DNA damage tolerance pathway choice in Drosophila

果蝇DNA损伤耐受途径的选择 项目摘要 当复制分支遇到DNA损伤时，它们必须绕过这些路障 DNA损伤耐受机制或DNA合成将停止，导致基因组出现缺口， 基因组的不稳定性，以及最终的细胞死亡。细胞用于旁路的策略包括 易出错的跨损伤合成聚合酶的招募，模板切换 复制叉子，并在病变下游重新启动，然后缝隙填充。选择 损伤耐受机制很重要，因为某些途径比其他途径更具突变性。 然而，后生动物对病变旁路选择的控制还不是很清楚， 尤其是在不同组织的情况下。这样的知识对于理解 正常细胞和癌细胞如何处理由 用化疗药物治疗。 我们最近发现Rev1蛋白是DNA损伤耐受性的关键介质 在果蝇身上。Rev1促进跨损伤聚合酶的募集以绕过受损的 碱基，我们已经证明了这是快速分裂组织的首选耐受机制 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，我们将使用区域特定的突变体， 遗传分析和幼虫成像盘中的细胞分析以表征Rev1如何 协调各种损伤耐受途径，以促进DNA复制的连续性。在……里面 此外，我们将使用遗传和生化方法来鉴定参与 损害容忍度。我们的调查将得到我们开发的新技术的帮助 通过收集丰富的DNA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，整个果蝇的使用提供了 美国有机会在组织特异性和 发展。总之，我们提出的研究将推进我们的长期目标，以了解 为什么不同的DNA损伤耐受机制在不同的环境中被优先使用 以及这种旁路策略的选择如何影响细胞生存和基因组稳定性。

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.