Mechanisms of subtelomere recombination in telomerase deficient tumors

描述（由申请人提供）：端粒维持在端粒酶缺失的情况下可以通过各种机制发生，统称为ALT，作为替代的延长机制。近10%的人类肿瘤，通常是肉瘤或胶质母细胞瘤，缺乏端粒酶来维持端粒。为了研究这些非端粒酶的端粒维持机制，我们使用E5myc+mTR-/-小鼠，基因上删除端粒酶（mTR）的RNA成分，并将其杂交到发展为b细胞淋巴瘤的E5myc+小鼠。为了测试重组在这些非端粒酶端粒维持机制中的作用，我们目前使用shRNA方法抑制各种重组基因，然后检查肿瘤生长速度的变化。为了了解更多的机制，我们分析肿瘤的亚端粒重组。在这个提议中，我们打算检查参与复制叉停滞的基因的贡献，以及DNA修复的作用，通过一种被称为断裂诱导复制的新机制。为了监测断裂诱导复制在哺乳动物细胞中，我们将开发检测断裂诱导复制的标志的方法。这些特征包括杂合性丧失、非互易易位和片段重复。最后，在独立阶段，我们打算研究LINE-1逆转录在断裂诱导复制和端粒维持中的作用。非LTR逆转录转座子，被称为LINE-1或L1，占小鼠和人类基因组的很大一部分。这些可移动的遗传元件通过一种被称为靶引逆转录的机制通过RNA中间体移动。此外，我们发现逆转录也可以发生在内源性DNA断裂和功能失调的端粒。此外，果蝇的非ltr逆转录转座子完全参与了端粒维持机制。因此，我们打算研究非ltr逆转录转座子是否也有助于人类和小鼠细胞的端粒维持，从而解释缺乏端粒酶的肿瘤的发生。

DESCRIPTION (provided by applicant): 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 E5myc+mTR-/- mice, genetically deleted for the RNA component of telomerase (mTR), and crossed to E5myc+ 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. PUBLIC HEALTH RELEVANCE: Telomere maintenance can occur without telomerase and 10% of human tumors lack telomerase activity. These additional mechanisms can also contribute to telomere maintenance in tumors expressing telomerase and likely are selected in tumors inhibited for telomerase. These studies are relevant to understanding tumorigenesis that occurs in the absence of telomerase, including human sarcomas and potentially certain types of brain tumors. )