Mechanisms of subtelomere recombination in telomerase deficient tumors

项目摘要 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， 称为ALT，用于替代延长机制。近10%的人类肿瘤，通常是肉瘤或 胶质母细胞瘤缺乏维持端粒的端粒酶。为了研究这些非- 端粒酶端粒维持机制，我们使用E？myc+mTR-/-小鼠，基因删除， 端粒酶（mTR）的RNA成分，并与E？myc+小鼠杂交，后者发展为B细胞淋巴瘤。 为了测试重组在这些非端粒酶端粒维持机制中的作用， 目前使用shRNA方法来抑制各种重组基因，然后检查 肿瘤生长速度的变化。为了了解更多的机制，我们对肿瘤进行了分析， 进行亚端粒重组。在这个建议中，我们打算研究参与基因的贡献， 复制叉停滞，以及DNA修复的作用，通过一种新的机制，称为断裂诱导 复制的为了监测哺乳动物细胞中断裂诱导的复制，我们将开发检测方法， 断裂诱导复制的标志。这些特征包括杂合性丢失、非互惠性 易位和片段性复制。最后，在独立阶段，我们打算检查 LINE-1逆转录转座在断裂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，占小鼠的很大一部分， 人类基因组这些移动的遗传元件通过RNA中间体移动， 称为靶启动逆转录。此外，我们发现逆转录转座也可以发生在 内源性DNA断裂和端粒功能障碍。此外，非LTR反转录转座子在 果蝇完全参与了端粒的维持机制。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的端粒维持 细胞，从而导致缺乏端粒酶的肿瘤的发生。

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.