Mechanisms of subtelomere recombination in telomerase deficient tumors

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PROJECT SUMMARY Telomere maintenance in the absence of telomerase can occur by various mechanisms, collectively termed ALT, for alternative lengthening mechanisms. Nearly 10% of human tumors, often sarcomas or glioblastomas, lack the enzyme telomerase for telomere maintenance. In order to study these non- telomerase telomere maintenance mechanisms we are using E¿myc+mTR-/- mice, genetically deleted for the RNA component of telomerase (mTR), and crossed to E¿myc+ mice, which develop B-cell lymphoma. To test the role of recombination in these non-telomerase telomere maintenance mechanisms we are currently using an shRNA approach to inhibit various recombination genes, and then examining the change in growth rate of tumors. To understand more about the mechanisms we then assay the tumors for subtelomere recombination. In this proposal we intend to examine the contribution of genes involved in replication fork stalling, and the role of DNA repair by a novel mechanism known as break-induced replication. To monitor break-induced replication in mammalian cells, we will develop assays to detect the hallmarks of break-induced replication. These characteristics include loss of heterozygosity, non-reciprocal translocations, and segmental duplications. Finally during the independent phase we intend to examine the role of LINE-1 retrotransposition during break-induced replication and telomere maintenance. Non- LTR retrotransposons, referred to as LINE-1 or L1, account for a significant fraction of the mouse and human genomes. These mobile genetic elements move by an RNA intermediate using a mechanism called target-primed reverse transcription. In addition we found that retrotransposition can also occur at endogenous DNA breaks and dysfunctional telomeres. Furthermore, non-LTR retrotransposons in Drosophila entirely contribute to the mechanism of telomere maintenance. Thus we intend to examine whether non-LTR retrotransposons also contribute to telomere maintenance in both human and mouse cells and thus account for the occurrence of tumors lacking telomerase.