This grant is focused on a molecular dissection of the mechanisms by which the yeast Ty1 elements replicate their nucleic acids, and how they integrate the newly made cDNA into very specific genomic target regions. Replication is mediated by host RNA polymerase II, the Ty1-encoded reverse transcriptase (RT) enzyme, and a cellular tRNA primer. Integration of the resulting cDNA is mediated by the Ty1-encoded integrase (IN). The process of integration is targeted to very specific regions of the host genome, namely """"""""integration windows"""""""" of several hundred base pairs immediately upstream of RNA polymerase III-transcribed genes. We seek to understand how this targeting is directed, presumably by a combination of Ty1-encoded and host functions. We will carry out genetic analyses of Ty1 retrotransposon and host functions, supplemented by biochemical studies that exploit the in vitro systems we have previously developed for the study of Ty1 reverse transcription and integration reactions.
The Specific aims are: 1) Molecular definition of the Ty1 reverse transcription pathway. We will comprehensively analyze the minimum cis-acting sites for Ty1 transposition, which are involved in packaging Ty1 RNA and priming its reverse transcription. We will analyze the mode of strong-stop DNA transfer, identify what plays the role of nucleocapsid protein in Ty1, and will continue efforts to develop a soluble in vitro system to study Ty1 reverse transcription. 2) Definition and characterization of the pathway leading to targeted integration of Ty1 upstream of pol III-transcribed genes. We will investigate the highly non-random nature of Ty1 integration, which occurs in specific """"""""integration windows"""""""". We will use a combination of genetic screens and biochemical studies using in vitro integration systems to identify important factors required for targeting. We will also biochemically analyze the Ty1 pre-integration complex and its localization to the nucleus. 3) Characterization of the host response to retrotransposition. We will begin new projects to explore the host transcriptional response to retrotransposition, and we will examine whether there is cell cycle regulation of transposition. We will examine the transcriptional interactions between Ty1 elements and the pol III genes that often lie adjacent to them.
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