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DNA damage tolerance pathway choice in Drosophila

果蝇 DNA 损伤耐受途径的选择

基本信息

批准号：
10809272
负责人：
Mitch McVey
金额：
$ 0.97万
依托单位：
TUFTS UNIVERSITY MEDFORD
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2025-04-30
项目状态：
未结题

项目摘要

DNA damage tolerance pathway choice in Drosophila Project Summary When replication forks encounter DNA lesions, they must bypass these roadblocks through DNA damage tolerance mechanisms or DNA synthesis will stop, leading to gaps in the genome, genomic instability, and eventual cell death. Strategies that cells use for bypass include the recruitment of error-prone translesion synthesis polymerases, template switching at the replication fork, and repriming downstream of the lesion followed by gap filling. The choice of damage tolerance mechanism is important, as some pathways are more mutagenic than others. However, the control of lesion bypass pathway choice in metazoans is not well understood, particularly in the context of different tissues. Such knowledge is critical in order to understand how both normal and cancerous cells deal with replication-blocking lesions that result from treatment with chemotherapeutic agents. We have recently discovered that the REV1 protein is a key mediator of DNA damage tolerance in Drosophila. REV1 promotes the recruitment of translesion polymerases to bypass damaged bases, which we have shown is the preferred tolerance mechanism in rapidly dividing tissues in the developing fly. In addition, REV1 appears to mediate template switching through an unknown mechanism. In the experiments described here, we will use domain-specific mutants, genetic analysis, and cellular assays in larval imaginal discs to characterize how REV1 coordinates various damage tolerance pathways to promote continuance of DNA replication. In addition, we will use both genetic and biochemical methods to identify new proteins involved in damage tolerance. Our investigations will be aided by novel techniques that we have developed to assess DNA repair and mutagenesis in both cells and tissues and by a rich collection of DNA repair and replication mutants. The use of whole Drosophila in our experimental design provides us with an opportunity to study damage tolerance in the context of tissue specificity and development. Together, our proposed studies will advance our long-term goal to understand why different DNA damage tolerance mechanisms are preferentially used in different contexts and how this choice of bypass strategy impacts cellular survival and genome stability.

果蝇 DNA 损伤耐受途径的选择项目概要当复制叉遇到 DNA 损伤时，它们必须绕过这些障碍 DNA损伤耐受机制或DNA合成将停止，导致基因组中出现缺口，基因组不稳定，最终导致细胞死亡。细胞用于旁路的策略包括招募容易出错的跨损伤合成聚合酶，在复制叉，并在病变下游重新启动，然后填充间隙。的选择损伤耐受机制很重要，因为某些途径比其他途径更具诱变性。然而，后生动物中病变旁路途径选择的控制尚不清楚，特别是在不同组织的情况下。这些知识对于理解至关重要正常细胞和癌细胞如何处理由复制引起的病变用化疗药物治疗。我们最近发现 REV1 蛋白是 DNA 损伤耐受的关键介质在果蝇中。 REV1促进跨损伤聚合酶的募集以绕过受损的碱基，我们已经证明这是快速分裂组织中的首选耐受机制发育中的苍蝇。此外，REV1 似乎通过一个未知的机制。在这里描述的实验中，我们将使用域特异性突变体，幼虫成虫盘中的遗传分析和细胞测定来表征 REV1 协调各种损伤耐受途径以促进 DNA 复制的持续性。在此外，我们将使用遗传和生化方法来鉴定参与的新蛋白质损伤容限。我们开发的新技术将有助于我们的调查通过丰富的 DNA 集合来评估细胞和组织中的 DNA 修复和突变修复和复制突变体。在我们的实验设计中使用整个果蝇提供了我们有机会在组织特异性和发展。我们提出的研究将共同推进我们的长期目标，以了解为什么在不同的情况下优先使用不同的 DNA 损伤耐受机制以及这种旁路策略的选择如何影响细胞存活和基因组稳定性。