much is known about the components of RT required for the polymerization of DNA and the degradation of RNA, little is known about which amino acids of RT recognize the primers or mediate the transfer events. To this end, we used assays of yeast genetics to identify residues of RT that are required for late steps in the pathway of cDNA synthesis. Ten of the mutations clustered within a small region of RNase H. Surprisingly, these mutations do not significantly reduce the production of full-length double stranded cDNA. Interestingly, most of these mutations occur in residues that correspond to the RNase H primer grip, amino acids that form direct contacts with critical nucleotides of the plus strand primer, the polypurine tract (PPT). Sequence analysis of the cDNA produced by two mutants reveals a defect in the removal of the PPT from the 5? end of the plus strand. These data demonstrate that the primer grip has the specific ability to process the PPT and this activity is not required for production of the full-length double stranded cDNA. Since the process of integration has the potential to compromise the fitness of the host, many transposons select specific sites for integration that avoid the disruption of genes. We wish to understand the balance between the ability of transposons to insert into the genome of S. pombe verses the efforts of this yeast to thrive. The recently completed sequence of the S. pombe genome allowed us to ask where transposon integrations have occurred. Surprisingly, the only transposons found in the genome were related to the Tf1/Tf2 family of LTR retrotransposons. Examination of all the 202 transposon sequences revealed that each element was located within intergenic regions of sequence. Since 60.2% of the genome of S. pombe is coding sequence, the positions of the LTRs was strongly biased. Surprisingly, the position of the insertions was associated with the 5? end of genes. This suggests that the preintegration complex may recognize some component of the transcription machinery. A genome-wide study of Tf1 transposition in S. pombe revealed that these new insertion events also occurred within intergenic regions and were associated the 5? end of genes. This result demonstrates that the location of the preexisting transposon sequences was due to integrase mediated selection. The systematic insertion of Tf1 into intergenic regions represents a novel method for protecting host genes from damage due to integration. The selection of these target sites may be due to the presence within integrase of a domain that is related to amino acid sequences known to bind to histones with specific modifications. Recent data from the lab of Dr. Shiv Grewal revealed an association between intergenic sequences next to pol II promoters and high levels of methylation of histone H3 at lysine 4. This suggested that the integrase of Tf1 may recognize the 5? end of genes by binding methylated histone H3. To test this possibility we measured the activity of Tf1 in a strain that lacks the methytransferase that methylates lysine 4 of histone H3. We observed that without the methytransferase, transposition frequencies were significantly reduced. In addition, an assay that detects cDNA in the nucleus demonstrated that the lack of the methyltransferase did not reduce the levels of cDNA or its import into the nucleus. We are currently testing whether the integrase of Tf1 can interact directly with specific forms of histone.
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