Mechanisms of subtelomere recombination in telomerase deficient tumors

说明书(由申请人提供)：在没有端粒酶的情况下，端粒的维持可以通过各种机制发生，统称为ALT，用于替代延长机制。近10%的人类肿瘤，通常是肉瘤或胶质母细胞瘤，缺乏端粒维持所需的端粒酶。为了研究这些非端粒酶端粒维持机制，我们使用了E5myc+MTR-/-小鼠，端粒酶RNA成分(MTR)基因缺失，并与患B细胞淋巴瘤的E5myc+小鼠杂交。为了测试重组在这些非端粒酶端粒维持机制中的作用，我们目前正在使用shRNA方法来抑制各种重组基因，然后检测肿瘤生长速度的变化。为了更多地了解这一机制，我们随后分析了肿瘤的端粒下重组。在这项建议中，我们打算研究复制分叉停滞中涉及的基因的作用，以及DNA修复通过一种被称为断裂诱导复制的新机制所起的作用。为了监测Break诱导的哺乳动物细胞复制，我们将开发检测Break诱导复制的特征的分析方法。这些特征包括杂合性丢失、非互换易位和节段性复制。最后，在独立阶段，我们打算研究LINE-1逆转录转座在中断诱导的复制和端粒维持中的作用。非LTR反转录转座子，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RNA中间体移动，使用一种称为靶标引发的反转录的机制。此外，我们发现逆转录转座也可以发生在内源性DNA断裂和功能失调的端粒上。此外，果蝇中的非LTR反转录转座子完全参与了端粒维持的机制。因此，我们打算研究非LTR反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.