Long terminal repeat (LTR)-retrotransposons are mobile elements that resemble retroviruses. RNA synthesized from LTR-retrotransposons is copied back into DNA, and this """"""""copy DNA"""""""" (cDNA) is then integrated at a new location in the genome. LTR-retrotransposons are widespread throughout eukaryotes, where they activate and inactivate host genes, promote genome rearrangements, and produce and disperse copies of host genes. Hence, they play an important role in the structure and evolution of the eukaryotic genome. Ty1 LTR-retrotransposons of the yeast, Saccharomyces cerevisiae, are typically functional and expressed at high levels, yet transposition occurs rarely because cDNA synthesis is inhibited by numerous host factors. The long-term goal of our work is to understand how the dormancy of functional LTR-retrotransposons is maintained. Our recent work has demonstrated that telomere erosion in the absence of telomerase triggers the """"""""lesion-induced Ty1 activation pathway"""""""", a DNA-damage signaling pathway that mobilizes Ty1 elements. In this proposal, we will test the hypothesis that a large group of genes necessary for genome stability and for the suppression of tumor formation in higher eukaryotes (known as caretaker genes) blocks the formation DNA lesions that trigger the """"""""lesion-induced Ty1 activation pathway"""""""". In their absence, the Ty1 activation pathway stimulates the synthesis of Ty1 cDNA, which results in excessive transposition. Moreover, the proposed experiments will test the hypothesis that enhanced Ty1 reverse transcriptase activity in telomerase-negative mutants results in the production and dispersal of cDNA copies of a subtelomeric repeat called Y'. Amplification of Y' repeats is one characteristic of telomerase-independent, recombination-dependent alternative telomere structures. Alternative telomere structures allow both yeast cells and human cancer cells to grow continuously in the absence of telomerase.
The specific aims are: 1. Screen previously identified inhibitors of Ty1 transposition for those that repress the lesion-induced Ty1 activation pathway. Identify and characterize the components of the lesion-induced Ty1 activation pathway. 2. Perform biochemical characterization of specific intermediates in retrotransposition in wild-type and mutant strains to identify the mechanism of stimulating cDNA synthesis in mutants in which the lesion-induced Ty1 activation pathway is activated. 3. Determine the structure of Y' cDNA in telomerase-negative mutants, and determine how and where Y' cDNA is inserted into the genome. Determine the role played by Ty1 gene products in the synthesis and mobility of Y' cDNA.
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